Supercritical fluid high oryzanol (SFE HO) and supercritical fluid low oryzanol (SFE LO) rice bran oils were obtained and compared with that extracted by the Soxhlet (SOX) method. Their composition and stability during storage were determined. The amount of unsaponifiable matter and gamma oryzanol of SFE samples were significantly (p < 0.05) higher than SOX. While the amount of tocopherol in SOX (4.0 mg·g-1) was higher than that reported in SFE HO and SFE LO, at 3.2 and 2.6 mg·g-1, respectively. After storage for 42 days at 70 °C the PV, FFA%, conjugated diene and
Rice bran is produced from the outer layer of brown rice. It contains high valued protein, fat and nutritional dietary fiber. In addition to phytonutrients, rice bran contains vitamins and minerals (Chatha
Solvent extraction is the conventional method used for extracting lipids from rice bran. This extraction practice uses highly toxic and flammable solvents like hexanes, petroleum ether, and isopropanol. These solvents also have problems associated with waste disposal due to increased environmental concerns and regulations. Toxic solvent residues in the final food product are also of concern (Patel,
Supercritical fluid extraction (SFE) of lipids has received attention as an alternative method to organic solvent extraction and has been shown to be useful for extracting certain lipids. Apart from carbon dioxide's non-toxic and non–flammable nature, supercritical carbon dioxide extraction also offers the most important advantage of varying the extraction power of the solvent by changing operating conditions such as pressure, temperature and flow rate. This provides selective extraction and fractionation capabilities to the process (Patel,
In Malaysia, rice is the main cereal that is consumed by the majority of its population. However, rice bran oil is still a new product in Malaysia, compared to several other countries such as Japan, India, Korea, China and Indonesia where rice bran oil is popular as a cooking oil. Rice bran oil actually has high potential availability in Malaysia and the research and development of rice bran oil is still in demand.
The nutritional and health qualities of rice bran oil have led researchers to be interested recognizing its oxidative stability. The oxidation of rice bran oil was evaluated after the addition of stabilized and unstabilized rice bran extracts (Mariod
This study aims to determine the oxidative stability of rice bran oil extracted by Soxhlet in comparison with SFE rice bran oil fractions (high and low oryzanol) during accelerated shelf-life storage. The oxidative stability of SOX, SFE HO, SFE LO during accelerated shelf-life storage was determined and measured by peroxide value, free fatty acids, conjugated dienes,
Rice bran was obtained from Bernas Rice Mills, Co. Tiram, Selangor, Malaysia. The rice bran samples were stabilized before the extraction process for 2 min at 2450MHz, using a national microwave/convection oven (model NN-C2003S, Matsushita Electric Industrial Co., Japan) (Mariod
Methanol, acetonitrile, dicholoromethane, isopropanol, acetic acid, chloroform, isooctane,
Chemicals such as potassium hydroxide, sodium chloride and sodium methoxide were obtained from Sigma - Aldrich (St. Louis, MO, USA). The standard for unsaponifiable matter (tocopherol) and neutral lipids were purchased from Sigma - Aldrich (St. Louis, MO, USA). The FAME reference standard was purchased from AccuStandard (New Haven, CT, USA). A pure standard of gamma-oryzanol (Standard compounds of oryzanols, cycloartenyl ferulate and 24-methylene cycloartanyl ferulate) was obtained from Sigma Chemical Co. Ltd, Japan.
The supercritical fluid extraction system was used (Thar Technologies, Pittsburgh, PA, USA, Laboratory Scale) with a rotary evaporator (Buchi, Flawil, Switzerland), universal and micro type centrifuge machine (Hettich Zentrifugen, Tuttlingen, Germany), ultrasonic water bath (HanShin, USA), and analytical balance (Shimadzu, Nakagyo Ku, Kyoto, Japan). Each component of lipids in rice bran oil was determined using Agilent Technologies HPLC series 1200 (Santa Clara, CA, USA).
Supercritical fluid high oryzanol rice bran (SFE HO) and supercritical fluid low oryzanol rice bran (SFE LO) oils were extracted using SFE with different pressures and temperatures. In brief, carbon dioxide (CO2) from a cylinder was pressurized and then fed into the solvent reservoir. Pressurized CO2 becomes the supercritical fluid by preheating in an equipped air-bath. After rice bran (180 g) was placed in the extractor, the supercritical fluid flowed into the extractor and mixed with rice bran. The extractor was connected to the collector vessel by a micro-metering valve. The temperature and pressure were controlled to obtain the SFE oil. After that the mixed solution of RBO and supercritical fluid from the extractor is depressurized and the RBO was collected (Bruhl and Matthaus,
In brief, 100 grams of ground rice bran were weighed and equally divided into 4 extraction thimbles. Each thimble was then transferred into a soxhlet extractor. Approximately, 300 mL of
Lipids extracted by SFE and Soxhlet were fractionated on an Solid Phase Extraction (SPE) system using a pre-packed 1-g silica gel cartridge column (Sigma - Aldrich St. Louis, MO, USA). Although vacuum could be applied to increase the flow rate of the eluent, generally gravity alone was sufficient to provide adequate flow. Initially, the cartridges were conditioned with 5 mL of hexane, followed by 5 mL of CHCl3. The neutral lipid (NL), glycolipid (GL), and phospholipid (PL) fractions were successively eluted with 10 mL of CHCl3 and acetone (4:1, v/v), 15 mL of acetone and methanol (9:1, v/v), and 10 mL of methanol. The purity of the fractions was checked by TLC. Solvents from the fractions were rotary evaporated, transferred to recovery vials using the eluting solvents, and evaporated to dryness at 37 °C. The recovery of the lipids was 100%. The amount of polar lipids was the sum of GL and PL (Hubbard
Unsaponifiable and wax contents were measured following AOCS Da 11-42, and AOCS Ch 8-02 Methods, respectively (AOCS,
The fatty acid compositions of the RBOs were determined by GLC. The oils were converted to their corresponding methyl esters according to the AOCS Official Methods (
Gamma oryzanol was quantified using a reversed-phase HPLC. A C18 column and a diode-array ultraviolet-visible detector (Hewlett-Packard, San Fernando, CA) were employed in the HPLC system. The mobile phase consists of methanol, acetonitrile, dichloromethane, and acetic acid (50:44:3:3) by volume. The flow rate was controlled at 1.4 mL·min-1. The analytes were detected at 330 nm. The samples were diluted with the mobile phase and analyzed. Peak identification was based on the comparison of retention time (RT) values with the authentic standards of cycloartenyl ferulate, 24-methylene cycloartanyl ferulate and a mixture of oryzanol standards. The total oryzanols were quantified based upon the peak area of 24-methylene cycloartanyl ferulate, the major oryzanol component in rice bran. The concentrations of individual components of γ-oryzanol were calculated and the concentration of γ-oryzanol was obtained by adding all individual components (Shahid
Tocopherols were measured by a reversed-phase HPLC system connected to a fluorescent detector at Ex = 298 nm and Em = 328 nm. A 2.1 mm narrow-bore analytical column packed with 5-m ODS (C18) hypersil silica used for the separation of tocols. The mobile phase acetonitrile/methanol/isopropanol/water (45:45:5:5 by volume), was programed to acetonitrile/methanol/isopropanol (50:45:5) for 10 min. These conditions were maintained for 15 min before returning to the original conditions. The total HPLC run time was 26 min. A fine separation was achieved for tocopherols but bands were broad for tocotrienols. The samples were quantified by comparing the retention times/peak areas with those of the obtained standards. The total tocopherol and tocotrienol contents were determined by summing up the Α, Β, γ and δ isomers (Shahid
For the oxidative stability studies, the antioxidant BHA (0.02%) was added to separate the samples of SFE HO, SFE LO, and SOX based on the concentration of antioxidant used and represents the maximum added antioxidant levels used in foods (0.02%). The antioxidant (0.02 g) was dissolved in 10 mL of ethanol and then added to 100 g of RBOs following the procedure of Mariod
CD value = A/(C * I)
Where CD = conjugated diene, A = absorbance at 234 nm, C = concentration (g·100 mL-1) and I = path length (cm).
TBARS value=(A*0.415)/m
Where A = absorbance at 532 nm and m = sample weight.
Free fatty acids as oleic acid percentages in the oil samples, were determined using an alkali titration method according to AOCS (
The analyses were performed with three replicates. The mean values and standard deviation (mean±SD) were calculated and tested using the Student's
Unsaponifiable, oryzanol, wax and lipid classes of RBOs extracted by SFE and Soxhlet methods
Sample | Unsaponifiable matters (%) | γ-Oryzanol (m·g-1) | Tocopherol (mg·g-1) | Wax (%) | Neutral lipid (g·g-1) | Glycolipid (g·g-1) | Phospholipid (g·g-1) |
---|---|---|---|---|---|---|---|
SFE HO | 8.2±0.2a | 7.7±0.3a | 3.2±0.3a | 0.45±0.1a | 0.97±0.01a | 0.03±0.1a | 0.01±0.1a |
SFE LO | 5.2±0.1b | 6.3±0.2b | 2.6±0.3b | 0.47±0.1a | 0.95±0.01a | 0.03±0.1a | 0.01±0.1a |
SOX | 4.7±0.1c | 4.7±0.2c | 4.0±0.1c | 0.32±0.1a | 0.70±0.4b | 0.01±0.1a | 0.06±0.1a |
SFE HO: Supercritical fluid extraction high oryzanol; SFE LO: Supercritical fluid extraction low oryzanol; SOX: Soxhlet;
Values are means of three replicates±standard deviation. Means within columns with common superscript letters are not different (
SFE and SOX extracted RBOs were analyzed for their tocopherol contents using the HPLC technique. The highest concentration of tocopherol was found in the sample of SOX (4.0 mg·g-1) which was higher than 3.2 and 2.6 mg·g-1 as reported in SFE HO and SFE LO, respectively as the concentration of tocopherol in rice bran oil was affected by the method of extraction. As shown in
In
The RBO obtained under different conditions by SFE HO, SFE LO and SOX extractions had similar fatty acid compositions. From
Fatty acids composition (weight %) of RBOs extracted by SFE and Soxhlet methods
Fatty acid | SFE HO | SFE LO | SOX |
---|---|---|---|
14:0 | 0.6±0.01a | 0.5±0.01a | 0.6±0.01a |
16:0 | 16.4±0.05a | 16.7±0.04a | 16.7±0.05a |
16:1n-7 | 0.2±0.01a | 0.2±0.01a | 0.2±0.01a |
16:1n-9 | 0.1±0.01a | 0.1±0.01a | 0.1±0.01a |
18:0 | 2.1±0.02a | 2.2±0.02a | 2.5±0.03b |
18:1n-9 | 43.8±0.04a | 43.3±0.03a | 42.2±0.05a |
18:1n-7 | 0.9±0.01a | 1.0±0.02a | 0.9±0.01a |
18:2n-6 | 33.0±0.04a | 33.0±0.04a | 32.8±0.03a |
20:1n-9 | 0.8±0.02ā | 0.9±0.02a | 0.9±0.02a |
18:3 | 1.3±0.02a | 0.0±0.01b | 0.0±0.01b |
20:1n-7 | 0.0±0.01a | 1.3±0.02b | 1.3±0.02b |
SFA | 19.1±0.04a | 19.4±0.04a | 19.8±0.05b |
MUFA | 44.9±0.1a | 46.8±0.04b | 45.6±0.05c |
PUFA | 34.3±0.2a | 33.0±0.1b | 32.8±0.04c |
For abbreviations, see
Values are means of three replicates±standard deviation. Values in the same row with different superscripts are significantly different (p = 0.05). SFA Saturated fatty acid, MUFA Monounsaturated fatty acid, PUFA Polyunsaturated fatty acid.
Using SFE, high oryzanol oil was obtained using 600 bars and 60 °C; these parameters were reduced to 200 bars and 40 °C to obtain low oryzanol oil with a similar 25g·min-1 flow rate and 150 min as duration of time. No change in fatty acid, wax of unsaponifaible contents of the two SFE extracted oils were detected so that SFE can profitably be applied in the extraction of HO and LO RBO as new functional ingredients of natural origin using an environmentally clean extraction technique that can be used by the food industry. However, LO seems to be economically better as it uses low pressure and temperature.
Oxidative stability (also known as the induction period) is a measurement of an oil or fat's resistance to oxidation. Because the process takes place through a chain reaction, the oxidation reaction has a period when it is relatively slow, before it suddenly speeds up. Oxidative stability is one of the most important indicators for maintaining the quality of edible oils. To date, many studies have been conducted to evaluate the oxidative stability of various seed oils.
PV is a measurement of the amount of peroxides formed in fats and oils through autoxidation and oxidation processes (Stier 2006). Generally, it is a measurement of the degree of initial oxidation of fats and oils. Measuring the peroxide value is well-established method for the determination of primary oxidation products in fats (Kulås and Ackman
Peroxide value (meq O2·kg-1 oil) of rice bran oil extracted by SFE and Soxhlet methods with and without BHA during storage
Time (day) | SOX | SOX + BHA | SFE(HO) | SFE(HO) + BHA | SFE(LO) | SFE(LO) + BHA |
---|---|---|---|---|---|---|
0 | 1.8±0.4 | 1.3±0.3 | 0.75±0.2 | 0.50±0.2 | 0.50±0.2 | 0.50±0.2 |
3 | 2.5±0.2 | 2.1±0.2 | 6.30±0.3 | 4.0±0.3 | 6.0±0.8 | 5.0±0.4 |
7 | 9.8±0.2 | 8.3±0.2 | 7.5±0.5 | 4.7±0.8 | 6.7±0.7 | 5.5±0.5 |
14 | 11.35±1.7 | 10.5±1.5 | 7.25±0.25 | 6.0±1.0 | 7.25±1.3 | 6.25±0.7 |
21 | 12.0±1.25 | 11.25±1.25 | 7.50±1.5 | 6.0±0.5 | 8.0±1.5 | 7.0±1.0 |
28 | 12.3±0.3 | 11.8±0.7 | 8.75±1.5 | 7.5±0.2 | 9.25±0.75 | 7.5±0.5 |
35 | 13.5±0.5 | 12.0±2.0 | 9.50±0.6 | 9.3±1.3 | 10.0±0.05 | 8.0±1.3 |
42 | 14.0±1.2 | 13.3±2.3 | 11.25±0.25 | 10.5±0.1 | 10.25±0.1 | 8.25±0.8 |
For abbreviations, see
Values are means of three replicates±standard deviation.
The free fatty acids (%) of different RBOs during storage are shown in
Free fatty acid (%) of rice bran oil extracted by SFE and Soxhlet methods during storage
Time (day) | SOX | SOX + BHA | SFE(HO) | SFE(HO) + BHA | SFE(LO) | SFE(LO) + BHA |
---|---|---|---|---|---|---|
0 | 4.13±0.06 | 2.75±0.07 | 3.10±0.28 | 2.54±0.28 | 3.57±0.09 | 2.65±0.05 |
3 | 12.69±1.41 | 11.28±2.82 | 11.28±1.69 | 11.28±0.56 | 12.69±0.28 | 10.01±0.14 |
7 | 12.97±0.10 | 12.69±1.41 | 12.4±0.10 | 10.99±0.84 | 20.17±0.43 | 19.32±0.71 |
14 | 13.53±0.57 | 12.97±0 | 18.05±2.54 | 18.76±5.2 | 21.72±0.57 | 21.15±0.56 |
21 | 18.33±1.40 | 16.64±0.85 | 18.33±1.41 | 19.45±0.84 | 22.42±0.99 | 21.72±0.57 |
28 | 21.99±0.50 | 21.42±0.56 | 20.86±2.25 | 20.02±0.84 | 22.59±0.29 | 21.98±1.27 |
35 | 21.99±0.50 | 21.71±0.28 | 21.24±0.55 | 21.16±0.54 | 22.88±0.36 | 22.11±0.75 |
42 | 23.04±1.26 | 21.78±2.26 | 21.99±0.29 | 21.25±1.41 | 23.00±1.16 | 22.34±0.50 |
For abbreviations, see
Values are means of three replicates±standard deviation.
The measurement of conjugated dienes is a useful index of the early stages of peroxidation in studies with pure lipids and isolated lipoproteins (Dekkers
Conjugated dienes (234 nm and 270 nm) of rice bran oil extracted by SFE and Soxhlet methods during storage
Time (day) | SOX | SOX + BHA | SFE(HO) | SFE(HO) + BHA | SFE(LO) | SFE(LO) + BHA |
---|---|---|---|---|---|---|
234 nm | ||||||
0 | 0.921±0.20 | 0.802±0.11 | 0.550±0.03 | 0.63±0.19 | 0.675±0.006 | 0.651±0.05 |
3 | 1.021±0.20 | 0.864±0.03 | 0.703±0.30 | 0.688±0.30 | 0.691±0.09 | 0.656±0.05 |
7 | 1.025±0.02 | 0.950±0.09 | 0.779±0.12 | 0.739±0.04 | 0.764±0.07 | 0.664±0.06 |
14 | 1.100±0.10 | 1.060±0.11 | 0.982±0.02 | 1.00±0.11 | 0.879±0.21 | 0.731±0.11 |
21 | 0.937±0.18 | 0.807±0.14 | 1.045±0.09 | 1.025±0.01 | 1.079±0.11 | 1.041±0.14 |
28 | 1.062±0.14 | 0.992±0.09 | 1.02±0.18 | 0.992±0.08 | 1.10±0.020 | 1.105±0.17 |
35 | 1.106±0.17 | 1.053±0.15 | 1.08±0.11 | 1.06±0.02 | 1.100±0.02 | 1.107±0.11 |
42 | 1.143±0.24 | 1.065±0.13 | 1.101±0.18 | 1.08±0.01 | 1.130±0.05 | 1.126±0.09 |
270 nm | ||||||
0 | 0.602±0.12 | 0.602±0.11 | 0.603±0.40 | 0.646±0.30 | 0.713±0.04 | 0.715±0.05 |
3 | 0.752±0.15 | 0.655±0.05 | 0.820±0.20 | 0.792±0.23 | 0.890±0.04 | 0.763±0.09 |
7 | 1.081±0.16 | 0.790±0.20 | 0.851±0.21 | 0.738±0.07 | 0.950±0.07 | 0.900±0.21 |
14 | 1.253±0.12 | 1.168±0.08 | 1.253±0.04 | 1.080±0.13 | 1.07±0.11 | 0.920±0.07 |
21 | 1.266±0.29 | 0.942±0.08 | 1.095±0.04 | 1.080±0.80 | 1.169±0.17 | 0.940±0.01 |
28 | 1.266±0.06 | 1.199±0.04 | 1.176±0.02 | 1.157±0.01 | 1.180±0.11 | 1.155±0.11 |
35 | 1.318±0.08 | 1.165±0.04 | 1.239±0.11 | 1.179±0.02 | 1.253±0.13 | 1.236±0.25 |
42 | 1.438±0.04 | 1.299±0.03 | 1.255±0.07 | 1.197±0.25 | 1.270±0.36 | 1.246±0.13 |
For abbreviations, see
Values are means of three replicates±standard deviation.
The change in TBAR levels (µmol·g-1)2 in the different RBOs during storage is shown in
TBAR (μmol·g-1)2 of rice bran oil extracted by SFE and Soxhlet methods during storage
Time (day) | SOX | SOX + BHA | SFE(HO) | SFE(HO) + BHA | SFE(LO) | SFE(LO) + BHA |
---|---|---|---|---|---|---|
0 | 0.370±0.005 | 0.310±0.010 | 0.207±0.002 | 0.203±0.010 | 0.31±0 | 0.300±0.010 |
3 | 0.606±0.030 | 0.617±0.010 | 0.556±0.003 | 0.598±0.005 | 0.577±0.002 | 0.445±0.007 |
7 | 0.795±0.004 | 0.690±0.020 | 0.706±0.040 | 0.594±0.016 | 0.661±0.05 | 0.560±0.108 |
14 | 0.897±0.02 | 0.705±0.080 | 0.732±0.080 | 0.689±0.060 | 0.639±0.09 | 0.570±0.050 |
21 | 0.73±0.18 | 0.820±0.020 | 0.740±0.020 | 0.71±0.030 | 0.860±0.01 | 0.771±0.020 |
28 | 0.913±0.004 | 0.850±0.010 | 0.786±0.070 | 0.738±0.050 | 0.899±0.08 | 0.738±0.050 |
35 | 0.94±0.008 | 0.880±0.070 | 0.881±0.220 | 0.729±0.005 | 0.916±0.007 | 0.864±0.010 |
42 | 1.32±0.2 | 1.03±0.010 | 0.946±0.070 | 0.808±0.110 | 0.96±0.05 | 0.870±0.040 |
For abbreviations, see
Values are means of three replicates±standard deviation.
The
P-anisidine value of rice bran oil extracted by SFE and Soxhlet methods during storage
Time (day) | SOX | SOX + BHA | SFE(HO) | SFE(HO) + BHA | SFE(LO) | SFE(LO) + BHA |
---|---|---|---|---|---|---|
0 | 0.107±0.003 | 0.094±0.002 | 0.039±0.002 | 0.030±0.003 | 0.042±0.002 | 0.032±0.010 |
3 | 0.115±0.010 | 0.104±0.006 | 0.045±0.001 | 0.047±0.003 | 0.049±0.020 | 0.056±0.010 |
7 | 0.135±0.160 | 0.109±0.001 | 0.048±0.001 | 0.046±0.010 | 0.052±0.010 | 0.054±0.010 |
14 | 0.143±0.001 | 0.125±0.002 | 0.068±0.005 | 0.051±0.007 | 0.065±0.005 | 0.059±0.006 |
21 | 0.390±0.004 | 0.223±0.010 | 0.070±0.008 | 0.057±0.017 | 0.092±0.005 | 0.075±0.004 |
28 | 0.400±0.004 | 0.378±0.020 | 0.090±0.020 | 0.067±0.010 | 0.103±0.020 | 0.086±0.003 |
35 | 0.424±0.006 | 0.416±0.020 | 0.108±0.020 | 0.100±0.005 | 0.112±0.002 | 0.098±0.002 |
42 | 0.489±0.010 | 0.476±0.020 | 0.121±0.030 | 0.110±0.050 | 0.100±0.002 | 0.082±0.002 |
For abbreviations, see
Values are means of three replicates±standard deviation.
Tocopherol (mg·g-1) of rice bran oil extracted by SFE and Soxhlet methods during storage
Time (day) | SOX | SOX + BHA | SFE(HO) | SFE(HO) + BHA | SFE(LO) | SFE(LO) + BHA |
---|---|---|---|---|---|---|
0 | 4.13±0.1 | 4.13±0.2 | 3.2±0.3 | 3.2±0.2 | 2.6±0.2 | 2.6±0.2 |
3 | 2.78±0.1 | 3.9±0.3 | 3.1±0.2 | 3.1±0.2 | 2.5±0.1 | 2.5±0.2 |
7 | 2.21±0.2 | 3.8±0.2 | 3.0±0.1 | 3.1±0.1 | 2.3±0.1 | 2.4±0.1 |
14 | 3.15±0.3 | 3.6±0.2 | 3.0±0.1 | 3.0±0.1 | 2.1±0.1 | 2.3±0.1 |
21 | 2.79±0.2 | 3.2±0.1 | 2.9±0.2 | 2.9±0.3 | 2.0±0.1 | 2.2±0.1 |
28 | 2.56±0.1 | 3.1±0.1 | 2.7±0.1 | 2.8±0.2 | 1.8±0.1 | 2.0±0.1 |
35 | 2.46±0.1 | 2.8±0.1 | 2.5±0.1 | 2.7±0.1 | 1.6±0.1 | 1.9±0.1 |
42 | 2.21±0.1 | 2.7±0.1 | 2.4±0.1 | 2.6±0.1 | 1.5±0.1 | 1.8±0.1 |
For abbreviations, see
Values are means of three replicates±standard deviation.
To obtain the HO from SFE the pressure was 600 bars and the temperature was 60 °C. Meanwhile, to obtain the LO the pressure was 200 bars and temperature was 40 °C. The flow rate and duration of time to extract HO and LO were similar with 25 g·min-1 of flow rate and 150mins duration of time. The present work indicates the importance of using SFE in the edible oil extraction process. The extraction of RBO using the SFE technique produces oils with high oxidative stability and free of solvents. SFE can be adjusted to produce high or low oryzanol RBOs and as oryzanol is an effective antioxidant these oils can be stored for up to 42 days under successive heating and show better stability than oils extracted by the Soxhlet conventional method. Peroxide, TBARS, CD and