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top oil phase was withdrawn by suction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.