2.2. Aqueous enzymatic extraction (AEE) process
Fresh rice bran was screened through a 40-mesh sieve (450 μm aperture size), the powders were sealed in plastic containers and stored in a refrigerator at 4 ℃ for 24 hours before use. The ground rice bran was mixed with distilled water at a ratio of 1:6 w/v using a beaker. The mixture was subjected to boiling for 5 min at 90 ℃ and allowed to cool down to room temperature (Mat Yusoff et al., 2016). The pH of the mixture was adjusted to the optimum pH of enzyme with 2 M NaOH and 2 M HCl. Then 2% (w/w) of enzyme (Celluclast 1.5L, Hemicellulase, Pectinex Ultra SP-L, Viscozyme L, Alcalase 2.4L) was added to the mixture, respectively. The samples were incubated at the optimum temperatures (50 ℃, 50 ℃, 50 ℃, 45 ℃, 60 ℃) of various enzymes for 120 min followed by centrifugation (Hexi Instrument Equipment Co. Ltd., H/T 16MM, Hunan, China) at 10000 rpm, 30 ℃ for 20 min. After centrifugation, the enzymatic hydrolysate was divided into four phases: oil phase, creaming phase, aqueous phase and residue phase. The top oil phase was withdrawn by suction tube, the creaming phase was separated and demulsified with ethanol solution and further centrifuged to obtain residual oil. The free oil was combined and used for analysis. Then the residue phase was baked at 60 ℃ for 4 hours and analysed for residual oil according to the 2.3 method. The extraction yield was calculated according to the method reported by Hanmoungjai et al. (2002). In addition to individual carbohydrase, the combined effect of each carbohydrase with Alcalase 2.4L on the extraction of RBO was also studied. The rice bran was first incubated with 1% (w/w) carbohydrase for 1 h and then with 1% (w/w) Alcalase 2.4L for another hour. All other treatments were identical to single enzyme treatments. Control samples underwent the same extraction process except for the enzyme addition.