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Microwave-assisted extraction of silkworm pupal oil and evaluation of its fatty acid composition, physicochemical properties and antioxidant activities
Accepted Manuscript Microwave-assisted extraction of silkworm pupal oil and evaluation of its fatty acid composition, physicochemical properties and antioxidant activities Bin Hu, Cheng Li, Zhiqing Zhang, Qing zhao, Yadong Zhu, Zhao Su, Yizi Chen PII: DOI: Reference:
S0308-8146(17)30548-4 http://dx.doi.org/10.1016/j.foodchem.2017.03.152 FOCH 20856
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
18 December 2016 22 March 2017 28 March 2017
Please cite this article as: Hu, B., Li, C., Zhang, Z., zhao, Q., Zhu, Y., Su, Z., Chen, Y., Microwave-assisted extraction of silkworm pupal oil and evaluation of its fatty acid composition, physicochemical properties and antioxidant activities, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.03.152
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1
Microwave-assisted extraction of silkworm pupal oil and evaluation of its fatty acid composition,
2
physicochemical properties and antioxidant activities
3 Bin Hu a, Cheng Li a, Zhiqing Zhang a, Qing zhao a, Yadong Zhu b, Zhao Su a, Yizi Chen a *
4 5
a
College of Food Science, Sichuan Agricultural University, Ya’an 625014, Sichuan, China
6
b
College of Literature, Sichuan Agricultural University, Ya’an 625014, Sichuan, China
7 8
*Corresponding author Tel./fax: : +86 835 2882187 (Y.-Z. Chen).
9
Email address: [email protected] (Y.-Z. Chen).
10 11
Running Title: Microwave-assisted extraction of oil
1
12
ABSTRACT
13
Microwave-assisted extraction (MAE) of oil from silkworm pupae was firstly performed in the present
14
research. The response surface methodology was applied to optimize the parameters for MAE. The
15
yield of oil by MAE was 30.16% under optimal conditions of a mixed solvent consisting of ethanol and
16
n-hexane (1:1, v/v), microwave power (360 W), liquid to solid ratio (7.5/1 mL/g), microwave time (29
17
min). Moreover, oil extracted by MAE was quantitatively (yield) and qualitatively (fatty acid profile)
18
similar to those obtained using Soxhlet extraction (SE), but oil extracted by MAE exhibited favourable
19
physicochemical properties and oxidation stability. Additionally, oil extracted by MAE had a higher
20
content of total phenolic, and it showed stronger antioxidant activities. Scanning electron microscopy
21
revealed that microwave technique efficiently promoted the release of oil by breaking down the cell
22
structure of silkworm pupae. Therefore, MAE can be an effective method for the silkworm pupal oil
23
extraction.
24 25
Keywords
26
Microwave-assisted extraction; Silkworm pupal oil; Fatty acid composition; Physicochemical
27
properties; Antioxidant activities
2
28
1. Introduction
29
Silkworms are well known as an efficient large-scale producer of silk thread. Among the various
30
species of silkworms, the mulberry silkworm (Bombyx mori L.), the oak silkworm (Antheraea pernyi)
31
and the eri silkworm (Samia cynthia ricina) are widely reared around the world for use in sericulture.
32
The former two silkworms come from China, with the third originating in India (Mishra, Hazarika,
33
Narain & Mahanta, 2003). Silkworm pupae are the main by-product obtained after extracting silk threads,
34
and they constitute 60% of dry cocoon weight. Based on statistics from the Ministry of Agriculture of
35
People’s Republic of China (http://english.agri.gov.cn/), the output of dry mulberry cocoon in China was
36
reported as 650,800 tons in 2014. Hence, 390,480 tons dry silkworm pupae are available in China per
37
year. However, the abundant silkworm pupae resources have not been fully utilized. Most of the
38
silkworm pupae are used only as fertilizer and feed, or even regarded as industrial waste. Indeed, the
39
disposal of silkworm pupae is a serious problem because the putrilage of waste had deleterious effects
40
on environment (Wang, Wu, Liang & Wang, 2010). The nutritional value of silkworm pupae is mainly
41
manifested in the high protein and fat content. The fat alone constitutes about 30% of the total dry pupae
42
weight. The oil extracted from silkworm pupae contains more than 70% unsaturated fatty acids,
43
particularly the α-linolenic acid and oleic acid accounting for a high percentage (Rao, 1994). Therefore,
44
the silkworm pupal oil is considered a good source of oil and could be used for various
45
applications including food, medicines and cosmetics (Longvah, Mangthya & Ramulu, 2011). So far the
46
extraction of silkworm pupal oil is mainly performed by traditional methods, including
47
mechanical pressing extraction, organic solvent extraction, aqueous enzymatic extraction and
48
supercritical fluid extraction developed lately (Zhu, 2012; Kotake-Nara, Yamamoto, Nozawa, Miyashita,
49
& Murakami, 2002; Jia, Wu, Du, He, Gui, & Yan, 2014; Wei, Liao, Zhang, Liu, & Jiang, 2009). Among
50
them, the yield of mechanical pressing extraction is relatively low, while organic solvent extraction and
51
aqueous enzymatic extraction take a relatively long time (zhang, Yao, Luo, Zhao, & Fu, 2016). In
52
addition, because of the operational complexity and high cost of the apparatus, supercritical fluid
53
extraction is limited in practice (Chen, Du, Zu, Yang, & Wang, 2016). Consequently, searching for an
54
efficient technique with low cost and high extraction of oil is vitally important for the extensive
55
utilization of silkworm pupae.
56
Recently, as an efficient extraction technique from oil, microwave-assisted extraction (MAE) has
57
attracted attention in recent years. Compared with conventional extraction methods, MAE has many
3
58
advantages, such as shorter extraction duration, higher extraction yield and lower energy consumption
59
(Leone, Tamborrino, Zagaria, Sabella, & Romaniello, 2015; Tsukui, Júnior, Oigman, Souza, Bizzo, &
60
Rezende, 2014). Response surface methodology (RSM) is an effective and powerful statistic method for
61
optimizing experimental conditions with a reduced number of experimental trials. Over the past several
62
years, RSM has been successfully applied to optimize the MAE of edible oil (Taghvaei, Jafari, Assadpoor,
63
Nowrouzieh, & Alishah, 2014; Jiao et al., 2014).
64
As we know, the application of MAE of silkworm pupal oil has not been reported, so the objective of
65
this study is to optimize MAE of silkworm pupal oil using RSM. Then, the fatty acid compositions,
66
physicochemical properties, total phenolic content and antioxidant activities of oil by MAE are evaluated
67
and compared with those extracted with soxhlet extraction. In addition, the microscopic structures of
68
material before and after various extraction methods are observed to clarify extraction mechanisms.
69 70
2. Materials and methods
71 72
2.1. Materials and Reagents
73 74
The mulberry silkworm pupae were acquired from sericulture farm in Ya’an district (Sichuan
75
Province, China) in June 2014, and were identified by Prof. Huimin Hu from the College of Science,
76
Sichuan Agricultural University, China. The Samples were dried in a vacuum drier at 70 ℃ and 25 kPa
77
for 24 h to moisture content of less than 5%, then ground into fine powder by a disintegrator (FW135,
78
Taisite, Tianjin, China). After passing through a set of standard-mesh sieves, the powder was sealed in
79
plastic containers and stored in a refrigerator at 4 ℃ for future experiments.
80
Gallic
acid,
Folin–Ciocalteu’s
phenol
reagent,
β-carotene,
Linoleic
acid
and
81
2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich Co. (Steinheim, Germany).
82
All other reagents and chemicals were of analytical grade and purchased from Chendu medicine group
83
Co., Ltd (Chengdu, China).
84 85
2.2. Soxhlet extraction (SE)
86 87
A conventional Soxhlet extraction was implemented with 20 g of silkworm pupae powder (40
4
88
mesh) and 200 mL of n-hexane in a Soxhlet extractor at 80 ℃ for 6 h through a water bath. After
89
extraction, n-hexane was removed at 50 ℃ under reduced pressure using a rotary evaporator
90
(RE-52A,Yarong, Shanghai, China). The oil was dried at 100 ℃ ± 5 ℃ for 15 min in a drying oven. The
91
yield of oil was calculated gravimetrically.
92 93
2.3. Microwave-assisted extraction (MAE)
94 95
MAE was conducted by microwave extraction apparatus (WBFY-205, Yuhua China). The
96
apparatus is equipped with a time controller, a power sensor, an electromagnetic stirrer and a
97
circulating water-cooling system. Its maximum power was 1000 W variable in 20 W increments with
98
2.45 GHz microwave frequency. The dimensions of inner cavity were 330 mm × 365 mm × 235 mm.
99
During the experiment, time, irradiation power, and stirring speed could be easily controlled by an
100
electronic control panel.
101
For each extraction, 20 g of silkworm pupae powder (40 mesh) and extraction solvent of
102
specified volume were added into a flask. The flask was placed in the microwave oven cavity and
103
connected to the cooling system through a hole at the top of microwave extractor. Then, the MAE
104
device was turned on, and the conditions including microwave power and extraction time were set by
105
the digital panel. After extraction, the extractive was filtrated through a qualitative filter, and then it
106
was collected and concentrated with a rotary evaporator at 50 ℃ to obtain oil. The amount of extracted
107
oil was calculated gravimetrically after collection, and then the extraction yield is expressed as follows:
108
mass of extracted oil Extraction yield of oil(%) 100 mass of dried material
109 110
2.4. Experimental design of MAE
111 112
A three-level, three-factor Box-Behnken design (BBD) combined with response RSM was
113
applied to have research on the optimal combination of variables in extraction of silkworm pupal oil.
114
On the basis of preliminary experimental data (data not shown), the independent variables were
115
microwave power (X1: 200–400 W), liquid to solid ratio (X2: 4–8 mL/g) and microwave time (X3:
116
20–30 min), while the response variable was the extraction yield of oil. Table 1 shows the arrangement
117
of the BBD in this research, 17 experimental runs including 5 replicates at the centre point were 5
118
119
employed to fit the full second-order polynomial equation model. The general equation is: 3
3
i 1
i 1
3
Y 0 i X i2 iiX 2i
3
X X ij
i
j
i 1 j i 1
120
where Y is the predicted response, β0, βi, βii and βij are the regression coefficients for intercept, linearity,
121
square and interaction, respectively, while Xi and Xj are the independent coded variables. The actual
122
variables were transferred to a range from 1 to -1 for the evaluation of the factors. The actual and coded
123
levels of the independent variables used in the experimental design were shown in Table 1. The
124
relevant mathematical equations were presented as follows:
125
126
Z0 j
j
xj 127
Z 2 j Z1 j 2
Z 2 j Z1 j 2
Z j Z0 j j
,
j 1, 2, 3.
128
where j was the experimental variable. Z2j was the actual value of the independent variable j at high level
129
point. Z1j was the actual value of the independent variable j at low level point. Z0j was the actual value of
130
the independent variable j at the center point. Zj was the actual value of the independent variable j. Δj was
131
the step change of the actual value of variable j at high level point and low level point. xj was the coded
132
value of the independent variable j.
133 134
2.5. Gas chromatography (GC) analysis of fatty acid compositions
135 136
To analyze the fatty acid composition of silkworm pupal oil, the oil was firstly converted into fatty
137
acid methyl esters (FAME) via esterification reaction. Silkworm pupal oil (1 g) was transfered into a
138
100 mL reaction flask, and the methanolic sodium hydroxide solution (10 mL) was added at the same
139
time. The mixture was boiled under reflux until the droplets of fat disappeared. The methanolic boron
140
trifluoride solution (12 mL) and the isooctane (10 mL) were added through the top of the condenser,
141
continuously boiling for 3 min. The flask was removed from the reflux condenser. Immediately, the
142
saturated sodium chloride solution (20 mL) was added into the flask, shaking it vigorously for 30 s.
143
Then, the saturated sodium chloride solution was added to the liquid level of the neck of the flask. After
144
stratification, the upper isooctane layer (1 mL) was transfered into a vial, and the anhydrous sodium
6
145
sulfate was added. The mixture was injected into the packed column for gas chromatography.
146
The compositional analysis of FAME was performed by using an gas chromatograph (7890A,
147
Agilent) equipped with a flame ionization detector and a capillary column (HP-FFAP, 30 m × 0.25 mm
148
I.D, 0.25 μm film thickness, Agilent). The carrier gas, He, was used at a flow rate of 2 mL/min. Sample
149
were injected (1 μL) with a split mode (ratio 10:1). Injector temperature and detector temperature were
150
set at 220 °C and 300 °C, respectively. Column oven temperature increased from 60 °C (1 min) to
151
220 °C at a rate of 10 °C/min, and remained at 220 °C for 10 min. Fatty acid were identificated with
152
retention times obtained from commercial FAME standards (Sigma Chemical, St. Louis, MO). The
153
relative amount of each FAME was calculated from the integrated area of each peak and expressed as a
154
percentage of the total area of all peaks and modified in comparison with those of each authentic
155
sample.
156 157
2.6. Physicochemical properties analysis
158 159
The refractive index and specific gravity were examined at room temperature of 25 °C using an
160
Abbe refractometer and a specific gravity bottle, respectively. Acid value, peroxide value and iodine
161
value of the extracted oil were examined by American Oil Chemists’ Society standard methods Cd 3d–63,
162
Cd 8–53 and Cd 1–25, respectively (AOCS, 1997).
163 164
2.7. Assay for total phenolic (TP) content
165 166
TP content were examined spectrophoto-metrically using Folin-Ciocalteu’s reagent according to
167
the method described by Bail, Stuebiger, Krist, Unterweger, and Buchbauer (2008). A calibration curve
168
of gallic acid in methanol was implemented in the concentration ranges of 0.05–0.50 µg/mL. The
169
concentration of phenolic compounds was calculated according to the following equation obtained from
170
the standard gallic acid curve: absorbance = 0.0185 × gallic acid (µg) − 0.0052, with R2 of 0.9949.
171
Results were expressed in mg of gallic acid equivalents (GAE) per kilogram of oil.
172 173
2.8. Antioxidant activity evaluation
174
7
175
The antioxidant activity of silkworm pupal oil was assessed using 2,2-diphenyl-1-picrylhydrazyl
176
(DPPH) radical scavenging assay and β-carotene bleaching test. The DPPH free radical scavenging
177
activity of oil was examined adopting the methods reported by Zhang et al. (2010). The β-carotene
178
bleaching test was measured as described by Liu et al. (2009). The results were expressed by IC50 values
179
which corresponded to the concentration of oil (mg/mL) neutralizing 50% of DPPH radicals or
180
inhibiting 50% of β-carotene bleaching.
181 182
2.9. Scanning electron microscopy (SEM) observation
183 184
The morphological alteration of sample using different extraction methods was observed by a SEM
185
(JEOL JSM-7500F, Tokyo, Japan). Samples were dried and coated by gold, and then micrographs were
186
taken at 500 magnification.
187 188
2.10. Statistical analysis
189 190
All experiments were performed in triplicate, and the mean of extraction yields were used for
191
statistical analysis. Multiple regression analyses, analysis of variance (ANOVA) and significance test
192
were performed by a software (Design-Expert 9.0 Trial, State-Ease, Inc., Minneapolis MN, USA).
193
Differences between mean were analyzed by t-test on the significance level of p < 0.05, using the SPSS
194
statistical software, version 17.0 (SPSS Inc., Chicago, IL, USA).
195 196
3. Results and Discussion
197 198
3.1. Selection of appropriate solvent for MAE
199 200
The type of extraction solvent is a critical factor that governs the extraction efficiency in MAE
201
process. Ethanol (boiling point 78 ℃), ethyl acetate (boiling point 77 ℃), petroleum ether (boiling range
202
60~90 °C), diethyl ether (boiling point 35 ℃) and n-hexane (boiling point 69 ℃) were chosen as
203
potential extraction solvent in this study. They were tested under the same condition using the same
204
method (20 g dried samples, 200 mL extractant, microwave power 300 W for 30 min). Fig. 1a shows that
8
205
compared with other solvents, the relatively high yields were obtained when using ethanol and n-hexane
206
respectively, but the yield was still limited. In MAE process, it requires that the extraction solvent not
207
only has a certain polarity to better absorb the microwave radiation energy, but also has
208
excellent dissolving capacity for the extractive. Ethanol has strong polarity, and n-hexane has a better
209
solubility for oil. Thus, a mixed solvent consisting of ethanol and n-hexane at a proper ratio was used in
210
MAE process. According to Fig. 1b, compared with single solvent, the yield of oil increased when a
211
mixed solvent consisting of ethanol and n-hexane was used, especially the volume ratio of ethanol and
212
n-hexane in 1 to 1 can providing the highest yield of oil. Therefore, a mixed solvent consisting of
213
ethanol and n-hexane (1:1, v/v) was selected as the ideal extraction solvent for the subsequent
214
experiments.
215 216
3.2. Optimisation of MAE
217 218
MAE parameters including microwave power, liquid to solid ratio and microwave time were
219
optimised by RSM using BBD to obtain the maximum yield of silkworm pupal oil. All the
220
experimental data from the established response surface analysis model are shown in Table 1.
221 222
3.2.1. Fitting the model
223 224
The results of ANOVA for the quadratic model were shown in Table 2, and the significance of
225
each coefficient was determined by F-value and p-value. Generally, The larger magnitude of F-value
226
and the smaller p-value had more significant effect on the corresponding coefficient. The model F-value
227
of 654.36 implied that the model was significant. There was only a 0.01% chance that a model F-value of
228
this size could occur due to statistical noise.
229
The lack-of-fit was used to measure how well the model fitted the data. The lack-of-fit with
230
non-significant (p > 0.01) could indicate that the model fitted the data well. In this model, the
231
lack-of-fit F-value of 2.28 implied that the lack-of-Fit was not significant relative to the pure error. There
232
was a 22.16% chance that a lack-of-fit F-value of this size could occur due to statistical noise. The
233
coefficient of determination (R2) meant the proportion of the total variation in the response expected by
234
the model. Higher coefficient of determination (R2) of model implied that the response surface model
9
235
was reasonable. R2 of 0.9898 indicated that model appropriately represented the experimental data, and
236
98.98% of the variation could be elucidated by the fitted model. Meanwhile, the adjustment coefficient
237
of determination (Adj R2) was used to analyze the model adequacy, and the value of Adj R2 (0.9731)
238
implied a high correlation between the experimental data and the predicted values. The coefficient of
239
variation (CV), a ratio of the standard error of estimate value to the mean value of observed response,
240
was used to measure of reproducibility of the model. The smaller the coefficient of variance was, the
241
more reliable the model would get. The coefficient of variation of 0.79% indicated a high degree of
242
precision and reliability of experiments conducted. The adequate precision was used to measure the
243
signal to noise ratio. The ratio of 80.82 was greater than 4, implying an adequate signal obtained from
244
model. Therefore, the model with desirable coefficient of determination, non-significant p-values of
245
lack of fit and highly significant levels suggested that it was accurate and applicable. As the results in
246
Table 2 illustrates, factors with significant effects on oil yield (p < 0.05) were linear terms of X1, X2 and
247
X3, interaction terms of X1X2, X1X3 and X2X3, and quadratic terms of X12, X22 and X32. The second order
248
polynomial model was applied to express the oil yield as the following equation:
249
Y=27.49 + 1.50X1 + 2.80X2 + 3.23X3 + 0.57X1X2 − 0.24X1X3 + 0.58X2X3 − 1.47X12 − 2.30X22 − 2.11X32。
250
Based on the mathematical model established, the optimal experimental conditions were as
251
follows: microwave power (359.58 W), liquid to solid ratio (7.57 mL/g) and microwave time (29.19
252
min). Considering the feasibility of operation, microwave power, liquid to solid ratio, microwave time
253
were modified to 360 W, 7.5 mL/g and 29 min, respectively. The yields of oil were 30.16% under the
254
optimum parameters, which were a good fit for the value forecasted (30.39%) by the regression model.
255
Therefore, the oil extraction conditions achieved by RSM were reliable and practical.
256 257
3.2.2. Analysis of the response surface
258 259 260
Three-dimensional response surfaces were applied to test the mutual effects of different variables, so as to better understand the independent variables’ effect on oil yields.
261
Fig. 2a shows the mutual effects between microwave power and liquid to solid ratio on the oil
262
yields extracted at fixed microwave time (25 min). It can be found that oil yields obviously increased
263
with growth of liquid to solid ratio at a designated microwave power, and the growth was increasingly
264
slow when liquid to solid ratio increased. But oil yields firstly increased and then decreased with the
10
265
growth of microwave power, which had dual influences on oil yields. The advantageous effect was that
266
the growth with microwave power was favorable for cell rupture, solvent’s movement speed, and
267
diffusion rate of extractive into solvent, so as to promote higher oil yields. The disadvantageous aspect
268
was that excessively high microwave power could cause thermal decomposition of oil to some extent
269
(Chen, Du, Zu, Yang, & Wang, 2016). Hence, optimal microwave power should be dependent on the
270
balance between the dual influences. Fig. 2b indicates the mutual effects between microwave power and
271
microwave time on the oil yields extracted at fixed liquid to solid ratio (6 mL/g). The increase of
272
microwave power could improve oil yields to some degree, but further increase of microwave power
273
was together with decrease of oil yields. With the growth of microwave time, the oil yields were
274
increased obviously. Therefore, both proper increase of microwave power and liquid to solid ratio
275
could lead to high oil yields. Fig. 2c shows the mutual effects between liquid to solid ratio and
276
microwave time on the oil yields extracted at fixed microwave power (300 W). The initial rapid growth
277
in oil yields was together with the increase in liquid to solid ratio at any designated microwave time.
278
Additionally, the impact of microwave time on oil yields was a persistent growth with the increase of in
279
microwave time. By contrast, with the increase of microwave time, the variation tendency for oil yields
280
grew firstly, and then a slow rise was found with further increase of microwave time. Oil yields’ growth
281
with the changing microwave time was mainly ascribed to enough time for enhancing the effective
282
extraction of constituent. The effect of liquid to solid ratio on oil yields was that a few solvent volume
283
could reduce the contact chance between solvent and material, thus resulting in insufficient extraction,
284
while a more solvent volume could effectively extract and obtain higher yields.
285 286
3.3. Comparison of MAE with SE
287 288
Soxhlet Extraction (SE), a reference method for oil extraction, was implemented to make a
289
contrast with MAE. MAE process was performed under the optimized conditions. Fig. S1 shows
290
the process of the extraction yield according to the extraction time by MAE and SE methods. The
291
result that the oil yields of MAE for 29 min (30.16 ± 0.25%) were comparable to those of SE for 360
292
min (30.42 ± 0.33%). No distinct differences were found in oil yields with the both methods. In the MAE
293
process, the microwaves induced a sudden temperature increase inside the cellular structures, which may
294
cause the rupture of cell structure to promote the rapid release of oil. Meanwhile, the cavitation and
11
295
turbulence effect can also break down cell construction and improve the mass transfer. Thus, MAE was
296
more effective than the SE on the extraction of silkworm pupal oil.
297 298
3.4. Fatty acid composition of oils
299 300
Fatty acid composition of silkworm pupal oil obtained under optimum MAE and SE is shown in
301
Table 3. The principal fatty acids found in experiments were palmitic acid (16:0), stearic acid (18:0),
302
oleic acid (18:1), linoleic acid (18:2) and α-linolenic acid (18:3). The unsaturated fatty acids (USFA)
303
accounted for about 70% of the total acids, in which α-linolenic acid (18:3) and oleic acid (18:1) were the
304
main polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA), respectively. In
305
addition, main saturated fatty acid (SFA) in silkworm pupal oil is palmitic acid (16:0), followedby stearic
306
acid (18:0). These values obtained in our research are similar to those reported by Wei et al, 2009; Pan,
307
Liao, Zhang, Dong, & Wei, 2012. However, some authors have reported higher or lower values in fatty
308
acid proportions. Kotake-Nara et al (2002) reported higher oleic acid (18:1) contents in Japan silkworm
309
pupae. The results from Shanker et al. (2006) showed different proportions of fatty acids present in
310
silkworm pupal oil from India. This may be due to different species origin, season and geographical
311
regions.
312 313
Additionly, no significant difference in fatty acid composition was observed between MAE and SE. Therefore, MAE does not change the fatty acid composition of silkworm pupal oil.
314 315
3.5. Effect of MAE on physicochemical properties
316 317
The physicochemical properties of edible oil are the common indexes for its quality evaluation in
318
food industry. Specific gravity (0.92 ± 0.01 g/mL) and refractive index (1.47 ± 0.00) from oil extracted
319
by MAE were similar to specific gravity (0.91 ± 0.01 g/mL) and refractive index (1.48 ± 0.00) from oil
320
extracted by SE, respectively. No significant differences (p > 0.05) were observed for the specific
321
gravity and refractive index. The iodine value of oil extracted by MAE (121.83 ± 1.72 g I/100g oil) was
322
comparable with that of SE (120.06 ± 1.47 g I/100g oil), and there was no significant difference (p >
323
0.05) about iodine value between MAE and SE. We also can conclude that MAE did not have influence
324
on the double bonds of the oil. In addition, the acid value of oil extracted by MAE (1.32 ± 0.11 mg
12
325
KOH/g oil) was significantly (p < 0.05) lower than oil extracted by SE (1.86 ± 0.15 mg KOH/g oil),
326
which indicated that oil extracted by MAE showed less rancidity. The peroxide value of oil extracted
327
by MAE (5.67 ± 0.04 meq O2/kg oil) was found to be significantly (p < 0.05) lower than oil extracted
328
by SE (9.35 ± 0.08 meq O2/kg oil). Oil sample of low acid value and peroxide value can be regarded as
329
good quality. These results proved the previously reports by Tamborrino, Romaniello, Zagaria, &
330
Leoneetal.(2014). So, the oxidative stability of oil extracted by MAE was improved as against that of
331
SE. On one hand, the lower oxidative stability of oil extracted by SE is probably attributed to the
332
elevated temperature and prolonged extraction duration of SE, which can accelerate the oil oxidation
333
(Kittiphoom & Sutasinee, 2015). On the other hand, the higher oxidative stability of oil extracted by
334
MAE can be ascribe to the higher content of phenolics (see results shown in section 3.6), which
335
contribute to promoting the oxidative stability of oil (Besbes, Blecker, Deroanne, Drira, & Attia, 2004).
336
Furthermore, it was obviously found that the peroxide value of silkworm pupae oil was below the level
337
(15 meq/kg) established by Food and Drug Administration/World Health Organization under the Codex
338
Alimentarius Commission (Azlan et al., 2010). Hence, the results of physicochemical properties shows
339
that silkworm pupal oil obtained by MAE had good quality and could be utilized as a resource of edible
340
oil.
341 342
3.6. Total phenolic (TP) content
343 344
Phenolics are regarded as primary terminator breaking antioxidants in free radical chain reactions,
345
so they can improve antioxidant potentials of oil. In this study, the contents of TP (78.36±1.28 mg
346
GAE/kg oil) in MAE were significantly (p < 0.05) higher than SE (42.76±0.82 mg GAE/kg oil). The
347
increase of TP in MAE (relative to those in SE) may be attributed to the microwave treatment. The
348
rupture of silkworm pupae cell by microwave treatment may be beneficial to the release of greater
349
amounts of phenolics into oils in short time (Desai, Parikh, & Parikh, 2010). And SE take a long time,
350
which may have disadvantageous impact on TP content. Moreover, the microwave treatment can
351
reduce the interaction of TP with sample protein, polysaccharide and cellulose, thereby promoting TP
352
release into the oil (Mason, Chemat, & Vinatoru, 2011). Additionly, the difference of TP content is
353
partly attributed to the presence of a polar solvent (ethanol), which extracts more phenolics from the
354
silkworm pupae in MAE (Kozłowska, Gruczyńska, Ścibisz, & Rudzińska, 2016).
13
355 356
3.7. Antioxidant activity
357 358
Because of the complexity of antioxidative mechanism, an individual experiment is incomplete to
359
evaluate the total antioxidant activity of sample. So two complementary experiment, DPPH and
360
β-carotene bleaching test were used to this study. As shown in Fig. S3a, with the growth of oil
361
concentrations, their DPPH radical scavenging activity increased accordingly. MAE exhibited
362
significantly (p < 0.05) superior efficacy in scavenging the DPPH radicals (26.47±1.27 mg/mL)
363
compared with SE (34.53±1.45 mg/mL). As shown in Fig. S3b, the β-carotene bleaching inhibition
364
rates of both oil appeared the dosed-dependent relationship. The lipid peroxidation inhibitory activity
365
of MAE (25.24±1.08 mg/mL) was also significantly (p < 0.05) higher than SE (32.73±1.36 mg/mL). In
366
the current examination of both tested systems, oil extracted by MAE showed stronger antioxidant
367
activities than oil obtained by SE. Study has shown that antioxidant activity of oil was mainly attributed
368
to the fatty acid composition, especially the high level of USFA (Hayes, 2002). Additionally, other
369
studies have demonstrated that the content of TP was closedly related to the antioxidant effect of oil
370
(Ting, Hsu, Tsai, Lu, Chou, & Chen 2011; Fu, Qu, Yang, & Zhang, 2016). Thus, oil by MAE displayed
371
superior antioxidant activities than oil by SE, which could be partly explained by the higher content
372
of TP in oil by MAE than oil by SE.
373 374
3.8. Analysis of microscopic changes
375 376
To better understand the extraction mechanism of MAE, the silkworm pupae powder were
377
examined by SEM to elucidate the morphological changes of samples. Fig. 3 shows that obvious
378
structure rupture occurred in the treated samples compared with the untreated samples. As shown in Fig.
379
3a, the external surface of the untreated sample was intact and smooth. After SE, only partial rupture
380
took place on the surface of the sample (Fig. 3b). After MAE, the morphology of samples was
381
obviously broken down (Fig. 3c). This confirms that localized and rapid heating brought about by
382
microwave irradiation lead to obvious cell rupture. It is beneficial to oil flow from the biomass. Li et al
383
(2013) performed a microwave-assisted aqueous enzymatic extraction of oil from yellow horn seeds.
384
The SEM results indicated that the microwave technique efficiently promoted the release of oil by
14
385
breaking down the cell structure of silkworm pupae.
386 387
3.9. Cost, energy and environmental ecology
388 389
The reduced cost of oil extraction was clearly beneficial for MAE in terms of time, energy and
390
environmental impacts. The MAE procedure required an extraction time of 29 min versus 6 h for SE.
391
The energy needed to perform two extraction methods (energy emitted from the microwave oven and
392
electrical heater) was 0.80 kWh for MAE and 0.30 kWh for SE, respectively. The power consumption
393
has been examined with a Wattmeter at the microwave generator entrance and the electrical heater
394
power supply. With regard to environmental impact, the calculated quantity of carbon dioxide emitted
395
into the atmosphere was lower in the case of the MAE (309 g CO2 per gram of oil) than for the SE
396
(1440 g CO2 per gram of oil). These calculations have been made according to the literature: to produce
397
1 kWh by combustion of fossil fuel will reject 800 g of CO2 into the atmosphere (Chematand Cravotto,
398
2013). Therefore, MAE could be proposed as an extraction method in favour of environment.
399 400
4. Conclusions
401 402
In the present study, MAE was applied to the extraction of silkworm pupal oil for the first time.
403
The yield of silkworm pupal oil reached 30.16% under the optimal conditions of a mixed solvent
404
consisting of ethanol and n-hexane (1:1, v/v), microwave power (360 W), liquid to solid ratio (7.5/1
405
mL/g), microwave time (29 min). Compared with SE, the yield of oil and fatty acid composition with
406
MAE were similar, but oil extracted by MAE exhibited superior physicochemical properties and
407
antioxidant activities. Additionally, SEM micrographs confirmed that microwave technique efficiently
408
promoted the release of oil by breaking down the cell structure of silkworm pupae. So MAE is a
409
promising technique for silkworm pupal oil extraction in the food industry.
410 411
Acknowledgements
412
The authors gratefully acknowledge the financial supports by Science and technology Project for
413
Sichuan Provincial Department of Education of China (2011ZB062) and Scientific Research
414
Foundation of Sichuan Agricultural University (06070905).
415 15
416 417 418
Conflict of interest statement We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
16
419
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503
Figure captions
504 505
Fig. 1. Effect of different solvent on extraction yield of oil (a). Effect of a mixed solvent consisting of
506
ethanol and n-hexane at different volume ratio on extraction yield of oil (b).
507
Fig. 2. Response surface for mutual effects of microwave power and liquid to solid ratio on extraction
508
yield of oil (a), microwave power and time on extraction yield of oil (b), liquid to solid and time on
509
extraction yield of oil (c).
510
Fig. 3. Scanning electron micrographs of silkworm pupae: silkworm pupae without treatment (a),
511
silkworm pupae by MAE (b), silkworm pupae by SE (c).
512
20
(a)
30
Extraction yield of oil(% )
24
18
12
6
0 diethyl ether
petroleum ether
n-hexane
ethanol
ethyl acetate
1:2
1:3
Extraction solvent type
513 514
Extraction yield of oil(% )
(b)
30
24
18
12
6
0 3:1
515 516 517 518
2:1
1:1
Ethanol to n-hexane ratio(v/v)
Fig. 1. Effect of different solvent on extraction yield of oil (a). Effect of a mixed solvent consisting of ethanol and n-hexane at different volume ratio on extraction yield of oil (b).
21
(a)
519
(b)
520
(c)
521 522 523 524 525
Fig. 2. Response surface for mutual effects of microwave power and liquid to solid ratio on extraction yield of oil (a), microwave power and time on extraction yield of oil (b), liquid to solid ratio and time on extraction yield of oil (c).
22
(a)
526 527
(b)
528 529
(c)
530 531 532 533
Fig. 3. Scanning electron micrographs of silkworm pupae: silkworm pupae without treatment (a), silkworm pupae by MAE (b), silkworm pupae by SE (c).
23
534 535
536 537
Table 1 Results of BBD design for the extraction of silkworm pupal oil. a Runs Microwave power Liquid to solid ratio Microwave time (X1, W) (X2, mL/g) (X3, time) 1 1(400) -1(4) 0(25)
a
Extraction yield of oil (Y, %) 21.87
2
1(400)
0(6)
1(30)
28.52
3
1(400)
0(6)
-1(20)
22.46
4
1(400)
1(8)
0(25)
28.39
5
-1(200)
-1(4)
0(25)
20.18
6
0(6)
-1(20)
18.82
7
-1(200) -1(200)
0(6)
1(30)
25.82
8
-1(200)
1(8)
0(25)
24.43
9
0(300)
1(8)
1(30)
29.75
10
0(300)
1(8)
-1(20)
22.21
11
-1(4)
1(30)
22.76
12
0(300) 0(300)
-1(4)
-1(20)
17.56
13
0(300)
0(6)
0(25)
27.55
14
0(6)
0(25)
27.64
15
0(300) 0(300)
0(6)
0(25)
27.60
16
0(300)
0(6)
0(25)
27.29
17
0(300)
0(25)
27.35
0(6) The results were obtained with Design Expert 9.0 software.
24
538 539
540 541 542 543 544 545 546 547
Table 2 Results of ANOVA about BBD design. a Variables Sum of squares b Df c Model 222.65 9
Mean square d
F-value e
24.74
654.36
p-value f < 0.0001
Significance g significant
X1
17.97
1
17.97
475.32
< 0.0001
significant
X2
62.78
1
62.78
1660.46
< 0.0001
significant
X3
83.21
1
83.21
2200.82
< 0.0001
significant
X 1X 2
1.29
1
1.29
34.07
0.0006
significant
X 1X 3
0.22
1
0.22
5.84
0.0463
significant
X 2X 3
1.37
1
1.37
36.21
0.0005
significant
X1
2
9.06
1
9.06
239.60
< 0.0001
significant
X2
2
22.31
1
22.31
590.05
< 0.0001
significant
X3
2
18.82
1
18.82
497.83
< 0.0001
significant
0.2216 Lack of fit 0.17 3 0.06 2.28 The results were obtained with Design Expert 9.0 software. b Sum of the squared differences between the average values and the overall mean. c Degree of freedom. d Sum of squares divided by degree of freedom. e Test for comparing term variance with residual variance. f Probability of the observed F-value. g p-value less than 0.05 indicate model term is significant. a
25
Not significant
548 549
Table 3 Fatty acid composition (%) of silkworm pupae oil obtained from the different methods. No Fatty acid MAE SE 0.18±0.00 0.19±0.00 1 Myristic acid(14:0) 2
Palmitic acid(16:0)
23.18±0.52
23.04±0.58
3
Palm Acid(16:1)
1.07±0.09
1.05±0.07
4
Cydonic acid(17:0)
0.15±0.00
0.17±0.00
5
Seventeen carbon and one acid(17:1)
0.10±0.00
ND
6
Stearic acid(18:0)
4.69±0.17
4.68±0.19
7
Oleic acid(18:1)
28.32±0.63
28.15±0.54
8
Linoleic acid(18:2) α-linolenic acid(18:3)
3.88±0.13
3.85±0.15
38.25±0.75
38.06±0.68
Peanut acid(20:0)
0.16±0.00
0.16±0.00
Saturated fatty acids Monounsaturated fatty acids Polyunsaturated fatty acids
28.36 29.49 42.13
28.24 29.20 41.91
9 10 Total Total Total
550 551
ND: not detected.
26
552
Highlights
553
•MAE is an effective method in the extraction of silkworm pupal oil.
554
•MAE process is optimised by response surface methodology.
555
•Oil by MAE reveals similar fatty acid composition with oil by Soxhlet extraction.
556
•Oil by MAE exhibits superior physicochemical properties and antioxidant activities.
557
•SEM shows that MAE promotes the release of oil by breaking down the cell structure.
558 559
.
560 561
27
S0308-8146(17)30548-4 http://dx.doi.org/10.1016/j.foodchem.2017.03.152 FOCH 20856
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
18 December 2016 22 March 2017 28 March 2017
Please cite this article as: Hu, B., Li, C., Zhang, Z., zhao, Q., Zhu, Y., Su, Z., Chen, Y., Microwave-assisted extraction of silkworm pupal oil and evaluation of its fatty acid composition, physicochemical properties and antioxidant activities, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.03.152
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1
Microwave-assisted extraction of silkworm pupal oil and evaluation of its fatty acid composition,
2
physicochemical properties and antioxidant activities
3 Bin Hu a, Cheng Li a, Zhiqing Zhang a, Qing zhao a, Yadong Zhu b, Zhao Su a, Yizi Chen a *
4 5
a
College of Food Science, Sichuan Agricultural University, Ya’an 625014, Sichuan, China
6
b
College of Literature, Sichuan Agricultural University, Ya’an 625014, Sichuan, China
7 8
*Corresponding author Tel./fax: : +86 835 2882187 (Y.-Z. Chen).
9
Email address: [email protected] (Y.-Z. Chen).
10 11
Running Title: Microwave-assisted extraction of oil
1
12
ABSTRACT
13
Microwave-assisted extraction (MAE) of oil from silkworm pupae was firstly performed in the present
14
research. The response surface methodology was applied to optimize the parameters for MAE. The
15
yield of oil by MAE was 30.16% under optimal conditions of a mixed solvent consisting of ethanol and
16
n-hexane (1:1, v/v), microwave power (360 W), liquid to solid ratio (7.5/1 mL/g), microwave time (29
17
min). Moreover, oil extracted by MAE was quantitatively (yield) and qualitatively (fatty acid profile)
18
similar to those obtained using Soxhlet extraction (SE), but oil extracted by MAE exhibited favourable
19
physicochemical properties and oxidation stability. Additionally, oil extracted by MAE had a higher
20
content of total phenolic, and it showed stronger antioxidant activities. Scanning electron microscopy
21
revealed that microwave technique efficiently promoted the release of oil by breaking down the cell
22
structure of silkworm pupae. Therefore, MAE can be an effective method for the silkworm pupal oil
23
extraction.
24 25
Keywords
26
Microwave-assisted extraction; Silkworm pupal oil; Fatty acid composition; Physicochemical
27
properties; Antioxidant activities
2
28
1. Introduction
29
Silkworms are well known as an efficient large-scale producer of silk thread. Among the various
30
species of silkworms, the mulberry silkworm (Bombyx mori L.), the oak silkworm (Antheraea pernyi)
31
and the eri silkworm (Samia cynthia ricina) are widely reared around the world for use in sericulture.
32
The former two silkworms come from China, with the third originating in India (Mishra, Hazarika,
33
Narain & Mahanta, 2003). Silkworm pupae are the main by-product obtained after extracting silk threads,
34
and they constitute 60% of dry cocoon weight. Based on statistics from the Ministry of Agriculture of
35
People’s Republic of China (http://english.agri.gov.cn/), the output of dry mulberry cocoon in China was
36
reported as 650,800 tons in 2014. Hence, 390,480 tons dry silkworm pupae are available in China per
37
year. However, the abundant silkworm pupae resources have not been fully utilized. Most of the
38
silkworm pupae are used only as fertilizer and feed, or even regarded as industrial waste. Indeed, the
39
disposal of silkworm pupae is a serious problem because the putrilage of waste had deleterious effects
40
on environment (Wang, Wu, Liang & Wang, 2010). The nutritional value of silkworm pupae is mainly
41
manifested in the high protein and fat content. The fat alone constitutes about 30% of the total dry pupae
42
weight. The oil extracted from silkworm pupae contains more than 70% unsaturated fatty acids,
43
particularly the α-linolenic acid and oleic acid accounting for a high percentage (Rao, 1994). Therefore,
44
the silkworm pupal oil is considered a good source of oil and could be used for various
45
applications including food, medicines and cosmetics (Longvah, Mangthya & Ramulu, 2011). So far the
46
extraction of silkworm pupal oil is mainly performed by traditional methods, including
47
mechanical pressing extraction, organic solvent extraction, aqueous enzymatic extraction and
48
supercritical fluid extraction developed lately (Zhu, 2012; Kotake-Nara, Yamamoto, Nozawa, Miyashita,
49
& Murakami, 2002; Jia, Wu, Du, He, Gui, & Yan, 2014; Wei, Liao, Zhang, Liu, & Jiang, 2009). Among
50
them, the yield of mechanical pressing extraction is relatively low, while organic solvent extraction and
51
aqueous enzymatic extraction take a relatively long time (zhang, Yao, Luo, Zhao, & Fu, 2016). In
52
addition, because of the operational complexity and high cost of the apparatus, supercritical fluid
53
extraction is limited in practice (Chen, Du, Zu, Yang, & Wang, 2016). Consequently, searching for an
54
efficient technique with low cost and high extraction of oil is vitally important for the extensive
55
utilization of silkworm pupae.
56
Recently, as an efficient extraction technique from oil, microwave-assisted extraction (MAE) has
57
attracted attention in recent years. Compared with conventional extraction methods, MAE has many
3
58
advantages, such as shorter extraction duration, higher extraction yield and lower energy consumption
59
(Leone, Tamborrino, Zagaria, Sabella, & Romaniello, 2015; Tsukui, Júnior, Oigman, Souza, Bizzo, &
60
Rezende, 2014). Response surface methodology (RSM) is an effective and powerful statistic method for
61
optimizing experimental conditions with a reduced number of experimental trials. Over the past several
62
years, RSM has been successfully applied to optimize the MAE of edible oil (Taghvaei, Jafari, Assadpoor,
63
Nowrouzieh, & Alishah, 2014; Jiao et al., 2014).
64
As we know, the application of MAE of silkworm pupal oil has not been reported, so the objective of
65
this study is to optimize MAE of silkworm pupal oil using RSM. Then, the fatty acid compositions,
66
physicochemical properties, total phenolic content and antioxidant activities of oil by MAE are evaluated
67
and compared with those extracted with soxhlet extraction. In addition, the microscopic structures of
68
material before and after various extraction methods are observed to clarify extraction mechanisms.
69 70
2. Materials and methods
71 72
2.1. Materials and Reagents
73 74
The mulberry silkworm pupae were acquired from sericulture farm in Ya’an district (Sichuan
75
Province, China) in June 2014, and were identified by Prof. Huimin Hu from the College of Science,
76
Sichuan Agricultural University, China. The Samples were dried in a vacuum drier at 70 ℃ and 25 kPa
77
for 24 h to moisture content of less than 5%, then ground into fine powder by a disintegrator (FW135,
78
Taisite, Tianjin, China). After passing through a set of standard-mesh sieves, the powder was sealed in
79
plastic containers and stored in a refrigerator at 4 ℃ for future experiments.
80
Gallic
acid,
Folin–Ciocalteu’s
phenol
reagent,
β-carotene,
Linoleic
acid
and
81
2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich Co. (Steinheim, Germany).
82
All other reagents and chemicals were of analytical grade and purchased from Chendu medicine group
83
Co., Ltd (Chengdu, China).
84 85
2.2. Soxhlet extraction (SE)
86 87
A conventional Soxhlet extraction was implemented with 20 g of silkworm pupae powder (40
4
88
mesh) and 200 mL of n-hexane in a Soxhlet extractor at 80 ℃ for 6 h through a water bath. After
89
extraction, n-hexane was removed at 50 ℃ under reduced pressure using a rotary evaporator
90
(RE-52A,Yarong, Shanghai, China). The oil was dried at 100 ℃ ± 5 ℃ for 15 min in a drying oven. The
91
yield of oil was calculated gravimetrically.
92 93
2.3. Microwave-assisted extraction (MAE)
94 95
MAE was conducted by microwave extraction apparatus (WBFY-205, Yuhua China). The
96
apparatus is equipped with a time controller, a power sensor, an electromagnetic stirrer and a
97
circulating water-cooling system. Its maximum power was 1000 W variable in 20 W increments with
98
2.45 GHz microwave frequency. The dimensions of inner cavity were 330 mm × 365 mm × 235 mm.
99
During the experiment, time, irradiation power, and stirring speed could be easily controlled by an
100
electronic control panel.
101
For each extraction, 20 g of silkworm pupae powder (40 mesh) and extraction solvent of
102
specified volume were added into a flask. The flask was placed in the microwave oven cavity and
103
connected to the cooling system through a hole at the top of microwave extractor. Then, the MAE
104
device was turned on, and the conditions including microwave power and extraction time were set by
105
the digital panel. After extraction, the extractive was filtrated through a qualitative filter, and then it
106
was collected and concentrated with a rotary evaporator at 50 ℃ to obtain oil. The amount of extracted
107
oil was calculated gravimetrically after collection, and then the extraction yield is expressed as follows:
108
mass of extracted oil Extraction yield of oil(%) 100 mass of dried material
109 110
2.4. Experimental design of MAE
111 112
A three-level, three-factor Box-Behnken design (BBD) combined with response RSM was
113
applied to have research on the optimal combination of variables in extraction of silkworm pupal oil.
114
On the basis of preliminary experimental data (data not shown), the independent variables were
115
microwave power (X1: 200–400 W), liquid to solid ratio (X2: 4–8 mL/g) and microwave time (X3:
116
20–30 min), while the response variable was the extraction yield of oil. Table 1 shows the arrangement
117
of the BBD in this research, 17 experimental runs including 5 replicates at the centre point were 5
118
119
employed to fit the full second-order polynomial equation model. The general equation is: 3
3
i 1
i 1
3
Y 0 i X i2 iiX 2i
3
X X ij
i
j
i 1 j i 1
120
where Y is the predicted response, β0, βi, βii and βij are the regression coefficients for intercept, linearity,
121
square and interaction, respectively, while Xi and Xj are the independent coded variables. The actual
122
variables were transferred to a range from 1 to -1 for the evaluation of the factors. The actual and coded
123
levels of the independent variables used in the experimental design were shown in Table 1. The
124
relevant mathematical equations were presented as follows:
125
126
Z0 j
j
xj 127
Z 2 j Z1 j 2
Z 2 j Z1 j 2
Z j Z0 j j
,
j 1, 2, 3.
128
where j was the experimental variable. Z2j was the actual value of the independent variable j at high level
129
point. Z1j was the actual value of the independent variable j at low level point. Z0j was the actual value of
130
the independent variable j at the center point. Zj was the actual value of the independent variable j. Δj was
131
the step change of the actual value of variable j at high level point and low level point. xj was the coded
132
value of the independent variable j.
133 134
2.5. Gas chromatography (GC) analysis of fatty acid compositions
135 136
To analyze the fatty acid composition of silkworm pupal oil, the oil was firstly converted into fatty
137
acid methyl esters (FAME) via esterification reaction. Silkworm pupal oil (1 g) was transfered into a
138
100 mL reaction flask, and the methanolic sodium hydroxide solution (10 mL) was added at the same
139
time. The mixture was boiled under reflux until the droplets of fat disappeared. The methanolic boron
140
trifluoride solution (12 mL) and the isooctane (10 mL) were added through the top of the condenser,
141
continuously boiling for 3 min. The flask was removed from the reflux condenser. Immediately, the
142
saturated sodium chloride solution (20 mL) was added into the flask, shaking it vigorously for 30 s.
143
Then, the saturated sodium chloride solution was added to the liquid level of the neck of the flask. After
144
stratification, the upper isooctane layer (1 mL) was transfered into a vial, and the anhydrous sodium
6
145
sulfate was added. The mixture was injected into the packed column for gas chromatography.
146
The compositional analysis of FAME was performed by using an gas chromatograph (7890A,
147
Agilent) equipped with a flame ionization detector and a capillary column (HP-FFAP, 30 m × 0.25 mm
148
I.D, 0.25 μm film thickness, Agilent). The carrier gas, He, was used at a flow rate of 2 mL/min. Sample
149
were injected (1 μL) with a split mode (ratio 10:1). Injector temperature and detector temperature were
150
set at 220 °C and 300 °C, respectively. Column oven temperature increased from 60 °C (1 min) to
151
220 °C at a rate of 10 °C/min, and remained at 220 °C for 10 min. Fatty acid were identificated with
152
retention times obtained from commercial FAME standards (Sigma Chemical, St. Louis, MO). The
153
relative amount of each FAME was calculated from the integrated area of each peak and expressed as a
154
percentage of the total area of all peaks and modified in comparison with those of each authentic
155
sample.
156 157
2.6. Physicochemical properties analysis
158 159
The refractive index and specific gravity were examined at room temperature of 25 °C using an
160
Abbe refractometer and a specific gravity bottle, respectively. Acid value, peroxide value and iodine
161
value of the extracted oil were examined by American Oil Chemists’ Society standard methods Cd 3d–63,
162
Cd 8–53 and Cd 1–25, respectively (AOCS, 1997).
163 164
2.7. Assay for total phenolic (TP) content
165 166
TP content were examined spectrophoto-metrically using Folin-Ciocalteu’s reagent according to
167
the method described by Bail, Stuebiger, Krist, Unterweger, and Buchbauer (2008). A calibration curve
168
of gallic acid in methanol was implemented in the concentration ranges of 0.05–0.50 µg/mL. The
169
concentration of phenolic compounds was calculated according to the following equation obtained from
170
the standard gallic acid curve: absorbance = 0.0185 × gallic acid (µg) − 0.0052, with R2 of 0.9949.
171
Results were expressed in mg of gallic acid equivalents (GAE) per kilogram of oil.
172 173
2.8. Antioxidant activity evaluation
174
7
175
The antioxidant activity of silkworm pupal oil was assessed using 2,2-diphenyl-1-picrylhydrazyl
176
(DPPH) radical scavenging assay and β-carotene bleaching test. The DPPH free radical scavenging
177
activity of oil was examined adopting the methods reported by Zhang et al. (2010). The β-carotene
178
bleaching test was measured as described by Liu et al. (2009). The results were expressed by IC50 values
179
which corresponded to the concentration of oil (mg/mL) neutralizing 50% of DPPH radicals or
180
inhibiting 50% of β-carotene bleaching.
181 182
2.9. Scanning electron microscopy (SEM) observation
183 184
The morphological alteration of sample using different extraction methods was observed by a SEM
185
(JEOL JSM-7500F, Tokyo, Japan). Samples were dried and coated by gold, and then micrographs were
186
taken at 500 magnification.
187 188
2.10. Statistical analysis
189 190
All experiments were performed in triplicate, and the mean of extraction yields were used for
191
statistical analysis. Multiple regression analyses, analysis of variance (ANOVA) and significance test
192
were performed by a software (Design-Expert 9.0 Trial, State-Ease, Inc., Minneapolis MN, USA).
193
Differences between mean were analyzed by t-test on the significance level of p < 0.05, using the SPSS
194
statistical software, version 17.0 (SPSS Inc., Chicago, IL, USA).
195 196
3. Results and Discussion
197 198
3.1. Selection of appropriate solvent for MAE
199 200
The type of extraction solvent is a critical factor that governs the extraction efficiency in MAE
201
process. Ethanol (boiling point 78 ℃), ethyl acetate (boiling point 77 ℃), petroleum ether (boiling range
202
60~90 °C), diethyl ether (boiling point 35 ℃) and n-hexane (boiling point 69 ℃) were chosen as
203
potential extraction solvent in this study. They were tested under the same condition using the same
204
method (20 g dried samples, 200 mL extractant, microwave power 300 W for 30 min). Fig. 1a shows that
8
205
compared with other solvents, the relatively high yields were obtained when using ethanol and n-hexane
206
respectively, but the yield was still limited. In MAE process, it requires that the extraction solvent not
207
only has a certain polarity to better absorb the microwave radiation energy, but also has
208
excellent dissolving capacity for the extractive. Ethanol has strong polarity, and n-hexane has a better
209
solubility for oil. Thus, a mixed solvent consisting of ethanol and n-hexane at a proper ratio was used in
210
MAE process. According to Fig. 1b, compared with single solvent, the yield of oil increased when a
211
mixed solvent consisting of ethanol and n-hexane was used, especially the volume ratio of ethanol and
212
n-hexane in 1 to 1 can providing the highest yield of oil. Therefore, a mixed solvent consisting of
213
ethanol and n-hexane (1:1, v/v) was selected as the ideal extraction solvent for the subsequent
214
experiments.
215 216
3.2. Optimisation of MAE
217 218
MAE parameters including microwave power, liquid to solid ratio and microwave time were
219
optimised by RSM using BBD to obtain the maximum yield of silkworm pupal oil. All the
220
experimental data from the established response surface analysis model are shown in Table 1.
221 222
3.2.1. Fitting the model
223 224
The results of ANOVA for the quadratic model were shown in Table 2, and the significance of
225
each coefficient was determined by F-value and p-value. Generally, The larger magnitude of F-value
226
and the smaller p-value had more significant effect on the corresponding coefficient. The model F-value
227
of 654.36 implied that the model was significant. There was only a 0.01% chance that a model F-value of
228
this size could occur due to statistical noise.
229
The lack-of-fit was used to measure how well the model fitted the data. The lack-of-fit with
230
non-significant (p > 0.01) could indicate that the model fitted the data well. In this model, the
231
lack-of-fit F-value of 2.28 implied that the lack-of-Fit was not significant relative to the pure error. There
232
was a 22.16% chance that a lack-of-fit F-value of this size could occur due to statistical noise. The
233
coefficient of determination (R2) meant the proportion of the total variation in the response expected by
234
the model. Higher coefficient of determination (R2) of model implied that the response surface model
9
235
was reasonable. R2 of 0.9898 indicated that model appropriately represented the experimental data, and
236
98.98% of the variation could be elucidated by the fitted model. Meanwhile, the adjustment coefficient
237
of determination (Adj R2) was used to analyze the model adequacy, and the value of Adj R2 (0.9731)
238
implied a high correlation between the experimental data and the predicted values. The coefficient of
239
variation (CV), a ratio of the standard error of estimate value to the mean value of observed response,
240
was used to measure of reproducibility of the model. The smaller the coefficient of variance was, the
241
more reliable the model would get. The coefficient of variation of 0.79% indicated a high degree of
242
precision and reliability of experiments conducted. The adequate precision was used to measure the
243
signal to noise ratio. The ratio of 80.82 was greater than 4, implying an adequate signal obtained from
244
model. Therefore, the model with desirable coefficient of determination, non-significant p-values of
245
lack of fit and highly significant levels suggested that it was accurate and applicable. As the results in
246
Table 2 illustrates, factors with significant effects on oil yield (p < 0.05) were linear terms of X1, X2 and
247
X3, interaction terms of X1X2, X1X3 and X2X3, and quadratic terms of X12, X22 and X32. The second order
248
polynomial model was applied to express the oil yield as the following equation:
249
Y=27.49 + 1.50X1 + 2.80X2 + 3.23X3 + 0.57X1X2 − 0.24X1X3 + 0.58X2X3 − 1.47X12 − 2.30X22 − 2.11X32。
250
Based on the mathematical model established, the optimal experimental conditions were as
251
follows: microwave power (359.58 W), liquid to solid ratio (7.57 mL/g) and microwave time (29.19
252
min). Considering the feasibility of operation, microwave power, liquid to solid ratio, microwave time
253
were modified to 360 W, 7.5 mL/g and 29 min, respectively. The yields of oil were 30.16% under the
254
optimum parameters, which were a good fit for the value forecasted (30.39%) by the regression model.
255
Therefore, the oil extraction conditions achieved by RSM were reliable and practical.
256 257
3.2.2. Analysis of the response surface
258 259 260
Three-dimensional response surfaces were applied to test the mutual effects of different variables, so as to better understand the independent variables’ effect on oil yields.
261
Fig. 2a shows the mutual effects between microwave power and liquid to solid ratio on the oil
262
yields extracted at fixed microwave time (25 min). It can be found that oil yields obviously increased
263
with growth of liquid to solid ratio at a designated microwave power, and the growth was increasingly
264
slow when liquid to solid ratio increased. But oil yields firstly increased and then decreased with the
10
265
growth of microwave power, which had dual influences on oil yields. The advantageous effect was that
266
the growth with microwave power was favorable for cell rupture, solvent’s movement speed, and
267
diffusion rate of extractive into solvent, so as to promote higher oil yields. The disadvantageous aspect
268
was that excessively high microwave power could cause thermal decomposition of oil to some extent
269
(Chen, Du, Zu, Yang, & Wang, 2016). Hence, optimal microwave power should be dependent on the
270
balance between the dual influences. Fig. 2b indicates the mutual effects between microwave power and
271
microwave time on the oil yields extracted at fixed liquid to solid ratio (6 mL/g). The increase of
272
microwave power could improve oil yields to some degree, but further increase of microwave power
273
was together with decrease of oil yields. With the growth of microwave time, the oil yields were
274
increased obviously. Therefore, both proper increase of microwave power and liquid to solid ratio
275
could lead to high oil yields. Fig. 2c shows the mutual effects between liquid to solid ratio and
276
microwave time on the oil yields extracted at fixed microwave power (300 W). The initial rapid growth
277
in oil yields was together with the increase in liquid to solid ratio at any designated microwave time.
278
Additionally, the impact of microwave time on oil yields was a persistent growth with the increase of in
279
microwave time. By contrast, with the increase of microwave time, the variation tendency for oil yields
280
grew firstly, and then a slow rise was found with further increase of microwave time. Oil yields’ growth
281
with the changing microwave time was mainly ascribed to enough time for enhancing the effective
282
extraction of constituent. The effect of liquid to solid ratio on oil yields was that a few solvent volume
283
could reduce the contact chance between solvent and material, thus resulting in insufficient extraction,
284
while a more solvent volume could effectively extract and obtain higher yields.
285 286
3.3. Comparison of MAE with SE
287 288
Soxhlet Extraction (SE), a reference method for oil extraction, was implemented to make a
289
contrast with MAE. MAE process was performed under the optimized conditions. Fig. S1 shows
290
the process of the extraction yield according to the extraction time by MAE and SE methods. The
291
result that the oil yields of MAE for 29 min (30.16 ± 0.25%) were comparable to those of SE for 360
292
min (30.42 ± 0.33%). No distinct differences were found in oil yields with the both methods. In the MAE
293
process, the microwaves induced a sudden temperature increase inside the cellular structures, which may
294
cause the rupture of cell structure to promote the rapid release of oil. Meanwhile, the cavitation and
11
295
turbulence effect can also break down cell construction and improve the mass transfer. Thus, MAE was
296
more effective than the SE on the extraction of silkworm pupal oil.
297 298
3.4. Fatty acid composition of oils
299 300
Fatty acid composition of silkworm pupal oil obtained under optimum MAE and SE is shown in
301
Table 3. The principal fatty acids found in experiments were palmitic acid (16:0), stearic acid (18:0),
302
oleic acid (18:1), linoleic acid (18:2) and α-linolenic acid (18:3). The unsaturated fatty acids (USFA)
303
accounted for about 70% of the total acids, in which α-linolenic acid (18:3) and oleic acid (18:1) were the
304
main polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA), respectively. In
305
addition, main saturated fatty acid (SFA) in silkworm pupal oil is palmitic acid (16:0), followedby stearic
306
acid (18:0). These values obtained in our research are similar to those reported by Wei et al, 2009; Pan,
307
Liao, Zhang, Dong, & Wei, 2012. However, some authors have reported higher or lower values in fatty
308
acid proportions. Kotake-Nara et al (2002) reported higher oleic acid (18:1) contents in Japan silkworm
309
pupae. The results from Shanker et al. (2006) showed different proportions of fatty acids present in
310
silkworm pupal oil from India. This may be due to different species origin, season and geographical
311
regions.
312 313
Additionly, no significant difference in fatty acid composition was observed between MAE and SE. Therefore, MAE does not change the fatty acid composition of silkworm pupal oil.
314 315
3.5. Effect of MAE on physicochemical properties
316 317
The physicochemical properties of edible oil are the common indexes for its quality evaluation in
318
food industry. Specific gravity (0.92 ± 0.01 g/mL) and refractive index (1.47 ± 0.00) from oil extracted
319
by MAE were similar to specific gravity (0.91 ± 0.01 g/mL) and refractive index (1.48 ± 0.00) from oil
320
extracted by SE, respectively. No significant differences (p > 0.05) were observed for the specific
321
gravity and refractive index. The iodine value of oil extracted by MAE (121.83 ± 1.72 g I/100g oil) was
322
comparable with that of SE (120.06 ± 1.47 g I/100g oil), and there was no significant difference (p >
323
0.05) about iodine value between MAE and SE. We also can conclude that MAE did not have influence
324
on the double bonds of the oil. In addition, the acid value of oil extracted by MAE (1.32 ± 0.11 mg
12
325
KOH/g oil) was significantly (p < 0.05) lower than oil extracted by SE (1.86 ± 0.15 mg KOH/g oil),
326
which indicated that oil extracted by MAE showed less rancidity. The peroxide value of oil extracted
327
by MAE (5.67 ± 0.04 meq O2/kg oil) was found to be significantly (p < 0.05) lower than oil extracted
328
by SE (9.35 ± 0.08 meq O2/kg oil). Oil sample of low acid value and peroxide value can be regarded as
329
good quality. These results proved the previously reports by Tamborrino, Romaniello, Zagaria, &
330
Leoneetal.(2014). So, the oxidative stability of oil extracted by MAE was improved as against that of
331
SE. On one hand, the lower oxidative stability of oil extracted by SE is probably attributed to the
332
elevated temperature and prolonged extraction duration of SE, which can accelerate the oil oxidation
333
(Kittiphoom & Sutasinee, 2015). On the other hand, the higher oxidative stability of oil extracted by
334
MAE can be ascribe to the higher content of phenolics (see results shown in section 3.6), which
335
contribute to promoting the oxidative stability of oil (Besbes, Blecker, Deroanne, Drira, & Attia, 2004).
336
Furthermore, it was obviously found that the peroxide value of silkworm pupae oil was below the level
337
(15 meq/kg) established by Food and Drug Administration/World Health Organization under the Codex
338
Alimentarius Commission (Azlan et al., 2010). Hence, the results of physicochemical properties shows
339
that silkworm pupal oil obtained by MAE had good quality and could be utilized as a resource of edible
340
oil.
341 342
3.6. Total phenolic (TP) content
343 344
Phenolics are regarded as primary terminator breaking antioxidants in free radical chain reactions,
345
so they can improve antioxidant potentials of oil. In this study, the contents of TP (78.36±1.28 mg
346
GAE/kg oil) in MAE were significantly (p < 0.05) higher than SE (42.76±0.82 mg GAE/kg oil). The
347
increase of TP in MAE (relative to those in SE) may be attributed to the microwave treatment. The
348
rupture of silkworm pupae cell by microwave treatment may be beneficial to the release of greater
349
amounts of phenolics into oils in short time (Desai, Parikh, & Parikh, 2010). And SE take a long time,
350
which may have disadvantageous impact on TP content. Moreover, the microwave treatment can
351
reduce the interaction of TP with sample protein, polysaccharide and cellulose, thereby promoting TP
352
release into the oil (Mason, Chemat, & Vinatoru, 2011). Additionly, the difference of TP content is
353
partly attributed to the presence of a polar solvent (ethanol), which extracts more phenolics from the
354
silkworm pupae in MAE (Kozłowska, Gruczyńska, Ścibisz, & Rudzińska, 2016).
13
355 356
3.7. Antioxidant activity
357 358
Because of the complexity of antioxidative mechanism, an individual experiment is incomplete to
359
evaluate the total antioxidant activity of sample. So two complementary experiment, DPPH and
360
β-carotene bleaching test were used to this study. As shown in Fig. S3a, with the growth of oil
361
concentrations, their DPPH radical scavenging activity increased accordingly. MAE exhibited
362
significantly (p < 0.05) superior efficacy in scavenging the DPPH radicals (26.47±1.27 mg/mL)
363
compared with SE (34.53±1.45 mg/mL). As shown in Fig. S3b, the β-carotene bleaching inhibition
364
rates of both oil appeared the dosed-dependent relationship. The lipid peroxidation inhibitory activity
365
of MAE (25.24±1.08 mg/mL) was also significantly (p < 0.05) higher than SE (32.73±1.36 mg/mL). In
366
the current examination of both tested systems, oil extracted by MAE showed stronger antioxidant
367
activities than oil obtained by SE. Study has shown that antioxidant activity of oil was mainly attributed
368
to the fatty acid composition, especially the high level of USFA (Hayes, 2002). Additionally, other
369
studies have demonstrated that the content of TP was closedly related to the antioxidant effect of oil
370
(Ting, Hsu, Tsai, Lu, Chou, & Chen 2011; Fu, Qu, Yang, & Zhang, 2016). Thus, oil by MAE displayed
371
superior antioxidant activities than oil by SE, which could be partly explained by the higher content
372
of TP in oil by MAE than oil by SE.
373 374
3.8. Analysis of microscopic changes
375 376
To better understand the extraction mechanism of MAE, the silkworm pupae powder were
377
examined by SEM to elucidate the morphological changes of samples. Fig. 3 shows that obvious
378
structure rupture occurred in the treated samples compared with the untreated samples. As shown in Fig.
379
3a, the external surface of the untreated sample was intact and smooth. After SE, only partial rupture
380
took place on the surface of the sample (Fig. 3b). After MAE, the morphology of samples was
381
obviously broken down (Fig. 3c). This confirms that localized and rapid heating brought about by
382
microwave irradiation lead to obvious cell rupture. It is beneficial to oil flow from the biomass. Li et al
383
(2013) performed a microwave-assisted aqueous enzymatic extraction of oil from yellow horn seeds.
384
The SEM results indicated that the microwave technique efficiently promoted the release of oil by
14
385
breaking down the cell structure of silkworm pupae.
386 387
3.9. Cost, energy and environmental ecology
388 389
The reduced cost of oil extraction was clearly beneficial for MAE in terms of time, energy and
390
environmental impacts. The MAE procedure required an extraction time of 29 min versus 6 h for SE.
391
The energy needed to perform two extraction methods (energy emitted from the microwave oven and
392
electrical heater) was 0.80 kWh for MAE and 0.30 kWh for SE, respectively. The power consumption
393
has been examined with a Wattmeter at the microwave generator entrance and the electrical heater
394
power supply. With regard to environmental impact, the calculated quantity of carbon dioxide emitted
395
into the atmosphere was lower in the case of the MAE (309 g CO2 per gram of oil) than for the SE
396
(1440 g CO2 per gram of oil). These calculations have been made according to the literature: to produce
397
1 kWh by combustion of fossil fuel will reject 800 g of CO2 into the atmosphere (Chematand Cravotto,
398
2013). Therefore, MAE could be proposed as an extraction method in favour of environment.
399 400
4. Conclusions
401 402
In the present study, MAE was applied to the extraction of silkworm pupal oil for the first time.
403
The yield of silkworm pupal oil reached 30.16% under the optimal conditions of a mixed solvent
404
consisting of ethanol and n-hexane (1:1, v/v), microwave power (360 W), liquid to solid ratio (7.5/1
405
mL/g), microwave time (29 min). Compared with SE, the yield of oil and fatty acid composition with
406
MAE were similar, but oil extracted by MAE exhibited superior physicochemical properties and
407
antioxidant activities. Additionally, SEM micrographs confirmed that microwave technique efficiently
408
promoted the release of oil by breaking down the cell structure of silkworm pupae. So MAE is a
409
promising technique for silkworm pupal oil extraction in the food industry.
410 411
Acknowledgements
412
The authors gratefully acknowledge the financial supports by Science and technology Project for
413
Sichuan Provincial Department of Education of China (2011ZB062) and Scientific Research
414
Foundation of Sichuan Agricultural University (06070905).
415 15
416 417 418
Conflict of interest statement We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
16
419
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Figure captions
504 505
Fig. 1. Effect of different solvent on extraction yield of oil (a). Effect of a mixed solvent consisting of
506
ethanol and n-hexane at different volume ratio on extraction yield of oil (b).
507
Fig. 2. Response surface for mutual effects of microwave power and liquid to solid ratio on extraction
508
yield of oil (a), microwave power and time on extraction yield of oil (b), liquid to solid and time on
509
extraction yield of oil (c).
510
Fig. 3. Scanning electron micrographs of silkworm pupae: silkworm pupae without treatment (a),
511
silkworm pupae by MAE (b), silkworm pupae by SE (c).
512
20
(a)
30
Extraction yield of oil(% )
24
18
12
6
0 diethyl ether
petroleum ether
n-hexane
ethanol
ethyl acetate
1:2
1:3
Extraction solvent type
513 514
Extraction yield of oil(% )
(b)
30
24
18
12
6
0 3:1
515 516 517 518
2:1
1:1
Ethanol to n-hexane ratio(v/v)
Fig. 1. Effect of different solvent on extraction yield of oil (a). Effect of a mixed solvent consisting of ethanol and n-hexane at different volume ratio on extraction yield of oil (b).
21
(a)
519
(b)
520
(c)
521 522 523 524 525
Fig. 2. Response surface for mutual effects of microwave power and liquid to solid ratio on extraction yield of oil (a), microwave power and time on extraction yield of oil (b), liquid to solid ratio and time on extraction yield of oil (c).
22
(a)
526 527
(b)
528 529
(c)
530 531 532 533
Fig. 3. Scanning electron micrographs of silkworm pupae: silkworm pupae without treatment (a), silkworm pupae by MAE (b), silkworm pupae by SE (c).
23
534 535
536 537
Table 1 Results of BBD design for the extraction of silkworm pupal oil. a Runs Microwave power Liquid to solid ratio Microwave time (X1, W) (X2, mL/g) (X3, time) 1 1(400) -1(4) 0(25)
a
Extraction yield of oil (Y, %) 21.87
2
1(400)
0(6)
1(30)
28.52
3
1(400)
0(6)
-1(20)
22.46
4
1(400)
1(8)
0(25)
28.39
5
-1(200)
-1(4)
0(25)
20.18
6
0(6)
-1(20)
18.82
7
-1(200) -1(200)
0(6)
1(30)
25.82
8
-1(200)
1(8)
0(25)
24.43
9
0(300)
1(8)
1(30)
29.75
10
0(300)
1(8)
-1(20)
22.21
11
-1(4)
1(30)
22.76
12
0(300) 0(300)
-1(4)
-1(20)
17.56
13
0(300)
0(6)
0(25)
27.55
14
0(6)
0(25)
27.64
15
0(300) 0(300)
0(6)
0(25)
27.60
16
0(300)
0(6)
0(25)
27.29
17
0(300)
0(25)
27.35
0(6) The results were obtained with Design Expert 9.0 software.
24
538 539
540 541 542 543 544 545 546 547
Table 2 Results of ANOVA about BBD design. a Variables Sum of squares b Df c Model 222.65 9
Mean square d
F-value e
24.74
654.36
p-value f < 0.0001
Significance g significant
X1
17.97
1
17.97
475.32
< 0.0001
significant
X2
62.78
1
62.78
1660.46
< 0.0001
significant
X3
83.21
1
83.21
2200.82
< 0.0001
significant
X 1X 2
1.29
1
1.29
34.07
0.0006
significant
X 1X 3
0.22
1
0.22
5.84
0.0463
significant
X 2X 3
1.37
1
1.37
36.21
0.0005
significant
X1
2
9.06
1
9.06
239.60
< 0.0001
significant
X2
2
22.31
1
22.31
590.05
< 0.0001
significant
X3
2
18.82
1
18.82
497.83
< 0.0001
significant
0.2216 Lack of fit 0.17 3 0.06 2.28 The results were obtained with Design Expert 9.0 software. b Sum of the squared differences between the average values and the overall mean. c Degree of freedom. d Sum of squares divided by degree of freedom. e Test for comparing term variance with residual variance. f Probability of the observed F-value. g p-value less than 0.05 indicate model term is significant. a
25
Not significant
548 549
Table 3 Fatty acid composition (%) of silkworm pupae oil obtained from the different methods. No Fatty acid MAE SE 0.18±0.00 0.19±0.00 1 Myristic acid(14:0) 2
Palmitic acid(16:0)
23.18±0.52
23.04±0.58
3
Palm Acid(16:1)
1.07±0.09
1.05±0.07
4
Cydonic acid(17:0)
0.15±0.00
0.17±0.00
5
Seventeen carbon and one acid(17:1)
0.10±0.00
ND
6
Stearic acid(18:0)
4.69±0.17
4.68±0.19
7
Oleic acid(18:1)
28.32±0.63
28.15±0.54
8
Linoleic acid(18:2) α-linolenic acid(18:3)
3.88±0.13
3.85±0.15
38.25±0.75
38.06±0.68
Peanut acid(20:0)
0.16±0.00
0.16±0.00
Saturated fatty acids Monounsaturated fatty acids Polyunsaturated fatty acids
28.36 29.49 42.13
28.24 29.20 41.91
9 10 Total Total Total
550 551
ND: not detected.
26
552
Highlights
553
•MAE is an effective method in the extraction of silkworm pupal oil.
554
•MAE process is optimised by response surface methodology.
555
•Oil by MAE reveals similar fatty acid composition with oil by Soxhlet extraction.
556
•Oil by MAE exhibits superior physicochemical properties and antioxidant activities.
557
•SEM shows that MAE promotes the release of oil by breaking down the cell structure.
558 559
.
560 561
27