3.1 Selection of appropriate enzyme for AEE
Fig. 1 showed the effect of five different enzymes (Celluclast 1.5L, Hemicellulase, Pectinex Ultra SP-L, Viscozyme L and Alcalase 2.4L) on RBO extraction yields. It could be observed from Fig. 1a that more RBO was extracted using hydrolytic enzymes (28.59-50.37%) compared with the control (21.30-40.12%). Alcalase 2.4L was found to be the optimum enzyme obtaining the highest RBO yields with 50.37%. The efficiency of Alcalase 2.4L for RBO extraction was consistent with the results of Hanmoungjai et al. (2002). Indeed, protease treatment can hydrolyze proteins into peptides, leading to effective dissolution and hydrolysis of proteins, then break down the protein networks of cotyledon cells and protein based membranes surrounding the lipid bodies, thereby liberating more oils (Jiao et al., 2014; Latif & Anwar, 2011). It was also found that the oil extraction yields of control at pH 9.0 was significantly higher than that of the carbohydrase treated rice bran. This might be due to the increased solubility of proteins under alkaline conditions and saponification of available free fatty acids, which improved the separation efficiency of oil bodies from their original location at high pH and low surface tension (Amarasinghe et al., 2009).
The effect of the combination of each carbohydrase with Alcalase 2.4L on oil yields was shown in Fig. 1b. It exhibited that the combination of carbohydrases and protease did not significantly increase the extraction yields of RBO. The use of cellulose, pectinase and other carbohydrase can hydrolyze cellulose, hemicellulose, pectin and other components forming cell wall thus breaking down the structural integrity of the cell walls and improving the oil extraction yields. However, the rice bran material selected in this study had been expanded, which could damage the cell wall to a certain extent, therefore, the carbohydrases had little effect on the release and extraction of RBO as shown in the result.
3.2 Physicochemical properties of extracted RBO
The physicochemical properties of AEE-extracted oil (AEEO) and SE-extracted oil (SEO) were listed in Table 1. Acid value and peroxide value, respectively, indicated the oxidation and rancidity of RBO and oil samples with low acid value and peroxide value were considered to be a high quality. The acid value in the AEEO were found to be slightly lower than the SEO, but the difference were not significant (p> 0.05). Moreover, the PV of AEEO (8.15 ± 0.44 mmol/kg) was found to be significantly (p < 0.05) lower than 8.80 ± 0.00 mmol/kg for SEO. These values are slightly different from those reported by Phan, Junyusen, Liplap & Junyusen (2018), which may be due to climate conditions and different cultivars of rice bran. Additionally, iodine value (106.31 ± 0.44 g I2/100 g oil) in the AEEO was found to be higher than the SEO (102.22 ± 0.56 g I2/100 g oil) which can be due to a higher content of unsaturated fatty acids in the AEEO. The saponification value of SEO was significantly (p < 0.05) lower than AEEO and the values were similar to those reported by Amarasinghe et al. (2009) and Sengupta et al. (1996).
Wax content of AEEO was found to be significantly less than the SEO. Wax in RBO seriously affects the quality of RBO. However, as an important by-product of RBO refining process, rice bran wax is rich in octacosanol. Octacosanol has been widely used in various food production due to the function of lowering blood lipid or cholesterol (Chen et al., 2007). A significantly (p< 0.05) higher concentration of phospholipids was determined in the SEO compared with the AEEO. According to Balachandran, Mayamol, Thomas, Sukumar, Sundaresan & Arumughan (2008), the phospholipid content in RBO extracted with n-hexane ranged from 1% to 5%, which was agreement with the results of this study. While, no phospholipids were detected in RBO extracted by AEE. The result was consistent with the report of Rovaris et al. (2012) that more than 90% lipids existed in the residue phase and the rest in the aqueous phase during the enzymatic extraction of soybean oil. Because of the presence of phospholipids and effective emulsifiers such as lecithin, oils are emulsified and not easy to be separated from raw materials. Phospholipids have a polar phosphate group attached to a lipid group by electrostatic forces and hydrogen bonds, which can be transferred to the oil when the polar solvent, able to break these bonds and release them, is used as extraction medium (Capellini, Giacomini, Cuevas & Rodrigues, 2017). It can be concluded that the contents of wax and phospholipid in AEEO was low, which can simplify the following refining steps.
It was also found that the L-value and b-value of AEEO were higher than SEO. It indicated that AEEO were more yellow and lighter-coloured than SEO. It can be attributed to that the organic solvent extracted pigments such as carotenes and chlorophylls from the bran. Consequently, the results of physicochemical properties showed that the quality of AEEO was better than that of SEO.