Physico-Chemical Properties and GCMS Analyses of Indigenous Rice Bran and Mustard Seed Oils and their Blends

Blended edible oils were formulated to enrich fatty acid profile with balance fatty acid ratio. Indigenous Rice Bran Oil (RBO) and Mustard Seed Oil (MO) were mixed in the ratio of 60:40, 70:30 and 90:10 (RBO:MO). Moisture content, density, specific gravity, refractive index, viscosity, free fatty acid, acid value, iodine value, unsaponifiable matter and peroxide value of the single vegetable oils and their blends were determined. Fatty acid composition such as Saturated Fatty Acid (SFA), Mono-Unsaturated Fatty Acid (MUFA), Poly-Unsaturated Fatty Acid (PUFA) of all oils were analyzed by GCMS. GCMS analysis of rice bran oil identified 6 fatty acids with ratio SFA:MUFA:PUFA=1.43:1.46:1.0 and mustard seed oil identified 8 fatty acids with ratio SFA:MUFA:PUFA=1.72:1:2.06. All blended oils revealed 9 fatty acids with ratio SFA:MUFA:PUFA=1.24:2.1:1.0;SFA:MUFA:P -UFA=1.34:2.56:1.0andSFA:MUFA:PUFA=1.2:1.6:1.0; for 60:40, 70:30 and 90:10 (RBO: MO) respectively. Among three blendings, SFA:MUFA:PUFA=1.2:1.6:1.0 for 90:10 (RBO: MO) ratio was near World Health Organization (WHO) guidelines. According to WHO and other health agencies, nutritionally superior oil must fall in the ratio of 1:1-3:1; SFA; MUFA and PUFA respectively. The results suggest that 90:10 (RBO:MO) blended oil can be good alternative oil for edible purposes with improve nutrition as well as desirable physico-chemical properties. GCMS Analyses of

oxidation and also for the formation of undesirable compounds or flavours [5,6]. However, there is global increasing demand for the production of a blending oil for edible purposes because of improved fatty acids and nutritional benefits.
Blending is one of the methods used to modify oils and fats for wide range of applications. Evidences suggest that no single oil can provide the recommended dietary fatty acid ratio. According to WHO and other health agencies, nutritionally superior oil must fall in the ratio of 1:1-3:1; SFA, MUFA and PUFA respectively [7].
The objective of the present investigation was to design an oil blend which may provide an ideal recommended fatty acid ratio regarding nutrition.

Materials
Mustard seeds were procured from local market then fresh and healthy mustard seeds were stored for oil extraction. Freshly milled rice bran, outer layer of the grain was collected from rice milling industry, Rajshahi, Bangladesh and immediately processed after milling for stabilization. Primarily sieved with 100 mesh size to separate husk and broken rice grain. All solvents and chemicals used were analytical, HPLC and GC grade. Fatty acids standards were purchased from Sigma-Aldrich.

Extraction of Oils
Rice bran oil was extracted by solvent extraction method. The extraction process was conducted in the laboratory by Soxhlet apparatus for 6 hours using n-hexane as extracted solvent. The ratio of rice bran:hexane used was 1:3 (w/v). n-hexane has been selected because the solvent has better effect over other polar solvents like alcohol, ketone, aldehyde ether and ester etc. [8]. The oil was recovered by the evaporation of the solvent under reduced pressure using a Rotary vacuum evaporator and the percentage of oil content was calculated. Mustard oil was extracted by automated oil expelling machine. The refining process of the oils was carried out using the method as described by [9] with some modification.

Preparation of Blends
Rice bran oil and mustard seed oil were taken in the ratio of 90:10, 70:30 and 60:40 respectively. Each blend was placed in 250 ml beakers and were mixed by using a mechanical stirrer at 180 rpm for 15 min. Then the single oils and all blended oils were stored at room temperature in air tight glass bottle for physico-chemical and GCMS analyses.

Analysis of Physicochemical Properties
Physical properties such as moisture content, refractive index, density, specific gravity and viscosity of the single and blended oils were determined by the method described by Garba, et al. [10] and AOAC [11]. Acid value, saponification value, iodine value, peroxide value, unsaponifiable matter and free fatty acid were estimated according to the AOAC and AOCS Official Methods [12,13].
Viscosity of all oils were determined by Fungilab digital Rotational viscometer. They measure viscosity by sensing the torque required to rotate a spindle at constant speed while immersed in fluid.

Determination of Fatty Acid Composition
Preparation of Fatty Acid Methyl Ester: 200 mg of oil sample was taken in a 10 mL Pyrex test tube. 3.5 mL 0.5M Sodium methoxide was added to the test tube and heated using burner until removing the bubbles. Then, 1.5 mL n-hexane was added to the mixture and mixing by vortex mixture. Then around 5 mL deionized water was added to test tube slowly and waited for settle down. Organic upper layer was taken into the GC vial for GCMS analysis.

Results and Discussion
Moisture is the amount of water present in edible oils. Moisture should be as low as possible. The maximum allowed moisture content in edible oils is 0.2% [14]. Previous studies have shown that fungus species such as Aspergillus niger and Mucor species survive and reproduce when the moisture content value is higher than 0.2% / [15]. Moisture content of all oils and their blends were determined presented in Table 1. All values were very low than specified limit. Density, specific gravity and refractive index of all single oils and their blends were determined presented in Table 1.
Results were within the WHO/FAO permissible limits. Viscosity of oil is a measure of the oils resistance to flow. In the present study, it can be observed from Table 1 that viscosity decreased in the blended oils than single vegetable oils, this is because blending oil with different ratios decreases saturation as well as increases unsaturation fatty acids of oil. This phenomenon also verified by other researcher since oil viscosity depends on molecular structure and decreases with unsaturation of fatty acids [7]. It may due to double bonds that make bonding more rigid and rotation between c-c bonds become more strenuous.  Table 1 that the FFA value of all oils were very low and the percentages of AV for all oils were within the permissible level (0.6 mg KOH/g) [16]. All oils were of good quality because of the low percentage of FFA and acid value. Theoretically, AV is known to be high when FFA content is high. FFA content differs from AV, which can be due to the fact that acid phosphates as well as amino acids contribute to the acid level [17]. Mean peroxide value of the oil samples are shown in Table 1. Highest level of peroxide value was observed for mustard oil (2.84 mEq/kg oil) and lowest for RBO:MO, 90:10 blended oil (1.44 mEq/kg oil). But levels of peroxide value obtained for the all oil samples were below the acceptable limit (10 mEq/kg oil) recommended by Codex Standards [18]. The low values of peroxide are indication of low levels of oxidative rancidity of the oils. A rancid taste often begins to be noticeable when the peroxide value is above 20 meq/kg [19]. The iodine value is a measurement of the total unsaturation of vegetable oils, as well as an indicator of their susceptibility to oxidation [19]. In the present study shown in Table 1, the blends of mustard oil with rice bran oil shows increase in iodine value. Hence, the observed higher iodine value in the oils indicated that they are likely to be healthier for consumption. Studies have recommended to switch from saturated to unsaturated fats because of the risk of cardiovascular disease associated with high consumption of saturated fatty acids [20][21][22][23].
The unsaponifiable matters for all oils were within 0.35-0.62 % presented in Table 1 and the values were in good agreement with the reported result of Tan, et al. [24]. Note: All the experiments were performed in triplicate and the results were expressed as mean ± SD (standard deviation).
The results of GCMS analysis of rice bran oil are displayed in Figure 1 and presented in Table 2. They revealed the presence  Table   3. GCMS analysis of mustard seed oil are shown in Figure 2 and presented in Table 4.  Table 3. GCMS analysis of blended oil mixing ratio of RBO:MO;90:10 are displayed in Figure   3 and presented in can be a cost-effective practice to modify their fatty acid profile and physico-chemical properties [7]. Present work can meet the desired requirements.