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2.4. Other extraction methods
2.4.1. Ultrasound-assisted extraction (UAE)
The ultrasound-assisted extraction was performed in an ultrasonic
cleaning bath (KQ3200B, 40.0 kHz,150W, Kunshan Ultrasonic
Instrument Co., Ltd., Jiangsu, China) with a usable capacity of 2.5 L
(the internal dimensions: 30.0 15.0 15.0 cm). An in-water pipe
was added to the opposite out-water pipe in the bath, and the flux
ratio between in-water and out-water was regulated to keep solution
temperature stable in the test. Samples were placed into a
conical flask (150 mL), made up to the required volume with
distilled water, and sonicated at the required temperature for
different times. Then the mixture was filtered through Whatman
No. 1 filter paper (Whatman-Xinhua Filter Papers Co., Zhejiang,
China). The filtrate was used for further study.
2.4.2. Microwave-assisted extraction (MAE)
A domestic WP700TL 23-K5 microwave-assisted extraction unit
(Glanz Group Co., Ltd., Guangdong, China) with a 2450 MHz
magnetron was used in the extraction step. The maximum output
power of the oven was 700 W. The whole system was run at atmospheric
pressure. Dried ground bran powder sample (10.0 g)
was mixed with 150 mL of distilled water in a 200 mL flask, and
then the suspensions were irradiated under microwave heating.
After each extraction, the obtained extracts were cooled to 25 C
(Yang et al., 2012a). The filtrate was used for further study.
2.4.3. Conventional reflux extraction
A 10.0 g sample was extracted with 150 mL of distilled water
under reflux at 100 C for 2 h. Then the mixture was filtered
through Whatman No. 1 filter paper (Whatman-Xinhua Filter Papers
Co., Zhejiang, China). The filtrate was used for further study.
2.5. Preparation of crude b-glucan
A step for further purificationwas used to remove starch residue
using 1% a-amylase (W/V) at 55 C water bath for 2 h. The mixture
was centrifuged for 15 min at 21,000 g at 4 C to remove solids.
The supernatant was added with 0.5% protease (W/V) at 70 C for
2 h and centrifuged again (20 min at 21, 000 g, 4 C) to separate
precipitated proteins, which were discarded. The supernatant was
precipitated with anhydrous ethanol to produce crude b-glucan,
and then the supernatant was removed after vacuum filtration, and
the residue was washed with four volumes of anhydrous ethanol
and dried at 40 C overnight. The samplewas weighed and stored in
a desiccator until analysis. The extracted dry matter content was
measured to calculate the extraction yield. The yield of crude bglucan
was calculated as a percentage of the weight of dry bran
powder. The percentage of crude b-glucan yield (%) was calculated
as follow:
Crude b glucan yield ð%Þ ¼ ðWeight of crude b glucanÞ=
ðWeight of bran powderÞ
100%
2.6. Experimental design
After determining the preliminary range of ASE variables
through a single test, a three-variable-three-level BBD (Box and
Wilson, 1951) was applied to optimize the extraction condition in
order to obtain a high yield of crude b-glucan from bran of hull-less
barley. The three independent variables were extraction temperature
(C, A), extraction time (min, B), and number of cycles (C), and
each variable was set at three levels. A total of 17 experiments were
designed according to BBD. Each experiment was performed in
triplicate and the average yield of crude b-glucan (%) was taken as
the response, Y.
Regression analysis was performed for the experimental data
and was fitted into the empirical second order polynomial model,
as shown in Equation (1):where Y was the dependent variable, A0 was constant, and Ai, Aii,
and Aij were coefficients estimated by the model. Xi and Xj were
levels of the independent variables.
2.7. Infrared spectroscopy of crude b-glucan
The IR spectrum of the crude b-glucan was determined using a
Fourier transform infrared spectrophotometer (FT-IR). The crude bglucan
sample was ground with spectroscopic grade potassium
bromide (KBr) powder and then pressed into 1mmpellets for FT-IR
determination in the frequency range of 4000e400 cm1.
2.8. Statistical analyses
A software Design-Expert 7.1.3 (Trial version, State-Ease Inc.,
Minneapolis, MN, U.S.A.) was used to obtain the coefficients of the
quadratic polynomial model. The quality of the fitted model was
expressed by the coefficient (R2) of determination, and its statistical
significance was checked by F-test. Data are expressed as
mean standard deviation (n ¼ 3).
3. Results and discussion
3.1. Comparison of different extraction methods
The yields of crude b-glucan using different extraction methods
were shown in Table 1. The highest yield of crude b-glucan (8.8%)
was obtained under ASE, followed by reflux method (2.2%), MAE
method (0.3%) and UAE method (0.3%). Compared with reflux (2 h)
and ultrasonic-assisted extraction method (1 h), ASE method spent
only 6 min. As compared with reflux and UAE methods, ASE produced
much higher b-glucan and consumed shorter time. The
conventional solvent extraction method has distinct drawbacks
such as time-consuming and labor-intensive operations and
extended concentration steps which can result in the loss of
degradation of target analytes (Li et al., 2010).
3.2. Effect of extraction temperature on yield of crude b-glucan
The extraction process was carried out at different temperature
conditions (50, 60, 70, 80 and 90 C) while other extraction variables
were set as follow: extraction time of 5 min, extraction
pressure of 10 MPa, and number of cycles of 2. As shown in Fig. 1a,
the crude b-glucan yield reached a maximum at a temperature of
70 C and began to decrease. This tendency was in agreement with
the report of other authors in extracting polysaccharides (Shao
et al., 2011). This indicated that temperature enhanced the crude
b-glucan extraction from bran of hull-less barley into the water to a
certain level followed by their possible loss, due to decomposition
at a higher temperature.
3.3. Effect of extraction time on yield of crude b-glucan
The effect of extraction time on yield of crude b-glucan is shown
in Fig. 1b. The extraction time was set at 3, 5, 7, 9, and 11 min, other
experimental conditions were as follow: extraction temperature of
60
C, extraction pressure of 10 MPa, and number of cycles of 2. The
results indicated that the extraction yield increased with time until
7 min and began to decrease, and the maximum extraction yield
was 11.7% at 7 min. The decreases of crude b-glucan yield might be
due to longer extraction time inducing the degradation of b-glucan
(Li et al., 2012).
3.4. Effect of number of cycles on yield of crude b-glucan
The number of cycles was a factor that would influence the
extraction yield. To study the effect of different numbers of cycles
on the crude b-glucan yield, the extraction process was carried out
using different numbers of cycles (1, 2, 3, 4, and 5). Other extraction
variables were set as follow: extraction time of 5 min, extraction
pressure of 10 MPa, and extraction temperature of 60 C. It could be
found that the yield of crude b-glucan increased by increasing the
number of cycles from 1 to 3, and then decreased when the number
of cycles ranged from 3 to 5 (Fig. 1c). The crude b-glucan yield
reached a maximum when the number of cycles was three.
3.5. Model building and statistical analysis
There were a total of 17 runs for optimizing the three individual
parameters in current Box-Behnken design. The current designwas
applied to the production of crude b-glucan by ASE. Table 2 show
the process variables and experimental data. The results of the
analysis of variance (ANOVA), goodness-of-fit and the adequacy of
the models were summarized. By applying multiple regression
analyses on the experimental data, the response variable and the
test variables were related in the following second-order polynomial
Equation (2):
The fit statistics of extraction yield (Y) for the selected quadratic
predictive model is shown in Table 3. For the model fitted, the
coefficient of determination (R2) was 0.9973, indicating that only
0.27% of the total variations was not explained by the model. Fvalue
for the lack of fit was insignificant (p > 0.05), thereby
confirming the validity of the model. The value of the adjusted
determination coefficient (adjusted R2 ¼ 0.9939) was almost 1,
indicating a high degree of correlation between the observed and
predicted values. At the same time, a low value 1.56 of coefficient of
the variation (CV) clearly indicated a very high degree of precision
and a good deal of reliability of the experimental values. The model
p-value (Prob > F) was very low (<0.0001), which implied that the
model was highly significant.
The model was found to be adequate for prediction within the
range of experimental variables. The regression coefficient values of
Eq. (2) were listed in Table 4. The p-valuewas used as a tool to check
the significance of each coefficient, which in turn might indicate
the pattern of the interaction between the variables. The smaller
the value of p was, the more significant the corresponding coefficient
was (Guo et al., 2010; Zhu and Liu, 2013). It can be seen from
Table 4 that the linear coefficients (C), cross product coefficients
3.6. Optimization of procedure
The graphical representations of the regression Eq. (2), called
the response surfaces and the contour plots were obtained using
Design-Expert software, and the results of extraction yield of crude
b-glucan affected by extraction temperature, extraction time and
number of cycles are presented in Fig. 2. The relationship between
independent and dependent variables was illustrated in 3D representation
of the response surface plots generated by the model of
extraction yield, two variables were depicted in one 3D surface plot
while the other variables were kept at level zero. As shown in
Fig. 2a, when the number of cycles (C) was fixed at zero level,
extraction temperature (A) and extraction time (B) showed reciprocal
interaction on extraction yield
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