comparative studies between conventional and microwave

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Journal of Oleo ScienceCopyright ©2017 by Japan Oil Chemists’ Societydoi : 10.5650/jos.ess17067J. Oleo Sci. 66, (9) 973-979 (2017)

Comparative Studies between Conventional and Microwave Assisted Extraction for Rice Bran OilHimanshu S. Shukla and Amit Pratap＊

Department of Oils, Oleochemicals and Surfactants Technology, Institute of Chemical Technology, Mumbai, INDIA

1 IntroductionRice（Oryza Sativa）is one of the most important grains in

the world and India is the second largest producer in the world. The rice bran oil is obtained from the bran compos-ite that is removed ` during the milling of brown rice to produce white rice. In actuality, the oil is found primarily in the germ, which represents ～2％ of the rice kernel. However, in typical rice milling operations it is not possible to separate the germ from the bran, so the resulting product, commonly called rice bran, contains the germ as well. Rice bran, which represents ～8％ of the whole grain weight, contains ～20％ lipid, with a range from 15％ to 25％1）.

Rice bran oil（RBO）extracted from rice bran by different methods. Rice bran wax is the value added by product of RBO refinery. It is hard non sticky wax and contains higher fatty alcohols and esters2）. The refining of RBO includes degumming, neutralization, bleaching, dewaxing and de-oderization2）. RBO is typically extracted with mechanical expression3）and solvent extraction4－6）. The various re-searches are going on the advanced methods to extract oil from rice bran such supercritical CO2 extraction6）, Micro-wave Assisted Extraction（MAE）7, 8）and Ultrasound Assisted Extraction（UAE）9）. RBO is used by many industries because of its unique component i.e. it contains natural antioxidant oryzanol and vitamin E10）. The unsaponifiable constituents of RBO include mainly tocols（vit E-0.01 to 0.14％）and y-oryzanol（ester of trans ferulic acid with sterols and triterpenic alcohols, 0.9-2.9％）. gamma-oryza-

＊Correspondence to: Amit Pratap, Department of Oils, Oleochemicals and Surfactants Technology, Institute of Chemical Technology, Mumbai, INDIAE-mail: [email protected], [email protected] May 8, 2017 (received for review March 14, 2017)Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 onlinehttp://www.jstage.jst.go.jp/browse/jos/　　http://mc.manusriptcentral.com/jjocs

nol can be extracted and can be further used in food, cos-metics, and pharmaceuticals industries11, 12）. Gamma-oryza-nol can be obtained from the soap-stock of oil refinery process11）. The tocotrienol forms of natural vitamin E pos-sesses powerful hypocholesterolemic, anti-cancer and neu-roprotective properties13）.

The different methods are employed to extract oil from bran. In cold press or hydraulic press, it was observed that oil has better nutritive property and natural antioxidant than those after refining. This method is simple, eco-friendly and does not require much energy. Cold pressed oil exhibit shelf stability, improved safety without added synthetic antioxidants and contaminated free（as no organic solvent is being used3）. In Case of solvent extrac-tion different food grade solvents can be used like hexane, acetone, isopropanol, ethanol etc.4, 6）. Microwave assisted extraction and Ultrasonication is the advanced methodology to extract oil and it is a green process for oil extraction7－9）.

In comparison with other vegetable crude oil, it contains higher level of non-glycerides component including wax ester（WE）. This rice bran wax is hard and non tacky. It is generally present in 2-5％ of the bran oil. It is recovered as sludge wax and further processed to slack wax, pressed wax. It is white to yellowish in color and can be made avail-able in flakes form. It can be obtain by dewaxing step in re-finery process14）. It can be again separated from oil using filtration and centrifuge process15）.

RBO was extracted by hydraulic press, solvent extrac-tion, and microwave assisted extraction（MAE）process

Abstract: The present work deals with comparison of microwave assisted extraction to that of conventional solvent extraction for the extraction of rice bran oil (RBO); focusing on extraction yield and oil composition. Microwave assisted extraction act as a green process over other method and proved that it is effective method for extraction of oil. The investigation also focuses on the study of functional group and component present in oil. Natural antioxidant component; its activity was confirmed by DPPH assay. The oryzanol content was also determined by measuring the optical density of the sample at 315 nm in n-heptane using UV visible spectrophotometer.

Key words: rice bran oil (RBO), hydraulic press, solvent extraction, microwave assisted extraction, oryzanol, DPPH assay

H. S. Shukla and A. Pratap

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were employed. Different solvent systems were used in solvent extraction process to get highest possible yield. Extraction was also carried out on MAE with isopropanol as a solvent. Wax separation and purification were per-formed for its value addition.

2 Materials and Methods 2.1 Material

Rice bran was purchased from M/s Vidarbha Rice Mill Ltd., Gondia, M.S.（India）. Solvents – hexane（LR）, isopro-panol（AR）, ethanol（absolute）and acetone（AR）were ordered from M/s Thomas Baker Ltd., Mumbai（India）. α,α-diphenyl-β-picrylhydrazyl（DPPH）was purchased from M/s Sigma Aldrich, Mumbai.

2.2 Methods to extraction of RBO2.2.1 Hydraulic press

Dried rice bran was used for hydraulic pressing to recover the oil. The rice bran was filled into the hydraulic cylinder in three equal parts. Pressure was increased grad-ually and kept constant until the oil was obtained. The oil was collected in a beaker, centrifuged and filtered. This method gives virgin rice bran oil.2.2.2 Solvent extraction

The extraction processes start with raw material prepa-ration. 200 g of Rice Bran were weighted and heat treat-ment was provided to it for 5 min in a hot air oven. It is then filled in a thimble and placed in a soxhlet apparatus, which was connected to the Round Bottom Flask at the bottom and condenser at the top. 500 ml of solvent was used to extract oil. In total 100 cycles were performed. As the time for extraction was increase the yield％ also in-creased. After completion of extraction, the solvent was separated from oil using rotary evaporator; where tempera-ture of the batch maintained at the boiling point of solvent under vacuum. Oil then filtered and weighted. This proce-dure was repeated for different solvent. Oil then stored in refrigerator until analysis.2.2.3 Microwave assisted extraction

It is the advanced method by which rice bran oil can be extracted in less time than that of solvent extraction and yield is high as that of solvent method. Extraction was per-formed using high performance microwave reactor, Ulta Clave, Milestone.

13.5 g of rice bran were weighted and filled in a 3 tube vessels used for microwave extraction, and then 37 mL of isopropanol was added into it. One tube was filled with solvent alone. The extraction was set for 30 min at pres-sure 2.1 bar, temperature 82℃（boiling temperature of iso-propanol）and power of 95 W. After completion of extrac-tion process, oil sample was filtered and solvent separated using rotary evaporator. Oil sample was stored in refrigera-

tor at 4℃.

2.3 Analysis using FTIRFurrier transfer infrared spectroscopy was used to

analyze the different functional groups in RBO. The equip-ment used was MIRacle 10, IR Affinity-1. The oil samples were placed on attenuated total reflectance（ATR）accesso-ry and analyzed using transmission mode, taking 64 scan per sample.

2.4 Analysis using GC-MSThe components present in RBO are analyzed using

GC-MS. GC column was used for the analysis has the speci-fication as 5％ diphenyl/ 95％ dimethyl poly sloxane with 30 m×0.25 mm×0.25 μm dimension. Oven temperature was started at 110℃ and held for 2 min, then it was raised at the rate of 10℃/min to 200℃ and then at the rate of 5℃/ min to 280℃ and held at this temperature for 9 min and the injector temperature was set at 250℃. N2 was used as a carrier gas in the system as a mobile phase with a rate of 1 mL/min.

2.5 DPPH scavenging assayThis test was used to find out the free radical scavenging

activity i.e antioxidant activity. Since the rice bran oil con-sists of gamma-oryzanol, the test was carried out to find its activity against methanolic DPPH solution. The solution was prepared by mixing 1 μL of oil solution into 3.9 ml of DPPH solution. The absorbance of test materials was cal-culated using UV- spectroscopy at 515 nm after incubation for a period of 30 min at 28℃. The blank test is also carried out at same condition without test material（oil）and absor-bance was recorded. The antioxidant activity was then cal-culated as percent inhibition according to the equation as follow16）

％ Inhibition＝｛（Ablank－Asample）/Ablank｝*100

2.6 Gamma-oryzanol estimation by spectrophotometerGamma–oryzanol content of each sample was deter-

mined by measuring the optical density（OD）of the sample at 315 nm in n-heptane in cuvvette using spectrophotome-ter. An accurately weighed amount of RBO（about 0.03-0.05 g）was dissolved in n-heptane and the volume was made up to 25 mL. After thorough mixing, the O.D. was recorded at 315 nm by spectrophotometer. Gamma–oryzanol content was calculated as

Gamma－oryzanol（ppm）＝（O.D * 25）/（359* sample wt）

2.7 Oxidation stabilityRBO oxidation stability（induction period）was measured

by a Metrohm 743 Rancimat. For oxidation stability index measurement, 3 g was transferred into a reaction vessel.

Comparative Studies between Conventional and Microwave Assisted Extraction for Rice Bran Oil

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The heater temperature was set at 120℃ and rate of air flow through the sample was adjusted to 20 L/h. The vola-tile components were collected into a glass vessel contain-ing 60 mL distilled water. The resulting curves were evalu-ated automatically by the software of the equipment17）.

3 Results and Discussions 3.1 Yield and property of oil

Yield and composition of oil is the function of time（Fig. 1）. The yield was calculated using following equation.

Oil％＝（mHE / mS）*100

Where, mHE＝oil mass（g）, mS＝Rice bran mass（g）

It was observed that hexane and isopropanol gave the highest yield at optimum time of 4 hour and 500 ml solvent. At highest temperature isopropanol proved as a better ex-traction solvent. When comparing microwave with conven-tional solvent extraction, it was found that microwave as-sisted extraction increased the yield percentage by 4-5％. MAE oil yield with hexane was 19.29％ and isopropanol was 23.6％. The other advantage of MAE over solvent is that it takes less time to extract oil. These results are illus-trated in Fig. 2.

The acid value, saponification value, iodine value and％ of non saponificable matter of rice bran oil extracted using different solvent were found out using AOCS procedure2）. The result of analysis is shown in Table 1.

3.2 FTIR analysis The FTIR results confirmed the presence of different

type of functional groups on the basis of％ of absorbance of UV- rays（wavelength range 4000-1000 cm－1）. The region 1500-750 cm－1（fingerprint）of the IR spectrum con-tains absorption bands that characterize the entire molecu-lar structure by vibrations of the spectrum: deformation, combining, harmonic bands that cannot generally be attrib-uted to normal vibrations. Fingerprint region can be used

to identify the structure compounds with the standard sample. The spectra for oil extracted with different solvent system, MAE and mechanical expeller are shown in Figs. 3-8. The spectra of the oil extracted were compared with the standard sample, which indicated the same type of spectra when compared with standard. Typical band are observed in the entire sample at different range are repre-sented in Table 2.

Fig. 1　Effect of solvent on the yield.

Fig. 2　Comparison of yield by different methods.

Table 1　Properties of rice bran oil.

Methods Acid Value Saponification Value Iodine Value % of Non

Unsaponifiable MatterSE (Ethanol) 4.5±0.5 180 86±1 4.4SE (Acetone) 4.5±0.5 180 85±1 5.3SE (Hexane) 4.5±0.5 180 85±1 4.5SE (Isopropanol) 4.5±0.5 180 85±1 4.3Hydraulic Press 4.5±0.5 180 85±1 5.7MAE (Hexane) 4.5±0.5 180 86±1 4.2MAE (Isopropanol) 4.5±0.5 180 86±1 4.2


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3.3 Identification of the components of the RBO using GC-MS

The identification of the components of the oil extracted by isopropanol solvent was used for analysis. The table showed the fatty acid composition of analyzed RBO. The saturated fatty acid detected in the extract were dodeca-noic acid（lauric acid, RT 6.961 min）, hexadecanoic acid（palmitic acid, RT 10.578 min）, n-hexadecanoic acid（pal-mitic acid, RT 11.428 min）, octadecanoic acid（stearic acid RT 13.244 min）and docosanoic acid（behenic acid, RT 19.409 min）. It also contain polyunsaturated fatty acid（es-pecially 9,12 -octadecadienoic acid（Z, Z）- ）i.e linoleic acid

at a RT of 13.990 min. i-Propyl hexadecanoate is the ester of palmitic acid and isopropyl alcohol. The chromatograph is illustrated in Fig. 9 and its ten major fragments of the compounds are also represented in Table 3.

3.4 DPPH radical-scavengingDPPH is a stable radical which showed a maximum ab-

sorbance at 515 nm. It can readily undergo reduction by antioxidant（AH）thus use in free radical scavenging assess-ment. The disappearance of the DPPH radical by the action of antioxidants was taken as a measure of antioxidant ac-tivity. It can be well expressed by below reaction.

Fig. 4　FTIR result of RBO extracted using ethanol. Fig. 7　FTIR result of RBO extracted using hydraulic press.

Fig. 5　FTIR result of RBO extracted using hexane. Fig. 8　FTIR result of RBO extracted using MAE.

Fig. 3　FTIR result of RBO extracted using acetone. Fig. 6　FTIR result of RBO extracted using isopropanol.

Table 2　Absorbance range and functional groups.

Sr. No. Absorbance Range Type Of Vibration Functional Group1 2852.72 - 2924.09 Stretching Alkane (C-H)2 1411.89 -1462.04 Bending Alkane (-C-H)3 1708.93 - 1710.86 Stretching Acid (C=O)4 1280.80 - 1282.66 Stretching Amine (C-N)5 1008 - 1053 Stretching Alkyl halide (C-F)6 935.48 - 948 Bending Alkene (=C-H)7 723.31 Stretching Alkyl Halide (C-Cl)


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DPPH・＋AH→DPPH-H＋A・

This test showed that oil extracted using hexane had a maximum antioxidant activity as compared to others. The％ of the antioxidant present in oil is illustrated in the Fig. 10. MAE showed the higher scavenging activity than that of solvent extraction using isoporpanol as solvent. Oil from hydraulic press showed moderate antioxidant activity.

3.5 Gamma-oyzanol content in oilOil extracted with different solvents and using different

procedure was checked for the oryzanol content in oil. For this 0.05 g of oil was weighed and dissolved in n-hexane and make solution of 25 mL. The concentration of gamma-

oryzanol using different methods is illustrated in the Table 4.

3.6 Oxidation Stability IndexThe oxidation stability index is highly affected by the

storage conditions. The oil sample were stored for 6 month in dark, the OSI of rice bran oil by each methods were reduced. It was notice that OSI was affected in first few days of storage. This may indicate that the degradation of components present in the oil, this may be due to influence of light on the oil. The result of the OSI is well shown in Fig. 11.

Fig. 9　GC-MS result.

Table 3　Components identified in the oil extracted using isopropanol.

Sr. NoRetention

Time (min)

Name of Compound Molecular formula and MW Largest Peak (m/z Values and Intensities)

1 6.954 Dodecanoic acid, methyl ester C13H26O2 (857) 74 999 | 87 657 | 55 271 | 43 218 | 41 201 | 143 153 | 75 131 |

59 125 | 57 120 | 69 106

2 10.578 Hexadecano ic ac id , methyl ester C17H34O2 (270) 74 999 | 87 725 | 55 229 | 143 200 | 75 195 | 227 135 | 69

133 | 57 130 | 270 113 | 83 88

3 11.428 n-Hexadecanoic acid C16H32O2 (256) 60 999 | 73 980 | 57 840 | 43 817 | 55 767 | 41 574 | 129 435 | 71 373 | 69 351 | 83 267

4 11.852 i-Propyl hexadecanoate C19H38O2 (298) 256 999 | 102 569 | 257 509 | 60 469 | 57 459 | 239 399 | 55 359 | 71 279 | 73 259 | 61 249

5 12.995 14,17-Octadecadienoic acid, methyl ester C19H34O2 (296) 68 999 | 55 840 | 81 820 | 82 680 | 41 670 | 67 650 | 69 500 |

95 500 | 96 480 | 54 390

6 13.244 O c t a d e c a n o i c a c i d , methyl ester C19H38O2 (298) 74 999 | 87 744 | 43 338 | 55 303 | 75 240 | 143 235 | 41 228 |

57 202 | 69 164 | 298 140

7 13.990 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 (280) 67 999 | 55 954 | 81 793 | 41 708 | 69 560 | 95 545 | 68 530 |

54 486 | 43 480 | 82 479

8 19.409 Docosanoic acid, methyl ester C23H46O2 (354) 74 999 | 87 734 | 43 534 | 55 383 | 57 344 | 75 284 | 41 266 |

143 230 | 69 180 | 354 167

Fig. 10　% Inhibition of DPPH.


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ConclusionThis study was carried out to compare different process-

es for extraction of RBO. MAE increased the oil yield up to 20％ and the amount of gamma-oryzanol contained in oil extracted was 2.256 ppm. This study demonstrated that MAE technology is the better alternative to conventional processes regarding energy consumption at laboratory scale. Hexane and isopropanol proved to be better solvents for conventional extraction. In case of MAE, Isopropanol gave the maximum yield than hexane. GC-MS analysis showed the presence of saturated and unsaturated fatty acid in the oil. The presence of omega 6 compound i.e lin-oleic acid improved the oil quality since it helps in decreas-ing blood cholesterol. Also isopropanol at higher tempera-ture resulted into higher percent of antioxidant compound which reveled 95.62％ of DPPH scavenging capability. Hence, MAE performed better than conventional solvent extraction due to its faster heating rate and more yield. The quantity of gamma-oryzanol varied with the type of solvents and methods used. Acetone extraction and MAE（using isopropanol）gave maximum oryzanol concentration in oil. The Oxidation stability indexes（OSI）of the entire sample were initially decreases during a period of 10 days; after that there is no significant decrease in the value.

AcknowledgementsThis research was financially supported by Technology

Education Quality Improvement Programme（TEQIP-II）funded by World Bank.

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Table 4　Conc. of gamma-oryzanol（ppm）content in oil.

Solvent Conc. of gamma-oryzanol (ppm)

Ethanol 2.6086

Acetone 2.636

Hexane 2.229

Isopropanol 2.0947

Method Conc. of oryzanol (ppm)

Hydraulic Press 1.681

Solvent Extraction (Isopropanol) 2.0947

MAE (Iso-propanol) 2.256

Fig. 11　OSI during 150 days of storage.


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