Journal of Oleo Science Copyright ©2018 by Japan Oil Chemists’ Society J-STAGE Advance Publication date : September 13, 2018 doi : 10.5650/jos.ess18078 J. Oleo Sci. Lipase – catalyzed Modification of Rice Bran Oil Solid Fat Fraction Patchara Kosiyanant1, Garima Pande2, Wanna Tungjaroenchai1, and Casimir C. Akoh2＊ 1 Faculty of Agro-Industry, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, THAILAND 2 Department of Food Science and Technology, The University of Georgia, Athens, Georgia, 30602, USA Abstract: This study used a rice bran oil solid fat fraction (RBOSF) to produce cocoa butter alternatives via interesterification reaction catalyzed by immobilized lipase (Lipozyme® RM IM) in hexane. Effects of reaction time (6, 12, and 18 h), temperature (55, 60, and 65℃), mole ratios of 3 substrates [RBOSF:palm olein:C18:0 donors (1:1:2, 1:2:3, and 1:2:6)] were determined. The substrate system was dissolved in 3 mL of hexane and 10% of lipase was added. Two sources of C18:0 donors, stearic acid (SAd) and ethyl stearate (ESd) were used. Pancreatic lipase – catalyzed sn-2 positional analysis was also performed on both substrates and structured lipids (interesterification products). Structured lipids (SL) were analyzed by gas – liquid chromatography (G40.35LC) for fatty acid composition. Major fatty acids of RBOSF were C18:1, oleic acid (OA, 41.15±0.01%), C18:2, linoleic acid (LA, 30.05±0.01%) and C16:0, palmitic acid (PA, 22.64±0.01%), respectively. A commercial raw cocoa butter (CB) contained C18:0, stearic acid (SA, 33.13±0.04%), OA (32.52±0.03%), and PA (28.90±0.01%), respectively. Fatty acids at sn-2 position of RBOSF were OA (46.52±0.63%) and LA (42.98±1.1%), while major fatty acid at sn-2 position of CB was OA (85.24±1.22%). The RBOSF had low SA (2.40±0.01%) compared to CB (33.13±0.04%). The content of OA (46.52±0.63%) at sn-2 position in RBOSF was half of that found in CB (85.24±1.22%). Optimal reaction was 1:2:6 mole ratio of the substrate (RBOSF:PO:SAd), at 65℃ for 12 h. Fatty acid compositions of the SL were 31.72±0.99% SA, 30.91±0.53% LA, 23.18±0.32% OA, and 13.26±0.34% PA, respectively. Fatty acids at sn-2 position of the SL were 53.72±4.21% OA, 25.11±3.69% LA, 14.18±1.58% PA, and 6.99±0.02% SA, respectively. DSC curves showed the melting point of CB at 20.94℃, while those of the SL were 14.15 and 40.35℃, respectively. The melting completion temperature (Tmc) of CB was 25.5℃ while that of SL was 43.9℃, respectively. Key words: rice bran oil, solid fraction, cocoa butter, interesterification, lipases, structured lipids 1 INTRODUCTION ols, and tocotrienols have antioxidant activity to reduce Rice bran oil（RBO）is an oil extracted from rice bran, a risk of cancer. Cholesterol lowering properties of RBO are by-product of rice milling process. This oil has been recog- reported as effects of these bioactive compounds more nized as a healthy cooking oil in Asian countries and it has than by its fatty acid composition in human3－5）and animal6）. been called Heart oil in Japan. Typically, RBO contains Several studies have been conducted in search of more ap- about 22％ saturated fatty acid（SFA）, 43％ monounsatu- plications of RBO in food industry7, 8）. rated fatty acid（MUFA）, and 35％ polyunsaturated fatty Cocoa butter（CB）is an important ingredient in chocolate acid（PUFA）, respectively. Fatty acid composition of and confectionary products. Fatty acid composition and natural RBO is close to the recommendations on edible oils physico-chemical properties of CB have influence on by American Heart Association（AHA）and World Health sensory and physico-chemical qualities of these products9, 10）. Organization（WHO）1）. Rice bran oil contains 37.6％ OA Chemical compositions of CB are predominant triacylglyc- （C18:1）, 33.6％ LA（C18:2）, 24.8％ PA（C16:0）, and 2.0％ erols（TAGs）of 15％ 1,3-dipalmitoyl-2-oleoylglycerol of SA（C18:0）. Common fatty acids at sn-2 position typical- （POP）, 40％ 1（3）-palmitoyl-3（1）-stearoyl-2-monoolein ly are found 43％ OA and 50.1％ LA2）. Unsaponifiable com- （POS）, and 27.5％ 1,3-distearoyl-2-oleoylglycerol（SOS）11）. ponents of RBO such as γ-oryzanol, phytosterols, tocopher- Major fatty acids of CB are 34.8％ OA, 34.4％ SA, and ＊Correspondence to: Casimir C. Akoh, Department of Food Science and Technology, The University of Georgia, Athens, Georgia, 30602, USA E-mail: [email protected] Accepted June 13, 2018 (received for review April 30, 2018) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs 1 P. Kosiyanant, G. Pande, W. Tungjaroenchai et al. 26.0％ PA, respectively9）. Due to high price and uncertainty were added accordingly to respective molar ratios and 3 in supply of CB while world demand is high, cocoa butter mL of n-hexane was added and heated to dissolve all the alternatives or replacers from cheaper fat and oils through substrate before 10％（ by weight based on total substrate modifications（fractionation, hydrogenation, and chemical weight）of Lipozyme® RM IM was added. The reaction or enzymatic interesterification）is challenging. temperature and time were controlled in a water-bath Enzymatic interesterification was used to modify physi- shaker at 200 rpm. Reaction products were filtered through 12） co-chemical properties of fat and oil with exchange of a Pasteur pipette that contained anhydrous Na2SO（4 anhy- 10, 13） fatty acids within and among TAGs , by using enzyme drous Na2SO4 column）to remove the enzymes. The filtrate as a catalyst14）. Several research used lipase as a catalyst to was concentrated under nitrogen, the extract was further produce a cocoa butter substitute from low value fat and separated and identified for TAGs by thin layer chromatog- oil15）. Another researcher found thermal behavior of a raphy（TLC）with silica gel as a stationary phase, and a de- product from enzymatic interesterification of tea seed oil veloping solvent system of petroleum ether:ethyl was comparable to cocoa butter at 20 - 30℃10）. In addition, ether:acetic acid（80:20:0.5, mL/mL/mL）16）. Identification of Pande and Akoh produced a trans-free structured marga- TAG bands was aided by spraying of 0.2％ 2, 7-dichloroflu- rine fat analog with high stearate soybean oil and palm orescein in methanol, and visualized under UV light, and stearin16）. triolein was used as the standard TAG. An identified TAG The objective of this study was to modify rice bran oil band scraped from TLC plates was dissolved in 2 mL of solid fat fraction by enzymatic modification and to choose diethyl ether and the mixture was vortexed for 2 min, and proper condition to produce CBA. then centrifuged at 1000 rpm for 5 min. Upper layer was filtered through anhydrous Na2SO4 column and dried under nitrogen before determination of fatty acid composition of TAG from SL synthesis. Synthesis of SL product containing 2 MATERIALS AND METHODS similar amounts of stearic acid and oleic acid, to those of 2.1 Materials cocoa butter would be considered optimal reaction. Rice bran oil solid fat fraction（RBOSF）was obtained by dry fractionation at 1℃（ Thai Edible Co. Ltd., Bangkok, 2.3 Fatty acid composition analysis Thailand）. Palm olein（PO）was donated by Loders Croklaan Fatty acid composition of RBOSF was determined by North America（Channahon, IL, USA）. Stearic acid was using Hewlett-Packard 6890 series II gas chromatograph from SAFC Supply Solution（St. Louis, MO, USA）. Lipo- （Agilent Technologies Inc., Palo Alto, CA, USA）. A Supelco zyme® RM IM with declared activity of 275 interesterifica- SP-2560 column（100 m×250 μm, 0.20 μm film）for sample tion unit/g［IUN/g］（ immobilized granulate）from Rhizomu- separation with a flame ionization detector（FID）. An injec- cor miehei was purchased from Novozymes（North tion of 1 L of sample（FAMEs）was made at a split ratio of America Inc., Franklinton, NC, USA）. An internal standard 5:1. Helium as a carrier gas was used at a constant flow fatty acid C15:0 was purchased from TCI（Tokyo, Japan）. rate of 1.1 mL/min. Inlet and detector temperature was Major organic solvents included n-hexane, diethyl ether, controlled at 250℃ and the oven was held at 140℃ for 5 and petroleum ether were from Fisher Scientific（Norcross, min, then increased up to 240℃ at a rate of 4℃/min and GA, USA）and Sigma-Aldrich Chemical Co.（ St. Louis, MO, held at 240℃ for 17 min. USA）. Ethyl stearate, triolein, 2-oleylglycerol（2-monoacyl- Preparation of FAMEs was followed18）with some modifi- glycerol, 2-MAG）, and lipid standards Supelco 37 compo- cation of the method17）. A sample of 100 mg was prepared nent fatty acid methyl esters（FAMEs）mix were purchased in a Teflon-lined screw-capped test tubes and 200 μl of an from Sigma-Aldrich Chemical Co.（ St. Louis, MO, USA）. internal standard（C15:0 20mg/mL hexane）was added and mixed well. Hexane was evaporated by N2 flushing, then 2 2.2 Determination of optimal condition for enzymatic mL of 0.5 N NaOH in methanol was added and vortexed for modication of RBOSF 1 min. The sample was heated for 5 min at 100℃ in an Factorial arrangement（2×33）in CRD experimental oven. The sample was cooled in an ice bath to room tem- design was used for enzymatic synthesis of SL, a modified perature before 2 mL of 14％ BF3 in methanol as a catalyst lipid.