Impact of Different Deacidification Methods on Quality Characteristics and Composition of Olein and Stearin in Crude Red Palm Oil

Transcription

1 Journal of Oleo Science Copyright 2014 by Japan Oil Chemists Society doi : /jos.ess14060 Impact of Different Deacidification Methods on Quality Characteristics and Composition of Olein and Stearin in Crude Red Palm Oil Prasanth Kumar Punathil Kannan and Gopala Krishna Ambale Gundappa * Department of Lipid Science & Traditional Foods, CSIR-Central Food Technological Research Institute, Mysore , India Abstract: Crude red palm oil of 8.7% free fatty acid content was deacidified using enzyme (lipase from Rhizomucor miehei), solvent (ethanol) and chemical (aqueous Sodium hydroxide) and its impact on chemical characteristics and composition were evaluated. Deacidification of oil using enzyme showed nearly 100% product yield. The neutral lipid loss after ethanol and sodium hydroxide deacidification of the oil was 13.6% and 19.5% respectively. The enzyme deacidified oil has shown a higher value in unsaponifiable matter (0.91%), monoacylglycerols (2.8%) and diacylglycerols (18.7%) contents as compared to the other two methods of deacidification. Also it showed a higher retention of nutraceuticals such as carotenoids (94%), phytosterols (57%), total tocopherols (71%), squalene (72%), coenzyme Q 10 (99%) and total phenolics (69%) with IC 50 value of 19.7 mg of oil/ml. Stearin content increased in the oil after deacidification with enzyme (47.4%) compared to the stearin content of crude red palm oil (28.6%). The olein fraction contained less saturated fatty acids (41.6%) than the fraction obtained by other two methods (47.2%). The enzyme catalyzed the esterification reaction of free fatty acids in crude red palm oil with added glycerol at 63 with a rotation speed of 150 rpm under vacuum of 5 mmhg for the period of 12 h showed that enzyme based deacidification can be effectively utilized for the preparation of low acidic nutraceutical retained red palm oil. Key words: crude Red palm oil, nutraceutical retention, enzymatic deacidification, palm oil fractions, nutraceutical composition 1 INTRODUCTION Palm oil is currently the most consumed vegetable oil in the world. It is derived from the oil palm fruit Elaeis guineensis mesocarp, which contains varieties of natural antioxidants viz., carotenoids, tocopherols, tocotrienols, phytosterols, squalene, coenzyme Q 10 and phenolics 1, 2. Edem has reviewed the beneficial effect of palm oil along with its nutraceuticals 3. The extraction and retention of these minor constituents in the oil is mainly dependent on the type of processing. The main steps in the processing of crude red palm oil CRPO include the removal of free fatty acids. Almeida et al. have reported the average free fatty acid FFA content of 9.92 and 9.03 for traditionally and industrially processed CRPO respectively in Bahia Brazil 4. High FFA content of CRPO makes it difficult to remove existing FFA by chemical refining due to the formation of emulsion and excessive loss of neutral oil. Oil refineries generally follow steam deacidification to remove ex- isting FFA from CRPO. Steam deacidification operates at under 5 mm vacuum. The high temperature dependent processing of CRPO leads to loss of heat labile minor components like carotenoids, tocopherols, tocotrienols, etc. from the oil. Some of the research work reported has focussed on the effect of processing on carotenoids, tocopherols, tocotrienols and squalene in oils 5 7. Different physical, chemical and biochemical methods are being used for refining high acid vegetable oils 8. Most of them are carrying out the removal of FFA from the oil and each has advantages and disadvantages. Chemical refining and physical steam refining are the techniques commonly applied in edible oil refineries. Chemical refining of oils with high acidity causes high loss of neutral oil due to saponification and emulsification 9. Steam refining removes most of the nutraceuticals from the oil along with free fatty acids. Membrane filtration is not feasible for high acid oil refining as it consumes more energy. High temperature * Correspondence to: Dr. A.G. Gopala Krishna, Chief Scientist and Head, Department of Lipid Science and Traditional Foods, CSIR- Central Food Technological Research Institute (CSIR-CFTRI), Mysore , India aggk_55@yahoo.com; lstf@cftri.res.in Accepted July 28, 2014 (received for review April 7, 2014) Journal of Oleo Science ISSN print / ISSN online

2 P. K. Prasanth Kumar and A. G. Gopala Krishna used in the chemical esterification process makes it expensive 10. High process cost of supercritical fluid extraction and molecular distillation makes the processes more expensive for edible oil deacidification. Choo et al. proposed molecular distillation to prepare red palm olein and the product branded as Carotino 11. This process used low acidic CRPO 5 FFA, consists of several steps and the product has become very expensive for common use. Enzymatic esterification is a safe, easy and cost-effective alternative to other deacidification methods. This process operates at mild reaction conditions without the use of chemicals and solvents. Hence, the process gives safer products. Several microbial lipases and alcohols/nucleophiles have been introduced in enzymatic deacidification process. Kurashige used diacylglycerol DAG and enzyme from Pseudomonas fluorescens for the esterification of free fatty acids in crude palm olein 12. Similarly, Sengupta and Bhattacharyya proposed Mucor miehei lipase for the esterification of rice bran oil free fatty acids with commercial monoacylglycerol MAG 13. All the reported studies dealt with the use of different enzymes and different esterifying agents and their efficiency to reduce the acidity of crude oil. Impact of enzymatic deacidification on the chemical characteristics and nutraceutical retention is scarce. Hence the present study was focused on the deacidification of CRPO with an emphasis on changes in chemical characteristics, composition, its impact on nutraceutical retention and radical scavenging activity, the results of which are presented in this paper. 2 MATERIALS AND METHODS 2.1 Materials Crude red palm oil extracted from Indian grown Elaeis guineensis was obtained from a palm oil industry M/s Oil Palm India Ltd., Kerala, India. Glycerol 99.9 of purity was obtained from Sisco Research Laboratories Ltd., Mumbai, India. Fucosterol, β-sitosterol, stigmasterol, campesterol, α-tocopherol, gallic acid, squalene, coenzyme Q 10 and 1-diphenyl-2-picrylhydrazyl DPPH were obtained from Sigma Chemical Co. St Louis, MO, USA and standard FAME mix from Supelco Inc. Bellefonte, PA, USA. All solvents and chemicals used were of analytical grade. 2.2 Methods Deacidification methods Three methods have been used for deacidification viz., enzymatic method to esterify free fatty acids, solvent extraction of free fatty acids and alkali treatment to remove free fatty acids as soap Enzymatic method Enzymatic deacidification of crude red palm oil was carried out according to recently developed process Indian patent filed 14. 1,3-specific Rhizomucur meihei lipase immobilized on a macroporous anion exchange resin Lipozyme RM IM was used for the esterification reaction. Glycerol was used as esterifying agent, based on lower cost and availability than MAG and DAG. The reaction was carried out with 100 g portion of CRPO taken in 500 ml capacity Soxhlet flask and glycerol was added in 1:2 molar ratio to FFA as palmitic acid. Lipozyme RM IM 5 w/w was introduced into the reaction medium and the reaction was operated at 63 with a rotation of 150 rpm under vacuum of 5 mmhg for the continuous removal of moisture from the reaction mixture. The reaction was carried out for 12 h. At the end of reaction, deacidified RPO was filtered under hot condition to remove enzyme and used for the next batch. The enzyme was used upto 20 times Solvent extraction method Deacidification was carried out in separating funnel with 100 g of CRPO, ethanol 90 and hexane in the ratio of 1:1:1 w/v/v were shaken for 15 min and allowed to separate into two phases. Oil phase settled at the bottom of the funnel was separated and continued the solvent extraction steps, if necessary. The use of hexane has reduced the viscosity of CRPO during deacidification and this has enhanced the separation of two phases and recovery of neutral lipid during the removal of FFA. Seven consecutive extractions were carried out to minimize the FFA level of the CRPO to the acceptable level of 1. Finally, the solvent in the oil phase was removed under vacuum at 40 using rotavapor Buchi Labortechnik, Switzerland Alkali treatment method Calculated amount of sodium hydroxide solution of strength 20 Be was added to 100 g portions of CRPO and stirred using mechanical stirrer at 150 rpm for 30 min at 63. Later, hexane 200 ml was added and mixed for a period of 10 min. The supernatant oil was decanted and it was washed with warm distilled water 50 until freed from soap. Samples were dried under vacuum at 50 using rotavapor Buchi Labortechnik, Switzerland Determination of neutral lipid loss during deacidification The neutral lipid loss during the deacidification reaction was calculated based on the following formula. Neutral lipid loss Neutral lipid in the CRPO yield of deacidified RPO 100 Neutral lipid in the CRPO Calculation of efficiency of different deacidification methods The efficiency of deacidification by different methods was calculated using the following formula. Final FFA 100 Deacidification efficiency 100 Initial FFA Fractionation of deacidified oils Initially the semi solid CRPO and deacidified RPO were 1210

3 Retention of nutraceuticals in crude red palm oil stirred for 30 min at 65 to ensure that the oil was completely free of irregular crystals before the cooling process. The oils were placed in a chamber at 25 and stirred slowly at 30 rpm over a period of 24 h. The separation of liquid olein and solid stearin fractions was carried out by vacuum filtration Chemical characteristics evaluation The chemical characteristics were evaluated by monitoring free fatty acids FFA AOCS O.M. No. Ca 5a-40, peroxide value PV AOCS O.M. No. Ca 8-53, unsaponifiable matter AOCS O.M. No.d Ca 6a , iodine value AOCS O.M. No. Cd 1d-92, 2004 and saponification value AOCS O.M. No. Cd 3-25, 2004 following the standard methods and practices recommended by the AOCS 16. The analysis was carried out in quadruplicate and the results shown in tables and in text are the arithmetic mean of these four sets of data Determination of weight ratio of olein and stearin Olein and stearin fractions obtained after the dry fractionation process was analysed for its weight ratios in the deacidified RPO. The weight ratio of the fraction was calculated using the formula weight of the representative fraction/weight of the representative red palm oil x Determination of partial glycerides content The MAG, DAG, and TAG contents of the samples were determined by using the standard column chromatographic method. A glass column i.d. 1.8 cm; length, 30 cm was used in which a silica gel mesh size bed was prepared from slurry of silica in petroleum ether. The samples 0.9 g in 5 ml chloroform portion was quantitatively loaded on the column and elution started at the rate of 2 ml/min. TAG, DAG and MAG were eluted with the standard solvent system and the quantity of each fraction was determined gravimetrically after evaporating the solvent as per AOCS O.M. No. Cd 11c Fatty acid composition The fatty acid composition of the oil samples was determined by preparing fatty acid methyl esters FAME and analysis by gas chromatography. FAME of the oil samples were prepared by transesterification, according to AOCS O.M. No. Ce 1b , using boron trifluride in methanol as catalyst 16. Analysis was carried out on a gas chromatograph model-gc-20a, Shimadzu Corporation, Japan equipped with FID detector and a glass capillary column 30 m 0.25 mm, coated with poly 90 biscyanopropyl/10 cyanopropylphenyl siloxane with a film thickness of 0.2 μm SP-2380 Supelco Analytical, Bellefonte, Pennsylvania, USA. The column temperature was maintained isothermal at 180, injector temperature at 220 and detector temperature at 230. A reference standard FAME mix Supelco Inc., Bellefonte, PA, USA was analyzed under the same operating conditions to determine peak identity. The FAMEs were expressed as relative area Triglyceride composition by HPLC The triglyceride composition was analysed on Shimadzu HPLC system consisting of LC-10A pump, fitted with 20 μl injector loop and RID-10A detector. The isocratic separation of triglycerides was achieved by reverse phase HPLC on C18 column Discovery C18 column, 15 cm 4 mm id, 5 μm, Sigma-Aldrich, Bellefonte, PA, USA. The mobile phase was acetone: acetonitrile 70:30, v/v. The TAG peaks were identified according to the retention times of standard triglycerides. The TAG was calculated as relative area percentage as per AOCS O.M. No. Ce 5b-89. TAG peaks were identified based on the theoretical carbon number TCN described by Bland et al 1991 and calculated according to the formula, Theoretical carbon number TCN ECN 0.7 x L 0.6 x O, where, Equivalent carbon number ECN CN 2 x ND, L linoleic acid, O oleic, CN carbon number, ND number of double bond Nutraceutical composition Determination of carotene by spectrophotometric method Carotene content was determined by diluting 1 g of palm oil melted at 65 to 10 ml using hexane and from this 1 ml aliquot further diluted to 10 ml with hexane and absorbance measured at 446 nm using a Uv-vis spectrophotometer Shimadzu UV-1601 Shimadzu Corporation, Kyoto, Japan followed by calculation using the diffusion coefficient of 383 and expressed as mg/kg oil Determination of phytosterols by HPLC Phytosterol content and composition of the oil samples was determined by HPLC according to Lopez-Hernandez et al. 19. A sample of 5 g was weighed in an Erlenmeyer flask to which KOH was added and saponified by refluxing and stirring for 1 h with constant shaking, after cooling to ambient temperature, the mixture was transferred to a separatory funnel to extract the non-saponifiable fraction with 6 50 ml portions of petroleum ether, then extracts pooled, washed with 10 ethanol to remove the alkali. Then the washed petroleum ether fraction was dried with anhydrous sodium sulphate and evaporated to dryness in a rotavapor at 50. The residue was dissolved with mobile phase 30:70 v/v, methanol: acetonitrile and filtered through 0.5 μm membrane Millipore, Bedford, MA, USA and used for HPLC analysis. The HPLC system comprised a liquid chromatograph equipped with a LC-20A pump Shimadzu, Tokyo, Japan, a 20 μl injection loop and a UV detector. Temperature was maintained at with a column heater. Separation was performed on a Kromasil C8, 5 μm column mm i.d.; Supelco Inc., Bellefonte, PA, USA, with 30:70 v/v methanol: acetonitrile at 1.2 ml/min as mobile phase, detector wavelength was set at 205 nm 20. Twenty microliters of the extract was injected into the HPLC column. 1211

4 P. K. Prasanth Kumar and A. G. Gopala Krishna Determination of tocopherol composition by HPLC Estimation of tocopherol composition by HPLC was carried out using LC-10A pump, injector fitted with 20 μl loop and FLD detector. The analysis was achieved by normal phase HPLC separation on silica column. Mobile phase was hexane: isopropyl alcohol 99.5:0.5, v/v at the flow rate of 1ml/min and was detected by fluorescence at the excitation and emission wavelengths of 290 nm and 330 nm respectively. The tocopherols were quantified based on peak areas with an external standard α-tocopherol and the tocotrienols were expressed as α-tocopherol equivalent as suggested by AOCS O.M. No. Ce 8-86, Estimation of total phenolics by spectrophotometer The phenolics were extracted from crude and deacidified RPO samples 5.0 g with methanol/water 80:20 v/v 1 ml 4 by vortexing for 2 min twice. Each time, the mixture was centrifuged at 1080 g for 15 min and the resultant supernatant 1 ml 4 was separated, pooled and kept in dark till analysis 21. Total phenolics content of phenolic extracts were determined by using Folin-Ciocalteu reagent. The extracts of 0.3 ml were mixed with 0.2 ml of Folin-Ciocalteu reagent and after 3 min, 1ml of 15 Na 2 CO 3 solution was added. The final volume was made up to 7 ml with distilled water and incubated for 45 min. The mixture was centrifuged and absorbance was measured at 745 nm in a UV-visible spectrophotometer Model UV-1601, Shimadzu Corporation, Kyoto, Japan with respect to a blank without any added phenolic extract. The total phenolics content was expressed as mg gallic acid equivalent GAE / 100 g of sample Determination of squalene by HPLC Squalene in the samples was estimated by HPLC according to Nenadis and Tsimidou The samples 0.02g was dissolved in 1mL of hexane, and 20 μl of the mixture was injected into an HPLC 10A VP, Shimadzu Corporation, Kyoto, Japan equipped with a UV detector SPD-10AV VP, Shimadzu. The analysis was achieved by isocratic separation on C18 column, 10 μm, u-bondapack, 4.6 X 300 mm; Millipore, Milford, MA with a mobile phase of acetone: acetonitrile 40/60, v/v and at a flow rate of 1 ml/min. The analysis was performed at 25 with the detector wavelength set at 208 nm 23. Identification was confirmed through spiking with a squalene standard solution and was quantified using standard squalene Determination of coenzyme Q 10 by HPLC Estimation of coenzyme Q 10 content was carried out by HPLC Shimadzu instrument 10A VP, Shimadzu Corporation, Kyoto, Japan was equipped with UV detector SPD- 10AV VP, Shimadzu that was coupled with 20 μl loop injector. The isocratic separation of samples was achieved on a C18 column, 10 μm, u-bondapack, mm; Millipore, Milford, MA using methanol/n-hexane/2-propanol 80:15:5, v/v/v as mobile phase at a flow rate of 1ml/min. The detector wavelength was set at 275 nm 24. It was identified and quantified using standard coenzyme Q Estimation of DPPH radical scavenging activity RSA The stable 1,1-diphenyl-2- picrylhydrazyl radical DPPH was used for determination of free radical-scavenging activity of the samples. Different concentrations of palm oil samples were taken and mixed with an equal volume 4 ml of 10 4 M toluenic solution of DPPH M. The mixture was incubated at room temperature for 30 min in dark and the absorbance was read at 517 nm against blank. Radical scavenging activity was expressed as the inhibition percentage and was calculated using the following formula, radical scavenging activity A control A sample / A control 100. Where, A control is the absorbance of the control without extract, and A sample is the absorbance of reaction mixture. The DPPH activity IC 50 was calculated using a plot of percent radical-scavenging activity against concentration mg/ml to determine the concentration of sample necessary to reduce DPPH by Statistical analysis Data were expressed as the mean standard deviation of quadruplicate analysis. Analysis of variance and least square deviation tests were conducted to identify differences among means using Graph pad statistical computer package 26. Statistical significance was declared at p RESULTS AND DISCUSSION 3.1 Impact of deacidification method on oil yield, neutral lipid loss and efficiency The CRPO with more than 5 FFA is considered as highly acidic and of poor quality. The deacidification of such CRPO by the commercially popular methods like steam deacidification which operates at high temperature of 250 to 270 under vacuum of 3 to 5 torr, cause excessive nutraceutical loss. The other popular method viz., alkali deacidification leads to the excessive loss of neutral lipids due to emulsification. The proposed deacidification was based on the enzymatic esterification of FFA with glycerol under suitable conditions. Most of the deacidification methods remove FFA from the starting oil, but the enzyme based deacidification esterifies the FFA with glycerol and retains it as glycerides instead of removing these out of the oil. Thus enzymatic deacidifcation provides 100 deacidified oil yield. In this study the RMIM based deacidification showed nearly 100 deacidified oil yield with no neutral lipid loss Table 1. The other methods used in the study viz., solvent EtOH and chemical method NaOH showed deacidified oil yields of 78 and 62 re- 1212

5 Retention of nutraceuticals in crude red palm oil Table 1 The deacidified oil yield, neutral lipid loss and deacidification efficiency with different deacidification methods. Analysis RMRPO EtOHRPO NaOHRPO Deacidified oil yield [%] a b c Neutral oil loss [%] a b Deacidification efficiency [%] a b b The values in the row with a,, b,, c, indicated difference at p<0.05. Abbreviations: CRPO Crude Red Palm oil, RMRPO Lipozyme RM IM deacidified Red Palm Oil, EtOHRPO Ethanolic deacidified Red Palm Oil, NaOHRPO Sodium hydroxide deacidified Red Palm Oil. spectively. The neutral lipid loss of EtOHRPO 13.6 and NaOHRPO 19.5 indicates that NaOH deacidification was prominent in the neutral lipid loss among the methods used and this may be due to the entrapment of neutral lipid in the emulsion formed during soap-stock formation. Loss of neutral lipid during EtOH deacidification of CRPO was very minimal probably due to the insolubility of other neutral compounds in EtOH. Thus, the tested methods have deacidified the oil in different ways. The deacidification efficieny of EtOH and NaOH deacidified RPO was 98 and Lipozyme RM IM deacidified RPO was 95. The efficiency of EtOH and NaOH deacidification depends on the number of extraction and calculated amounts of NaOH required for the neutralization respectively. But, Lipozyme RM IM based deacidification showed maximum efficiency 95 during 12h of deacidification and further reaction did not show significant reduction in the FFA. 3.2 Chemical characteristics of deacidified oil FFA is the indicator for the acidity of an oil. The FFA content of the CRPO was 8.7 which was relatively high compared to specification for crude palm oil. Therefore special attention has been taken to reduce FFA with the retention of nutraceuticals. The acidity of the deacidified oil depends on the method used for refining the oil. The FFA of EtOH deacidified CRPO depends on the number of extractions used to remove FFA from the oil. But in the case of NaOH a calculated amount of NaOH required to neutralize the FFA in CRPO is used which removes all FFA in one step as soapstock. The Enzymatic deacidification of CRPO differs from the EtOH and NaOH methods, and depends on various factors and achieved a 95 of deacidification at 12 h of esterification. The acidity level of deacidified oils was the lowest, with EtOH 0.19 and NaOH 0.2 Table 2. The RMRPO has shown an FFA of 0.4. The lower FFA of all the deacidified samples indicates that they are within the acceptable limit of FFA for that of the frying oils reported 27. Saponification value indicates average molecular weight and fatty acid chain length of oils and fats. The CRPO showed an SV of 192 mg KOH/g and there was no significant change after deacidification with Lipozyme RM IM 190 mg KOH/g, EtOH 192 mg KOH/g and NaOH 191 mg KOH/g Table 2. The saponification of oils varies according to the kind of fatty acids present in the oil. Here the similar SV of all samples indicates that there was no change in the type of fatty acids after deacidification with different methods. There was no significant difference in IV shown by CRPO 55.7 I 2 g/100g after deacidification with EtOH 55.9 I 2 g/100g and NaOH 55.6 I 2 g/100g. Whereas, a slight reduction in IV 54.1 I 2 g/100g was observed for Lipozyme RM IM based deacidified RPO. This is probably due to the incorporation of a slightly more of saturated fatty acids during the Lipozyme RM IM based deacidification to the deacidified RPO. Peroxide value is one of the quality index of edible oils and indicates oxidation level in oils. The initial PV of CRPO 7.5 meq O 2 /kg, deacidified RPO by Lipozyme RM IM 7.5 meq O 2 /kg and NaOH 7.5 meq O 2 /kg showed no significant change p 0.05 during the deacidification Table 2. The vacuum provided throughout the deacidification reaction has prevented the oxidation in Lipozyme RM IM based deacidified RPO. The slightly higher PV of EtOH 8.2 meq O 2 / kg compared to other samples was probably due to the higher exposure to oxygen during the deacidification compared to Lipozyme RM IM and NaOH deacidification methods. The unsaponifiable matter of CRPO was The deacidified samples have shown the unsaponifiable matter of 0.91 for RMRPO, 0.58 for EtOHRPO and 0.63 for NaOHRPO Table 2. The lowest retention of unsaponifiable matters of EtOHRPO was most probably due to the solubility of unsaponifiable matter along with FFA in the extraction solvent system. The low unsaponifiable matter content 0.58 of NaOHRPO indicates the loss of unsaponifiable matter in soap-stock during deacidification. The higher retention of unsaponifiables was shown in RMRPO indicated lowest loss of antioxidants and could be due to the mild reaction conditions used for the deacidification. MAG and DAG are the partial glycerides present in the CRPO. The level of MAG and DAG in the starting oil, CRPO was 1.8 and 6.56 respectively Table 2. The MAG level 1213

6 P. K. Prasanth Kumar and A. G. Gopala Krishna Table 2 Chemical characteristics of crude and deacidified red palm oils. Analysis CRPO RMRPO EtOHRPO NaOHRPO Free fatty acid value (%) a b b b Saponification value (mg KOH/g) a b a a Iodine value (I 2 g/100g) a b a a Peroxide value (meq.o 2 /kg) a a b a Unsaponifiable matter (%) a a b b Glyceride composition Triacylglycerol (%) a b c c Diacylglycerol (%) a b a a Monoacylglycerol (%) a b c d Fatty acid composition C12: a a a a C14: a a ab ab C16: a ab ab ab C18: a ab abc abc C18:1n a a a a C18:2n a ab ab ab ΣSAFA ΣMUFA ΣPUFA The values in the row with a,, b,, c, indicated difference at p<0.05. Abbreviations: CRPO Crude Red Palm oil, RMRPO Lipozyme RM IM deacidified Red Palm Oil, EtOHRPO Ethanolic deacidified Red Palm Oil, NaOHRPO Sodium hydroxide deacidified Red Palm Oil. has decreased in deacidified oil to 1.0 for EtOH and 0.55 for NaOH deacidification. But the level of MAG in RMRPO showed an increase to 2.77 after deacidification with Lipozyme RM IM. Likewise, the DAG content of EtOHRPO 4.84 and NaOHRPO 4.80 has shown a significant loss, while, the RMRPO has shown an increase in DAG content A higher amount of MAG 2.77 and DAG in RMRPO than in other samples indicates the probable formation of new partial glycerides during the esterification of FFA with glycerol. The fatty acid composition of crude and deacidifed red palm oils are shown in Table 2. Two saturated fatty acids viz. palmitic acid 45.6 and stearic acid 3.8 and two unsaturated fatty acids viz. oleic acid 38.7 and linoleic acid 10.9 were found in good amounts in CRPO with lauric acid and myristic acid as minor fatty acids. The deacidification of CRPO with Lipozyme RM IM, EtOH and NaOH methods did not alter the fatty acid composition significantly. Hence, the level of total saturated fatty acids ΣSAFA, monounsaturated fatty acids ΣMUFA and polyunsaturated fatty acids ΣPUFA of deacidifed RPO prepared by different methods of refining remained similar to the ΣSAFA, ΣMUFA and ΣPUFA content of CRPO. 3.3 Impact on content and composition of deacidified oils Weight ratio of olein and stearin and fatty acid composition of the fractions Fractionation is one of the simple modification techniques used to separate the liquid olein and solid stearin from semisolid palm oil. In this study a dry fractionation was carried out at 25 to evaluate the effect of different deacidification methods on olein and stearin ratios Fig. 1. The CRPO has shown 71.4 olein and 28.6 stearin weight ratios indicating that olein was the major portion of the CRPO. But for enzyme based refining, an increase in stearin ratio was observed with a concomitant decrease in olein ratio The Lipozyme RM IM based deacidification modified the oil property through the formation of new glycerides mostly partial glycerides and includes MAG and DAG. The newer glycerides mostly have melting point higher than the room temperature 25. Hence the fractionation of RMRPO at room temperature 25 showed an increase in the stearin ratio to 47.4 as compared to stearin ratio of CRPO 28.6 Table 5. The EtOH based deacidification has shown entirely reverse effect which reduced the ratio of stearin to 21.0 and increased the olein ratio to 79 as compared to that of CRPO 28.6 and This was probably due to less emulsification 1214

7 Retention of nutraceuticals in crude red palm oil Fig. 1 Weight ratio of olein and stearin obtained after deacidification by different methods. during refining and removal of partial glycerides along with FFA due to increased solubility. But the NaOH based deacidification showed similar stearin and olein ratio 28.5 and 71.0 as compared to the stearin and olein ratio of CRPO 28.6 and The increase in the stearin ratio of RMRPO may help this product to be used as hard stock for some of the food formulations like margarine, food spreads, shortenings, etc.. instead of using hydrogenated fats which contain harmful trans fatty acids. The deacidification of CRPO with different methods did not alter the fatty acid composition of the oil. But the fractionation of CRPO provided two fractions with different fatty acid composition. The olein fraction of CRPO showed 41.0 of palmitic acid and 45.6 of oleic acid Table 5. Similarly the stearin fraction consisted of 62.2 palmitic acid and 26 oleic acid as the major fatty acids. The results of deacidification showed that both fractions of EtOH and NaOHRPO were not significantly different p 0.05 from the fatty acid composition of corresponding starting oil fractions. But, the olein fraction from RMRPO showed lower palmitic acid 37.3 and higher oleic acid 45.5 than the oleins fraction from the other samples. Similarly stearin fraction of RMRPO showed a decrease in palmitic acid 57.1 and oleic acid Variation in the fatty acid composition of RMRPO fractions indicated the incorporation of different fatty acids at various levels in newer glycrides. The results showed that Lipozyme RM IM based deacidification followed by the fractionation of deacidified palm oil helps to produce red palm olein with improved fatty acid composition Fatty acid composition of glycerides in deacidified oils Table 3 shows the fatty acid distribution in MAG, DAG and TAG components of crude and deacidified RPOs. The results showed that palmitic acid and oleic acid are the major fatty acids and stearic acid and linoleic acid are the minor fatty acids present in CRPO. The palmitic acid level in the MAG fraction 41.6 and TAG fraction 45.0 of CRPO did not change significantly after deacidification Table 3 Fatty acid distribution in the glycerides (MAG, DAG and TAG components) of crude and deacidified red palm oils. CRPO RMRPO EtOHRPO NaOHRPO MAG DAG TAG MAG DAG TAG MAG DAG TAG MAG DAG TAG C12: a a a a b a a a a b a a C14: a a a a a a a a a a a a C16: a a b ad c bc a a bc abc a bc C18: a b c ad bc bc acde bcde bce bcdef bc bcf C18:1n a ab abc abc d ace abc ab abce abce ae abce C18:2n a ab ab ab bc ab ab abc ab ab ab ab ΣSAFA ΣMUFA ΣPUFA The values in the row with a,, b,, c, indicated difference at p<0.05. Abbreviations: ΣSAFA total saturated fatty acids, ΣMUFA total monounsaturated fatty acids, PUFA total polyunsaturated fatty acids, CRPO Crude Red Palm oil, RMRPO Lipozyme RM IM deacidified Red Palm Oil, EtOHRPO Ethanolic deacidified Red Palm Oil, NaOHRPO Sodium hydroxide deacidified Red Palm Oil. MAGmonoacylglycerols, DAG Diacylglycerols and TAG triacylglycerols 1215

8 P. K. Prasanth Kumar and A. G. Gopala Krishna Table 4 TAG composition of crude and deacidified red palm oils obtained by different methods. TAG species (%) TCN CRPO RMRPO EtOHRPO NaOHRPO MOL a b a a MLP/MOM 43.3/ a ab a ab OOL a b a a POL a b a a PLP/MOP 45.3/ a a a a MPP a a a a OOO a b a a POO a a a a POP a b a a PPP a b a a SOO a ab a ab SOP a b a a PPS a b a a Other TAGs a b a a ΣStStSt ΣStUSt ΣStUU ΣUUU TAG content (%) The values in the row with a,, b,, c, indicated difference at p<0.05. Abbreviations: nd not detected, M Myristic acid, O oleic acid, L linoleic acid, P palmitic acid, S stearic acid, ΣStStSt trisaturated triacylglycerol, ΣUStSt monounsaturated disaturated triacylglycerol, ΣUUSt diunsaturated monosaturated triacylglycerol, ΣUUU triunsaturated triacylglycerol, TAG triacylglycerol, CRPO Crude Red Palm oil, RMRPO Lipozyme RM IM deacidified Red Palm Oil, EtOHRPO Ethanolic deacidified Red Palm Oil, NaOHRPO Sodium hydroxide deacidified Red Palm Oil with Lipozyme RM IM, EtOH and NaOH deacidification. But, the level of palmitic acid in DAG fractions of CRPO 42.5 has increased after deacidification with Lipozyme RM IM. The oleic acid level in the MAG fraction of CRPO 41.1 showed no significant change after deacidification with Lipozyme RM IM 41.7, EtOH 41.4 and NaOHRPO The oleic acid level in the TAG fraction of CRPO 39.1, RMRPO 39.1, EtOHRPO 39.0 and NaOHRPO 38.9 showed no significant difference, while the DAG fraction 43.0 of CRPO reduced to 30.5 by Lipozyme RM IM based deacidification. The reduction in the oleic acid 30.5 in the DAG fraction and increase of palmitic acid in the DAG fraction of RMRPO indicates the formation of palmitic acid based new DAGs during deacidification reaction and this reduced the level of oleic acid in DAG fraction. Probably the FFA was rich in palmitic acid and hence most of palmitic acid DAGs were formed TAG composition of deacidified oils Table 4 shows the TAG profile of CRPO and deacidified RPOs. The results showed that POP 27.8, POO 23.5 and POL 11.0 are the major TAGs in the CRPO and deacidified RPO. The major TAG present in the EtOHRPO and NaOHRPO has shown no significant difference p 0.05 compared to CRPO. The levels of POP 19.3, POO 18.0 and POL 10.1 were reduced in RMRPO. The level of PLP/MOP 9.6, PPP 4.6, SOP 5.2, OOO 3.8, MOL 3.0, OOL 1.7 and SOO 2.6 remained unchanged after deacidification of CRPO with EtOH and NaOH. While the levels of PLP/MOP 6.3, SOP 3.5 MOL 1.9 and SOO 1.7 decreased and PPP 6.0, OOO 3.8 and OOL 3.8 increased in RMRPO. The variation in the TAG profile of RMRPO indicates the occurrence of modification of CRPO during Lipozyme RM IM based deacidification. This modification can change the properties like slip melting point. The quantity of trisaturated TAG 5.0, monounsaturated TAG 43.1, diunsaturated TAG 40.1 and triunsaturated TAG 5.6 remained unchanged during EtOH and NaOH deacidification. Whereas the quantity of trisaturated TAG 6.3 and triunsaturated TAG 8.4 increased and 1216

9 Retention of nutraceuticals in crude red palm oil Table 5 Fatty acid composition of olein and stearin fractions obtained after deacidification by different methods. Fatty acids Olein fractions Stearin fractions CRPO RMRPO EtOHRPO NaOHRPO CRPO RMRPO EtOHRPO NaOHRPO C12: a b b b b b b b C14: a b b b ac d ac cd C16: a ab bc ab d d d d C18: a ab ab ab abc cd a acd C18:1n ab c b bc d d d d C18:2n ab b ab ab c c c c ΣSAFA ΣMUFA ΣPUFA Weight ratio (%) a b c a d e f d The values in the row with a,, b,, c, indicated difference at p<0.05. Abbreviations: ΣSAFA total saturated fatty acids, ΣMUFA total monounsaturated fatty acids, ΣPUFA total polyunsaturated fatty acids, CRPO Crude Red Palm oil, RMRPO Lipozyme RM IM deacidified Red Palm Oil, EtOHRPO Ethanolic deacidified Red Palm Oil, NaOHRPO Sodium hydroxide deacidified Red Palm Oil monounsaturated TAG and diunsaturated TAG decreased in RMRPO. The TAG profile indicates that the RMRPO was different from those of other samples due to the formation of new TAG molecular species during the deacidification process with Lipozyme RM IM TAG composition of fractions from deacidified oils TAG profile of the olein and stearin fractions of CRPO and deacidified RPOs are presented in Table 6. The results showed that POP 26.6, POO 26.0, POL 12.4 and PLP/MOP 10.1 were the major triacylglycerols present in the olein fraction and POP 31.9, PPP 24.9, POO 12.9 were the major triacylglycerols present in the stearin fraction. All the TAG present in olein fractions from EtOHRPO and NaOHRPO were similar to TAG composition of olein fractions from CRPO TAG of olein from RMRPO showed an increase in OOL 26.2, POL 15.2, OOO 7.0 and decrease in PLP/MOP 8.9 and POP Similarly the TAG of stearin from RMRPO showed an increase in OOL 3.5, POL 9.6, OOO 3.5 POO 15.6 and PPS 6.4 and decrease in PLP/ MOP 6.6, POP 23, PPP Braipson-Danthine and Gibon 2007 have reported trisaturated TAG ranges for palm stearin The trisaturated TAG content of stearin fraction from all the samples 31.1, 30.5, 30.9 and 30.7 for CRPO, RMRPO, EtOHRPO and NaOHRPO respectively agreed with literature report 28. According to the same report the monounsaturated TAG content of stearin was The obtained monounsaturated TAG content of stearin fraction of CRPO 44.4, EtOHRPO 43.7 and NaOHRPO 43.0 also agreed with this report 28. But the stearin fraction from RMRPO showed reduction in monounsaturated TAG content 34.6 indicating significant difference from other stearin samples. The diunsaturated TAG content of all stearin fractions 21.8, 28.1, 23.5, 23.2 for CRPO, RMRPO, EtOHRPO and NaOHRPO respectively were within the diunsaturated TAG limit reported by Braipson-Danthine and Gibon The triunsaturated TAG content of stearin reported ranges from and present study shows similar triunsaturated TAG content 2.3, 2.6 and 3.2 of all stearin fractions except stearin fraction from RMRPO 7.0 which was higher than other samples. The olein fraction of CRPO has shown 82.3 of TAG and TAG content of olein fractions obtained from deacidified samples were 82.1, 93.2 and 92.4 for RMRPO, EtOHRPO and NaOHRPO respectively. Similarly TAG of stearin fractions was 69.8, 79.8, 97.8 and 95.0 for CRPO, RMRPO, EtOHRPO and NaOHRPO respectively. The high TAG content of the EtOHRPO and NaOHRPO fractions olein and stearin was due to the removal of FFA and the loss of DAG and MAG during deacidification. The lowest TAG content of RMRPO fractions was due to the formation of newer MAG and DAG by the enzymatic esterification of FFA with added glycerol. Hence RMRPO olein has shown an increase in MAG 2.0 and DAG Similarly the RMRPO stearin has shown an increase in MAG 3.7 and DAG Nutraceutical composition of deacidified oils The presence of carotene in oil ppm is responsible for the orange-red color of CRPO. Steam deacidification destroys all the carotenoids present in the CRPO. Table 7 shows the retention of carotene after deacidification of CRPO by different methods. CRPO showed 514 mg/kg of carotene to start with and a retention of mg/kg, mg/kg and mg/kg have been observed for NaOH, Lipozyme RM IM and EtOH based deacidification respectively. These results indicate that all the deacidification methods were efficient in retaining carotene 1217

10 P. K. Prasanth Kumar and A. G. Gopala Krishna Table 6 TAG composition of the olein and stearin fractions obtained after deacidification by different methods. Olein fractions Stearin fractions TCN CRPO RMRPO EtOHRPO NaOHRPO CRPO RMRPO EtOHRPO NaOHRPO MOL a ab a b c c c c MLP/MOM 43.3/ a a a b b b b b OOL a b a a c d c c POL a b c ac d e d d PLP/MOP 45.3/ a b ac a bd e d d MPP 460 nd nd nd nd a b a c OOO a b a a c d c e POO a a a a b b b b POP a b ac ac c b ac ac PPP a a a nd b c b bc SOO a b a c d de d de SOP a b a a b b b b PPS a nd nd nd b c b d SOS 51.4 nd nd nd nd nd a a a PSS 52.0 nd nd nd nd nd a ab b Other TAG a b c c d e d d ΣStStSt ΣStUSt ΣStUU ΣUUU FFA (%) a b c c d b c c TAG (%) a b c c a d e f DAG (%) a b c c c d e f MAG (%) a b c d e f g h The values in the row with a,, b,, c, indicated difference at p<0.05. Abbreviations: M Myristic acid, O oleic acid, L linoleic acid, P palmitic acid, S stearic acid, ΣStStSt trisaturated triacylglycerol, ΣStUSt diunsaturated monosaturated triacylglycerol, ΣStUU monosaturated diunsaturated triacylglycerol, ΣUUU triunsaturated triacylglycerol, TAG triacylglycerol, CRPO Crude Red Palm oil, RMRPO Lipozyme RM IM deacidified Red Palm Oil, EtOHRPO Ethanolic deacidified Red Palm Oil, NaOHRPO Sodium hydroxide deacidified Red Palm Oil, nd not detected. in RPO during deacidification. Phytosterols constitute the bulk of unsaponifiable matter of palm oil and have bioactive properties such as anticancer and plasma total cholesterol lowering effects 29. Bonnie and Choo reported that sterol content of commercial red palm olein was higher than refined, bleached and deodorized palm oil 30. Analysis of individual phytosterols of CRPO and deacidified RPOs showed fucosterol, stigmasterol and β-sitosterol campesterol in these oils Table 7. Fucosterol was present in small amount which varied from 29 mg/kg for CRPO to 12 mg/kg for EtOHRPO. Stigmasterol content of 107 mg/kg was observed in CRPO, which was retained to a maximum extent of 65 mg/kg in RMRPO. The lowest retention of stigmasterol 32 mg/kg was found in EtOHRPO and the loss probably was due to the solubility of stigmasterol in ethanol which was used to remove FFA. The overall retention of phytosterols 72 was high in RMRPO 225 mg/kg and lowest 30 in EtOHRPO 95 mg/kg. The lowest retention of overall phytosterols in EtOHRPO was probably due to the extractability of sterols in EtOH during deacidification. Tocopherols and tocotrienols are the vitamin E precursors present in CRPO. Gee has reported that tocotrienols are potentially better chemo-prevention and chemotherapy agents for degenerative diseases 31. It is considered that tocotrienols are the most valuable components in CRPO because no other vegetable oil contains tocotrienols except oil from bran sources like rice bran, wheat bran etc. Natural source of tococtrienols are scarce. The tocotrienols-α, γ and δ were present in CRPO. The tocopherols include α 106 mg/kg and γ-tocopherols 11 mg/kg which together constituted 117 mg/kg in CRPO Table 7. The different decidification methods showed an influence on the retention of tocopherols. The content retention of tocopherols was 80 mg/kg 68, 50 mg/kg 44 and 17 mg/ kg 15 for RMRPO, EtOHRPO and NaOHRPO respectively. Similarly the tocotrienols including α 161 mg/kg, γ 401 mg/kg and δ 188 mg/kg together constituted 750 mg/kg. Different deacidification methods showed that retention of tocotrienols was mg/kg,

11 Retention of nutraceuticals in crude red palm oil Table 7 Nutraceuticals composition of crude and deacidified red palm oils. Minor components (mg/kg)* CRPO RMRPO EtOHRPO NaOHRPO β-carotene a b (94%) c (98%) b (93%) Fucosterol a b (93%) c (41%) d (69%) Stigmasterol a b (75%) c (30%) b (50%) β-sitosterol+campesterol a b (54%) c (29%) d (47%) Σ-Phytosterols (72%) 95 (30%) 156 (50%) α-tocopherol a b (69%) c (47%) d (10%) β+γ-tocopherol a b (61%) c (18%) d (55%) Σ-Tocopherol (67%) 50 (44%) 17 (15%) α-tocotrienol a b (67%) c (37%) d (59%) β+γ-tocotrienol a b (71%) c (26%) d (49%) δ-tocotrienol a b (74%) c (35%) d (56%) Σ-Tocotrienols (71%) 173 (36%) 207 (53%) Tocols (71%) 223 (36%) 224 (63%) Squalene a b (70%) c (59%) c (58%) Coenzyme Q a a (99.3%) b (70.4%) b (69.7%) Total phenolics a b (69%) c (57%) d (67%) IC 50 mg/ml a a b c The values in the row with a,, b,, c, indicated difference at p<0.05. * Values in brackets indicate percentage retention of nutraceuticals. Abbreviations: CRPO Crude Red Palm oil, RMRPO Lipozyme RM IM deacidified Red Palm Oil, EtOHRPO Ethanolic deacidified Red Palm Oil, NaOHRPO Sodium hydroxide deacidified Red Palm Oil mg/kg and mg/kg in RMRPO, EtOHRPO and NaOHRPO respectively. The tocol tocopherol tocotrienol content was 867 mg/kg for CRPO which was retained to different extents in RMRPO 612 mg/kg, EtOHRPO 223 mg/kg and NaOHRPO 224 mg/kg. The loss of tocols in EtOHRPO and NaOHRPO indicates low retention of tocols during deacidification of CRPO with EtOH and NaOH deacidification methods. Hence Lipozyme RM IM can be utilized to deacidify CRPO with maximum retention of tocols. Oil palm fruit is a rich source of water-soluble phenolics. Most of these phenolics are being removed along with waste stream during the milling process to extract the oil from palm fruit 32. Aleksandra et al. 2011, have reported the total phenolics content of crude palm oil and it is 91 mg/kg of oil 33. Audly, 1986 has found a number of acidic and neutral phenolic compounds in crude palm oil, totalling about 100 mg/kg 34. CRPO used for the study shows mg/kg which was slightly higher to the phenolic content of CRPO 100 mg/kg repororted by Audly 34. The phenolics retention of RMRPO was a higher mg/ kg than that of other samples mg/kg and mg/kg observed for NaOHRPO and EtOHRPO respectively Table 7. The CRPO has shown the squalene content of mg/ kg and the values are agreeing with the range of mg/kg reported by Gunstone 35. Commercial deacidification strips out squalene to PFAD and the PFAD has been considered as an alternative to traditional source of squalene like shark liver oils 36. The Lipozyme RM IM based deacidification of CRPO showed mg/kg retention of squalene in RPO. The EtOH and NaOH based deacidification showed retention of mg/kg and mg/kg of squalene respectively as compared to the squalene content of CRPO mg/kg Table 7. Hence, the Lipozyme RM IM deacidification of CRPO may be used to retain squalene and provide health benefits antioxidant and anti-carcinogenic effect to consumers. Han et al 2006 reported that the CRPO contains mg/kg of coenzyme Q The CRPO used for the study has shown 40.2 mg/kg of coenzyme Q 10. The deacidification with different methods showed that maximum retention of mg/kg of coenzyme Q 10 was with Lipozyme RM IM based deacidification Table 7. The EtOH and NaOH based deacidification has retained mg/kg and mg/kg of coenzyme Q 10 respectively. Han et al 2006 have reported that the coenzyme Q 10 is ten times more powerful as antioxidant than vitamin E 37. Hence the retention of coenzyme Q 10 in deacidified RPOs may provide more antioxidant capacity for example to the deacidified oil prepared by Lipozyme RM IM method Radical scavenging activity of deacidified oils The Radical scavenging activity of CRPO and deacidified 1219