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\begin{document}

\title{Aqueous enzymatic extraction of oil from rice bran and its quality
evaluation.}



\author[1]{Duoxia Xu}%
\author[1]{Jia Hao}%
\author[1]{Zhenhua Wang}%
\author[1]{Huanyue Yang}%
\author[1]{Qianru Gao}%
\author[1]{Ningning Ma}%
\author[1]{Junhai Wang}%
\author[1]{Yinsong  Ma}%
\author[1]{min Zhang}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Rice bran as a by-product of rice processing can be used to produce oil.
The main objective of this study was to select appropriate enzymes for
aqueous enzymatic extraction (AEE) and evaluated the quality of rice
bran oil (RBO) obtained by AEE and solvent extraction (SE). In addition,
the microstructure of rice bran before and after different extraction
processes were also observed. The results showed that Alcalase 2.4L was
the most effective in releasing RBO. Moreover, iodine value and
saponification value in the AEE-extracted oil (AEEO) were found to be
higher than the SE-extracted oil (SEO). AEEO with lower acid value and
peroxide value was considered to be a high quality and the content of
wax and phospholipid in AEEO were lower, which could simplify the
refining steps. Additionally, AEEO exhibited a higher content of
unsaturated fatty acids (76.31\%), vitamin E (1004.42 mg/kg), sterols
(7749.44 mg/100 g), squalene (2962.56 mg/kg) and oryzanol (2.43\%). In
comparison to the SEO, AEEO was found to have lower crystallization and
melting points. Scanning electron microscope (SEM) results showed the
destruction of cell walls and the decrease of substances in the cell by
AEE. In general, the results of this study showed improvement in the
quality of the AEEO and provided necessary data support for green
extraction of RBO in food industry.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Rice bran, as a raw material for rice bran oil (RBO) extraction, is a
by-product of rice processing, in which oil content can reach 12-22\%
(Amarasinghe, Kumarasiri \& Gangodavilage, 2009; Pandey \& Shrivastava,
2018). RBO is a new type of edible oil with high nutritional value. Its
fatty acid composition is reasonable and contains more than 80\% oleic
acid, linoleic acid and linolenic acid. In addition, RBO contains
significant amounts of \selectlanguage{greek}γ-\selectlanguage{english}oryzanol, tocopherols, squalene, phytosterol
and other important bioactive substances, so it is known as
''nutritional health oil'' by nutritionists (Khoei \& Chekin, 2016;
Trevisani Juchen, Nolasco Araujo, Hamerski, Corazza \& Pedersen Voll,
2019).

Current extraction methods of RBO used in oil industry mainly include
cold pressing method, solvent extraction (SE) method and pre-pressed
followed by SE. Cold pressing method can maintain the unique flavor of
oils (Liu et al., 2011). However, this method has many problems, such as
high residual oil rate, high cost, high labor intensity and power
consumption, and easy to cause protein denaturation (Dun et al., 2019).
SE method is often used in commercial RBO production, which is
relatively efficient in oil production, and the residue of oil is low
(Amarasinghe \& Gangodavilage, 2004), but there are also some
shortcomings such as its toxicity, no renewability and easy to cause
environmental pollution (Hanmoungjai, Pyle \& Niranjan, 2001).
Pre-pressed treatment followed by SE method can obtain high oil
extration yield. This process, however, has some problems in the protein
structure leading to protein denaturation. Furthermore, n-hexane has
been identified as an air pollutant that is emitted during extraction
and recovery.

Compared with the other two methods, aqueous enzymatic extraction (AEE)
process has the advantages of good oil quality, no chemical pollution,
low energy consumption and good retention of protein, polysaccharide and
other components. However, AEE has certain limitations. Due to the low
solubility of oil in water, the efficiency of AEE is lower than that of
SE. This disadvantage may be overcomed by using selected enzymes and
optimizing process parameters (Mat Yusoff, Gordon, Ezeh \& Niranjan,
2016). AEE has become a new oil extraction technology for simultaneous
extraction of oil and protein, polysaccharide and other components with
broad application prospects.

It was reported that Sengupta \& Bhattacharyya (1996) used pectinase and
cellulase for the first time to extract RBO by AEE combined with organic
solvent. Hernandez, Rodriguez-Alegr\selectlanguage{ngerman}ía, Gonzalez \& Lopez-Munguia (2000)
obtained glucose, protein hydrolysate, RBO in three steps by adding
glucoamylase and protease before extracting RBO with solvent. After
that, Hanmoungjai, Pyle \& Niranjan (2002) focused on the extraction
yield of RBO, protein and the level of reducing sugar in the enzymatic
hydrolysis process, and finally indicated that Alcalase 2.4L could
significantly improve the yield of oil and protein, while the use of
carbohydrase could improve the level of reducing sugar in the enzymatic
hydrolysate. Although these papers have published on the extraction of
RBO by AEE, no systematic studies have been conducted on the analysis of
quality, thermal behavior and extraction mechanism of the extracted oil.

The objective of this study was to investigate the extraction of oil
from rice bran using the AEE with different enzymes (Celluclast 1.5L,
Hemicellulase, Pectinex Ultra SP-L, Viscozyme L and Alcalase 2.4L) and
selected appropriate enzymes for AEE. The physicochemical properties,
fatty acid compositions, bioactive compounds and thermal behavior of
AEE-extracted oil (AEEO) were evaluated and compared with those of
Soxhlet-extracted oil (SEO). In addition, the microstructure of rice
bran before and after different extraction processes were observed, and
the mechanism of rice bran oil by enzyme extraction was clarified.

\textbf{2. Materials and methods}

\emph{2.1. Materials}

Expanded rice bran were purchased from Fushi Rice Industry Ltd,
Kiamusze, Heilongjiang Province, China. In order to prevent rancidity,
the raw materials were stored in the refrigerator at -20 \selectlanguage{english}. Celluclast
1.5L, Pectinex Ultra SP-L, Viscozyme L and Alcalase 2.4L were purchased
from Novozymes Bagsvaerd (Denmark). Hemicellulase and the standard of
tocopherols, tocotrienols, sterols and squalene were obtained from
Sigma-Aldrich Co. (Steinheim, Germany). Fatty acid methyl esters (FAMEs)
standards were obtained from the AccuStandard (New Haven, CT, USA).
Additionally, all other chemicals were of analytical grade.

\emph{2.2. Aqueous enzymatic extraction (AEE) process}

Fresh rice bran was screened through a 40-mesh sieve (450 \selectlanguage{greek}μ\selectlanguage{english}m 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.

\emph{2.3. Soxhlet extraction (SE) process}

Rice bran was placed into a cellulose paper cone and extracted for 6 h
in a Soxhlet extractor at 90  with n-hexane according to the Soxhlet
standard extraction method (AOAC, 1997). The oil was then recovered by
evaporating off the solvent at 50  under reduced pressure using a
rotary evaporator Model RE-2000A (Yarong Biochemical Instrument Factory,
Shanghai, China) and residual solvent was removed by flushing with
99.99\% nitrogen. This technique produced 18.45 \selectlanguage{ngerman}± 0.25 g of oil per 100
g of rice bran, which was set as 100\% oil recovery for comparison. The
recovered oil was stored at 4 \selectlanguage{english} until use.

\emph{2.4. Physicochemical properties}

AOCS Standard Methods (1997) were used for the determination of the acid
value (method Cd 3d-63), iodine value (method Cd 1-25), saponification
value (method Cd 3-25) and peroxide value (method Cd 8-53) of AEEO and
SEO.

The wax and phospholipid content of RBO were measured according to the
method reported by Pandey et al. (2018). Specifically, wax content in
RBO was measured as acetone insoluble fraction and phospholipid content
of RBO was measured by the Vanadomolybdate method.

The colour parameters of oil samples were measured using chroma meter
CR-400 (Konica Minolta Sensing, Inc., Osaka, Japan). Before using the
chroma meter, it was necessary to correct. A certain amount of oil
samples were taken in the cuvette, then used the chroma meter to
irradiation. The data of L-value, a-value, b-value and colour difference
(\selectlanguage{english}^E) were recorded.

\emph{2.5. GC--MS analysis of fatty acid compositions}

GC-MS was used for fatty acid composition of RBO analysis. The sample
pretreatment was carried out according to the method reported by Zhang
et al. (2010). GC-MS analysis of FAMEs according to the method of China
National Standard GB 5009.168-2016. The analysis were performed by a
QP2010 gas chromatography/mass spectrometer (Shimadzu, Kyoto, Japan),
equipped with a TR-WAXMS capillary column (30 m x 0.25 mm x 0.25 \selectlanguage{greek}μ\selectlanguage{english}m).
The specific operating conditions were as follows: injector temperature
250 ; carrier gas was high purity nitrogen; flow rate 1 mL/min; split
ratio 1:10; the column temperature from 80  to 210  at the rate of 15
/min; finally increased to 240  at the rate of 2 /min; ion source
temperature 200 ; detector temperature 240 ; mass range 40-500 m/z.
The components of oil were determined by comparison of their retention
indices with those of authentic standards of FAMEs.

\emph{2.6.Bioactive compounds analysis}

\emph{2.6.1. Chromatographic analysis of vitamin E}

The content of vitamin E in oil samples was determined by HPLC. Oil was
diluted with n-heptane and analyzed using Agilent Technologies 1260-HPLC
apparatus (Santa Clara, CA, USA) equipped with a fluorescent detector
from the same manufacturer. Separation was carried on a Venusil XBP
Silica column (4.6 \selectlanguage{ngerman}× 250 mm × 5 \selectlanguage{greek}μ\selectlanguage{english}m). The mobile phase was a 3.85\%
tetrahydrofuran in n-heptane solution with a flow rate of 1 mL/min. The
fluorescence detector was set at \selectlanguage{greek}λ\selectlanguage{english}\textsubscript{excitation}= 294 nm and
\selectlanguage{greek}λ\selectlanguage{english}\textsubscript{emission} = 330 nm.

\emph{2.6.2. Gas chromatography analysis of sterols}

According to the modified method of Nyam, Tan, Che Man, Lai \& Long
(2009), the content of sterols was determined by the gas chromatography.
Oil samples and 5\selectlanguage{greek}α-\selectlanguage{english}cholestane solution were refluxed with ethanolic
potassium hydroxide solution for 80 min. The unsaponifiables were first
extracted with diethylether for three times, the ether layers were
washed twice with 0.5 M KOH and deionized water, and then dried with
anhydrous sodium sulphate. The extract was evaporated in a vacuum
evaporator at 25 \selectlanguage{ngerman}°C. N, O-Bis (trimethylsilyl) trifluoroacetamide
(BSTFA) with 1\% trimethylchlorosilane (TMCS) was added to the dry
extracts, and the mixtures were heated at 60 °C for 60 min. The analysis
was carried out using the GC-2010 PLUS (Shimadzu, Kyoto, Japan). Sterols
were separated on a HP-1 capillary column (25 m × 0.32 mm × 0.17 \selectlanguage{greek}μ\selectlanguage{english}m;
Agilent, Santa Clara, CA, USA). The temperatures were as follows:
injector 250 \selectlanguage{ngerman}°C, column 190 °C increased to 230 °C at 20 °C/min, then to
255 °C at 40 °C/min, and maintained for 17 min.

\emph{2.6.3. Gas chromatography analysis of squalene}

The content of squalene was determined by the gas chromatography. 0.2-2
g oil samples and 300 \selectlanguage{greek}μ\selectlanguage{english}L squalane internal standard solution were
saponified with 50 mL ethanolic potassium hydroxide solution at 80 \selectlanguage{ngerman}°C
for 50 min, then were transferred to a separatory funnel containing
deionized water. The saponification solution was extracted three times
with n-hexane, and the extract was combined. The organic phase was
washed to neutral by adding 25 mL ethanol solution. Then the residual
water was removed by anhydrous sodium sulfate and the solvent was
evaporated in a vacuum evaporator at 40 °C. The analysis was carried out
using the GC-2010 PLUS (Shimadzu, Kyoto, Japan) and squalene was
separated on a DB-5 capillary column (25 m × 0.32 mm × 0.17 \selectlanguage{greek}μ\selectlanguage{english}m; Agilent,
Santa Clara, CA, USA). The operating conditions were as follows: split
ratio 1:10; injector 250 \selectlanguage{ngerman}°C; column 160 °C increased to 220 °C at 15
°C/min, increased to 280 °C at 5 °C/min, maintained for 20 min, and
finally to 300 °C at 5 °C/min for 2 min. Squalane was identified by
standard substances and the contents were determined by the
concentration of internal standard.

\emph{2.6.4.} \emph{Determination of the contents of oryzanol}

According to the method of China Grain Sector Standard LS/T 6121.1-2017,
the oryzanol content in RBO was calculated and analyzed by UV-VIS
spectrophotometer (UV2800-A, UNICO instruments Co. Ltd., Shanghai,
China). 0.02 g oils were weighed and diluted to 25 mL with n-heptane.
The absorbance of the solution was measured at 315 nm with n-heptane as
blank. The measured absorbance should be between 0.2-0.8, beyond which
further dilution was required. The content of oryzanol was calculated
according to the formula given below, and the specific extinction
coefficient of oryzanol was known (359 g / 100 mL).

Oryzanol (\%) =\(\frac{\mathrm{A\times N\times 25}}{\mathrm{m(g)\times 359}}\)\(\ \frac{\mathrm{\text{\ A\ }}\mathrm{\ }\mathrm{\text{N\ }}\mathrm{\ }\mathrm{25}}{\mathrm{m\ (g)\ }\mathrm{\ 359}}\)

where A, N, m, were the absorbance of samples, dilution factor after
samples volume determination (N=1 if not diluted) and weight of samples,
respectively. \(\ \)

\emph{2.7.Thermal behavior} \emph{analysis}

Thermal behavior of RBO was analyzed following the method given by
Samaram, Mirhosseini, Tan \& Ghazali (2014). The thermal properties of
the RBO were studied by differential scanning calorimetry (DSC) using a
DSC-60A (Shimadzu, Kyoto, Japan). The purge gas used was 99.99\%
nitrogen with a pressure of 0.5 MPa and a flow rate of 100 mL/min. 5-7
mg of the RBO was weighed in a aluminum pan, heated to 60 \selectlanguage{english} and held for
5 min, then cooled from 60 to -50 \selectlanguage{english} by a rate of 10 \selectlanguage{english}/min with 5 min
holding time, finally heated from -50 \selectlanguage{english} to 60 \selectlanguage{english} at a rate of 10 \selectlanguage{english}/min,
and maintained for 5 min.

\emph{2.8. Scanning electron micrographs (SEM)}

The morphological changes in bran before and after extraction were
studied by a Hitachi S-4800 scanning electron microscope system (Hitachi
Corporation, Japan). Samples were dried at 105 \selectlanguage{english} for 24 h, then were
fixed on a specimen holder with an aluminium tape and sputter-coated
with gold. The images were obtained under vacuum with an acceleration
tension of 15 kV.

\emph{2.9. Statistical analysis}

Results were expressed as means +- standard deviations. The data were
statistical analysis using the software package SPSS 17.0 for Windows
(SPSS Inc., Chicago, IL). Differences between means were determined by
analysis of variance (ANOVA) with Duncan multiple range test.
Differences were considered statistically significant at \emph{P
\textless{}} 0.05. All tests were performed in triplicate.

\textbf{3. Results and discussion}

\textbf{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.

\textbf{3.2 Physicochemical properties} \textbf{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
(\emph{p}\textgreater{} 0.05). Moreover, the PV of AEEO (8.15 +- 0.44
mmol/kg) was found to be significantly (\emph{p} \textless{} 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 I\textsubscript{2}/100 g
oil) in the AEEO was found to be higher than the SEO (102.22 +- 0.56 g
I\textsubscript{2}/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 (\emph{p} \textless{} 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 (\emph{p}\textless{} 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.

\textbf{3.3 Fatty acid compositions}

As shown in Table 2, fourteen major fatty acid components were detected
and identified, including eight saturated fatty acids, four
monounsaturated fatty acids and two polyunsaturated fatty acids.
Comparing the composition of AEEO and SEO fatty acids, it was found that
there were some differences presented in fatty acid contents, while no
obvious difference in their types was detected. The results showed that
the extraction method had no significant effect on fatty acid
composition of oils, which was in agreement with other reports (Jiao et
al., 2014; Li et al., 2013). The contents of fatty acid obtained were
different from the values obtained by Trevisani Juchen et al. (2019).
The difference in fatty acid contents might be attributed to the
cultivars, planting regions, climate conditions, and the posttreatment
process (Liu et al., 2019).

It could be observed that the preponderant fatty acid in RBO was
linoleic acid (38.26\%-38.84\%), oleic acid (33.83\%-34.31\%), followed
by palmitic acid (19.17\%-19.87\%). It also could be seen that the
content of unsaturated fatty acids accounting for about 75\% of total
fatty acids in AEEO and SEO. Moreover, the contents of monounsaturated
fatty acids and polyunsaturated fatty acids in AEEO were much higher
than that of SEO. It was likely because the relatively mild extraction
conditions of AEE was not easy to cause the oxidation and decomposition
of unsaturated fatty acids. According to the literature, the oil
contains a high level of monounsaturated fatty acid, which has a great
potential to produce biodiesel (Lovato, Pelegrini, Rodrigues, Braz De
Oliveira \& Piloto Ferreira, 2014). The high proportion of linoleic and
linolenic acids were precursors of polyunsaturated fatty acids from the
omega-3 and omega-6 series and were essential for human health and
disease prevention, which indicated their potential development
prospects in health products and pharmaceutical industries (Chen, Luo,
Qi \& Wan, 2014), but also reduced oil stability and shelf life (Haiyan,
Bedgood, Bishop, Prenzler \& Robards, 2007). Therefore, compared with
SE,~AEE can reduce costs and green production, while AEEO also has
health benefits.

\textbf{3.4.1 Vitamin E contents}

\begin{quote}
VE is a recognized antioxidant, mainly \selectlanguage{greek}α/γ-\selectlanguage{english}tocopherol and tocotrienol in
RBO (Capellini et al., 2017). In this experiment, the total amount of
vitamin E was expressed as the sum of \selectlanguage{greek}α-\selectlanguage{english}tocopherol, \selectlanguage{greek}α-\selectlanguage{english}tocotrienol,
\selectlanguage{greek}γ-\selectlanguage{english}tocopherol and \selectlanguage{greek}γ-\selectlanguage{english}tocotrienol. As shown in Table 3, the vitamin E
content in RBO ranged from 839.15 to 1004.42 mg/kg, which was similar to
that of Zigoneanu, Williams, Xu \& Sabliov (2008). A significantly
(\emph{p}\textless{} 0.05) higher concentration of total vitamin E was
determined in the AEEO as against the SEO. The higher concentration of
vitamin E in the AEEO could improve its oxidative stability. It also
could be seen that the content of tocotrienol ranged from 547.02 to
643.67 mg/kg, accounting for about 60\% of total vitamin E in RBO and
the tocotrienol content in AEEO was significantly (\emph{p} \textless{}
0.05) higher than that of SEO. Tocotrienols had more significant
physiological functions than tocopherol in antioxidant activity,
cholesterol and blood lipid reduction (Qureshi et al., 1991). Comparison
of AEEO and SEO showed significant (\emph{p} \textless{} 0.05)
difference of \selectlanguage{greek}α-\selectlanguage{english}tocopherol contents which were 315.87 mg/kg and 261.13
mg/kg. Karmowski, Hintze, Kschonsek, Killenberg \& B\selectlanguage{ngerman}öhm (2015) confirmed
that the antioxidant capacity of \selectlanguage{greek}α-\selectlanguage{english}tocopherol was slightly lower than
that of \selectlanguage{greek}α-\selectlanguage{english}tocotrienol. It can be seen that AEEO with higher natural
antioxidant is an important nutritional and healthy oil, and its
development and utilization prospects are very broad.
\end{quote}

\textbf{3.4.2 Sterols contents}

The content and composition of phytosterols in RBO extracted using
various methods were listed in Table 3. It was found that the levels of
phytosterols in RBO obtained by AEE was much higher compared with that
of SEO. It was in agreement with Konopka, Roszkowska, Czaplicki \&
Ta\selectlanguage{polish}ń\selectlanguage{english}ska (2016) who reported that higher phytosterols content in the
enzyme-extracted oil. While, it was different from the results of Nyam
et al. (2009) that the Kalahari melon seed oil extracted by organic
solvents contained higher sterols contents. As could be seen in Table 3,
the predominant sterols in RBO were \selectlanguage{greek}β-\selectlanguage{english}sitosterol (50\% of total
sterols), followed by campesterol, stigmastanol and stigmasterol. It
indicated that enzymatic treatment can release more antioxidant
compounds from rice bran due to the hydrolysis of cell walls. In
addition, enzymatic hydrolysis can reduce the degree of interaction
between antioxidants and proteins, polysaccharides and pectins in the
bran and promote their release into oils (Fang, Du, Luo \& Jin, 2015).
The lower content of unsaponifiable compounds in SEO might be due to the
low polarity of n-hexane.

\textbf{3.4.3Squalene contents}

Squalene is a highly unsaturated aliphatic hydrocarbon
(C\textsubscript{30}H\textsubscript{50}), which is widely found in olive
oil, soybean oil and peanut oil (Chanioti \& Tzia, 2019). Previous
studies have shown that squalene has natural antioxidant activity, which
makes it widely used in cosmetics. However, phenols and tocopherols are
the main antioxidants in oils, which makes the antioxidant activity of
squalene less obvious (Psomiadou \& Tsimidou, 1999). Comparison of AEEO
and SEO showed significant (\emph{p} \textless{} 0.05) differences of
squalene contents which were 2962.56 mg/kg and 2479.56 mg/kg,
respectively. It indicated that AEE promoted the release of squalene
compared with SE. The contents of squalene obtained by extraction were
much less compared with that by cold pressing (3189 mg/kg) (Pokkanta,
Sookwong, Tanang, Setchaiyan, Boontakham \& Mahatheeranont, 2019). It
illustrated that the content of squalene in crude oils depended on the
different extraction methods.

\textbf{3.4.4. Oryzanol contents}

Rice bran is one of the most abundant sources~of oryzanol. RBO contains
about 0.9-2.9\% oryzanol which can vary greatly according to the origin
of the rice bran (Arab, Alemzadeh \& Maghsoudi, 2011). Oryzanol is a
mixture of esters of ferulic acid with sterols and triterpene alcohols,
and has similar antioxidant activity as vitamin E (Lloyd, Siebenmorgen
\& Beers, 2007). In Table 3, the content of oryzanol in AEEO was
significantly higher (\emph{p \textless{}} 0.05) than in SEO, which can
be attributed to the degradation of oryzanol caused by the increase of
temperature and the prolongation of SE treatment time.

\textbf{3.5 Thermal behavior of AEEO and SEO}

DSC is to determine the thermal behavior of oils during heating or
cooling and it provides another method for studying the chemical
composition of oils. The crystallization and melting curves of the AEEO
and SEO were shown in Fig. 2 and their corresponding transition
temperatures were also given.

In Fig. 2a, the crystallization curves of RBO extracted by Alcalase 2.4L
and n-hexane were represented. The cooling curve of AEEO and SEO
exhibited three exothermic peaks, with a major peak (a\textsubscript{2},
b\textsubscript{2}) and two small shoulder peaks (a\textsubscript{1},
b\textsubscript{1}) and (a\textsubscript{3}, b\textsubscript{3}). It
could be clearly seen that the points of crystallization peaks of the
two extraction methods were obviously different. The initial
crystallization peaks of AEEO and SEO were 30.93  and 33.70 ,
respectively. Compared with the SEO, the crystallization point of AEEO
was lower. The various of crystallization peaks might be due to the
different extraction methods, which could influence the degree of
saturation in RBO (Tan, Chong, Hamzah \& Ghazali, 2018). Generally, each
kind of oil has unique characteristic of fatty acids and triacylglycerol
(TAG) profiles and the thermal transition of lipids is affected by the
compositional changes of the degree of saturation and fatty acid chain
length. The crystallization peaks of oils with high levels of saturated
fatty acids and tri-saturated TAGs would be at a relatively high
temperature (Mohammadpour, Sadrameli, Eslami \& Asoodeh, 2019).

The melting curves of RBO extracted by Alcalase 2.4L and n-hexane were
represented in Fig. 2b. It was found that SEO displayed one peak
(b\textsubscript{1}) at low-melting region (\textless{} 0 ) and the
other peak (b\textsubscript{2}) at high-melting region (\textgreater{} 0
). However, this phenomenon was not observed in the melting curves of
AEEO and all the melting peaks of AEEO were located in the low-melting
region. Results showed that the melting behavior of RBO was
significantly related to the extraction method. As discussed, the
possible reason of these differences might be the variations in the
proportional distributions of the identified fatty acid and TAG
components. It might be due to the saturated compounds of SEO were
slightly higher than AEEO. Therefore, higher temperatures were needed to
melt SEO completely.

\textbf{3.6. Microstructural analysis}

To better understand the influence of different extraction methods on
the characteristics of RBO, microstructure of native and samples after
extraction were investigated by SEM. The images showed that the internal
tissue of the rice bran seems to undergo a process of destructing and
restructuring. In Fig. 3a, natural rice bran had a relatively complete
structure, regular or dense shape and smooth surface, meanwhile, the
intact oil cells on the surface of bran tissues before extraction were
observed. Fig. 3b showed the expansion process caused the disruption of
cell walls and loose cell arrangement of expanded rice bran, making
conventional extraction or enzymatic extraction more accessible to
organelle. After AEE, the oil was almost extracted together with
aleurone grains and the substances in the cell gradually were decreased,
therefore, the structure of rice bran cell was further destroyed (Fig.
3c). In Fig. 3d, the surface of rice bran treated with n-hexane became
more fragmented, dried and cracked, and some lipids and starches were
dragged away (Alc\selectlanguage{ngerman}ázaralay, Cardenastoro, Santos \& Meireles, 2015). In
the process of SE, the chemical affinity between organic solvent and
oils affected the mass transfer process which caused a high oil
extraction yield, but it could also damage the protein and starch in the
extraction process. The process of enzymatic extraction was based on the
insolubility of oils in water and the efficiency was lower than that of
SE. However, AEE could simultaneously separate solid phase (mainly
starches and proteins) and an oil phase, so the development of these
by-products could also make full use of rice bran.

\textbf{4. Conclusion}

In the present work, AEE process proved to be effective in extracting
RBO. Alcalase 2.4L was found to give higher oil yields than Celluclast
1.5L, Hemicellulase, Pectinex Ultra SP-L, Viscozyme L. Comparisons of
the physicochemical characteristics of AEEO with those of SEO revealed
that different extraction methods had influence on the quality
parameters of RBO. Briefly, iodine value, saponification value in the
AEEO were found to be higher than SEO. AEEO had lower acid value and
peroxide value which was considered to be a high quality oil. The
content of wax and phospholipid in AEEO was lower, which could simplify
the refining steps. In addition, GC-MS results showed that AEE increased
the amounts of unsaturated fatty acids compared with SE. A comparison of
the content of bioactive compounds showed that RBO extracted by the AEE
was more abundant in vitamin E, sterols, squalene and oryzanol. DSC
results indicated~that the different methods used for oil extraction
caused the variations in the melting and crystallization behaviour of
RBO. In comparison to the SEO, AEEO were found to have lower
crystallization and melting points. Moreover, SEM results demonstrated
that the destruction of cell walls and the decrease of substances in the
cell by AEE. These results suggested that AEE offers an efficient,
environment friendly and easy procedure of RBO extraction and the oil
produced by this emerging method can be used as high-quality edible oil
in food industry.

\textbf{Acknowledgements}

This research was supported by the 13th Five-Year the State Key
Development Program (2018YFD0401100), National Natural Science
Foundation of China (31771976), Support Project of High-level Teachers
in Beijing Municipal Universities in the Period of 13th Five-year Plan
(CIT \& TCD201804018), Construction of Service Capability of Scienti\selectlanguage{english}fic
and Technological Innovation (PXM2019\_014213\_000010,
PXM2018\_014213\_000033, PXM2018\_014213\_000014,
PXM2018\_014213\_000041, 19005857058), Cultivation and Development of
Innovation Base (Z171100002217019), School Level Cultivation Fund of
Beijing Technology and Business University for Distinguished and
Excellent Young Scholars (BTBUYP2020), Beijing Key Laboratory of the
Innovative Development of Functional Staple and the Nutritional
Intervention for Chronic Disease.

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