- Email: [email protected]
Study on the mechanism of microwave modified wheat protein fiber to improve its mechanical properties
Accepted Manuscript Study on the mechanism of microwave modified wheat protein fiber to improve its mechanical properties Cuiqing Liu, Xiaojun Ma PII:
S0733-5210(16)30080-7
DOI:
10.1016/j.jcs.2016.05.018
Reference:
YJCRS 2147
To appear in:
Journal of Cereal Science
Received Date: 14 September 2015 Revised Date:
1 April 2016
Accepted Date: 11 May 2016
Please cite this article as: Liu, C., Ma, X., Study on the mechanism of microwave modified wheat protein fiber to improve its mechanical properties, Journal of Cereal Science (2016), doi: 10.1016/ j.jcs.2016.05.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Study on the mechanism of microwave modified wheat protein fiber to
2
improve its mechanical properties
3
Cuiqing Liu, Xiaojun Ma*
4
School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu
5
Province, PR China
7
*Corresponding authors
8
Tel/fax: 13861750315
9
Email address: [email protected]
10 11
15 16 17 18 19 20 21
EP
14
AC C
13
TE D
12
M AN U
SC
6
RI PT
1
22 23 24 25 1
ACCEPTED MANUSCRIPT
26
Abstract Wheat protein is widely used in food industry. In order to expand the scope of its application on non-food
28
field, we managed to apply the wheat protein to fiber production. To improve the mechanical properties of the
29
fibers, we used the method of microwave modification. The best process conditions obtained by response
30
surface analysis were a microwave power of 20.6 W/mL, microwave time of 3 min, and pH 8. Compared to
31
non-microwaved fibers, the breaking strength was 19% higher and the elongation was 302.43% higher which
32
indicated the microwaved fiber toughness was increased. To study the mechanism underlying the effect of
33
microwave treatment on the improvement of mechanical properties, changes in the -SH and -S-S- content
34
during wheat protein fiber preparation, a secondary structure study, X-ray diffraction, thermal performance
35
analysis, SEM, surface hydrophobicity, and standard moisture regain measurement were examined. The
36
microwaved fiber had increased –S-S- content, α-helices, crystallinity, which may be responsible for the better
37
mechanical properties. DSC and TG results showed that the thermal stability of microwaved fiber was
38
increased. Additionally, SEM micrographs revealed that the structure of microwaved fibers was smoother and
39
denser, and contained less pores than non-microwaved fibers. Although the surface hydrophobicity and
40
standard moisture regain were decreased, microwaved fiber had good hygroscopicity, which was close to that
41
of silk.
42
Keywords: wheat protein, fiber, microwave, mechanical properties, mechanism
44
SC
M AN U
TE D
EP
AC C
43
RI PT
27
45 46 47
2
ACCEPTED MANUSCRIPT
48
1. Introduction Wheat gluten, the endosperm storage protein, is a typical water-insoluble protein that makes up about 85% of
50
total wheat protein. Wheat gluten is formed by glutenin and gliadin through disulfide inter– and intra–molecule
51
linkage(Shewry & Tatham, 1997). The unique properties required for forming flour dough make wheat gluten
52
very popular in food industries, and also allow the development of biodegradable biomaterials(Veraverbeke &
53
Delcour,2002). Today, gluten is extensively used in human and pet food applications, but also used in non-food
54
industries, such as natural adhesives, films for food packing, and so on (Day, et al,2006).Besides, there also
55
have reports about wheat gluten fibers which have fineness similar to that of wool (Narendra Reddy & Yiqi
56
Yang, 2007).
M AN U
SC
RI PT
49
Wheat gluten is also a cheap, abundant, and renewable source for producing protein fibers(Lens, et al,1999;
58
Ye, et al,2006; Woerdeman & Veraverbeke,2004) . In addition, it has good resistance to water and heat,
59
excellent elasticity, and easy degradability, all of which are desirable properties for fibers(Woerdeman &
60
Veraverbeke,2004; Bietz & Lookhart,1996; Krull & Inglett,1971). Therefore, the application of wheat gluten on
61
fiber can not only expand its consumption market but also increase its value addition.
EP
TE D
57
Several attempts have been made to use plant proteins such as soybeans, corn, and peanut or milk proteins
63
(casein) for fiber production. However, pure plant protein fibers are scarce because of their poor properties. For
64
example, soyprotein fibers’ breaking tenacity is about 30% of wool and the breaking elongation is less than
65
10% of wool(Moncrieff, 1975; Farrow,1956). Zein fibers’ mechanical properity is similar to soyprotein
66
fiber(Yang,et al,1996; Farrow,1956).Modification of wheat gluten by physical, chemical, and enzymatic
67
methods can change the nature of the protein, such as the spatial structure, the electric charge as well as the
68
length of peptide chain. Chiou (Chiou, et al, 2013) used citric acid to modify wheat gluten to produce
AC C
62
3
ACCEPTED MANUSCRIPT
superabsorbent materials. It was reported that a sample with a gluten:citric acid ratio of 0.5:1 and a reaction
70
temperature of 120°C‐had the largest water uptake value. Additionally, all modified gluten samples had lower
71
thermal stability than neat gluten. The reaction of transglutaminase (TGase) with wheat gluten was reported to
72
improve the physical and mechanical properties of the fiber as well as its hydrolytic stability(Jun Li, et al,
73
2013).
RI PT
69
Microwave is an electromagnetic wave whose frequency of 300 MHz-300 GHz can cause molecular
75
vibrations at the molecular level. Microwave can also induce changes in water polarity orientation with the
76
external electric field in high frequency, thus further causing molecular movement and mutual friction.
77
Therefore, microwave energy converts into heat and increases the material temperature(Banik, et al,2003;
78
Thostenson & Chou,1999) . It is reported that microwave can change the materials' structure and permanently
79
change their properties. Accordingly, Byaruhanga (Byaruhanga, Emmambux, Belton, Wellner, Ng &
80
Taylor,2006) found that the kafirin content of β-folding increased after microwave treatment.
TE D
M AN U
SC
74
Application of microwave thermal energy is considered to be a mean for producing even heating
82
throughout the sample as compared with direct oven heating, and thus could be used to create uniform
83
cross-linking throughout the sample(Larhed &Hallberg ,2001). Therefore in the current study, we prepared a
84
pure protein fiber using microwave-modified wheat protein. We investigated the effect of microwave power,
85
time, and pH on the mechanical properties of the fiber and further optimized the process using response surface
86
methodology. We hoped that the microwave can improve the mechanical properties of fiber and shorten the
87
production time. Additionally, we also hoped to find the mechanism of the improved mechanical properties
88
observed with microwave treatment.
89
2. Materials and Methods
AC C
EP
81
4
ACCEPTED MANUSCRIPT
90
2.1. Materials Commercially available wheat gluten, Whetpro 80 with about 80% protein content was purchased from
92
Chinatex HuiZe Bio-technology (De Zhou) Co. Ltd. Analytical grade urea, sodium sulfite, sulfuric acid,
93
sodium hydroxide, absolute alcohol, and sodium sulfate were purchased from commercial sources in China.
94
2.2. Separation of glutenin and gliadin
RI PT
91
Wheat gluten was mixed with 65% alcohol in a solid: liquid ratio of 1:30 and stirred at 50°C for 3 h. The
96
mixture was then centrifuged at 4500 rpm for 20 min after the extraction. Precipitation was continued using
97
65% alcohol and extracted an additional 2 times. The supernatants were pooled and the alcohol was removed
98
using a rotary evaporator at 40°C. The gliadin-rich fraction was freeze-dried. The final precipitate was then
99
mixed with water in a material: liquid ratio of 1:20. The pH of the mixture was regulated to pH 11.5 and stirred
100
at 60 °C for 3 h, and then centrifuged at 4500 rpm for 20 min after the extraction. The alcohol (65%) was added
101
to the supernatant and regulated to pH 7. The solution was then stored at 4°C for 12 h and centrifuged (4500
102
rpm, 20 min). The subsequent deposits were the glutein, which were collected and freeze-dried. The glutenin
103
and gliadin were crushed and mixed with hexane in a solid: liquid ratio of 1:2 to remove the fat, and then
104
passed through an 80 mesh stand-by.
105
2.3. Fiber Preparation
AC C
EP
TE D
M AN U
SC
95
106
Protein solutions with wheat gluten concentrations of 15% (w/w), and a glutenin: gliadin ratio of 1:1 at pH 7
107
were prepared according to the Reddy and Yang( Reddy & Yang, 2007), by dissolving wheat gluten in 8 M urea
108
solution as a swelling agent and 1% (w/w) sodium sulfite on the total weight of the bath as the reducing agent.
109
Wheat protein solutions were treated by microwave under different conditions. Fibers were extruded into a
110
coagulation bath consisting of 10% (w/w) sodium sulfate and 10% (w/w) sulfuric acid using a normal syringe
5
ACCEPTED MANUSCRIPT
and needle. The fibers formed were allowed to stay in the coagulation bath for 20 min and later rinsed with
112
water and air-dried. Then the spun fiber was drawn and annealed. The effects of microwave power (five
113
different levels: 100,200,300,400, and 500 W), exposure time (five different times: 1, 2, 3, 4, and 5 min), and
114
pH (four different pH: 5, 6, 7, and 8) on the breaking strength and breaking elongation of the fiber were
115
examined.
116
2.4. Tensile Testing
SC
RI PT
111
All of the fiber samples were conditioned before testing in a standard testing atmosphere of 21°C and 65%
118
relative humidity for 24 h. Fibers were tested for their tensile properties using a texture analyzer (TA). A gauge
119
length of 40 mm and an extension speed of 2 mm/min were used for tensile testing. Approximately 15 fibers
120
were tested for each condition and the average values were obtained.
M AN U
117
To determine the optimal conditions for microwave technology, we chose microwave power, time, and pH as
122
single factors. Based on this experiment, we chose the breaking strength and elongation at break as the
123
evaluation index and determined the response surface experiment with these three factors at three levels.
124
2.5. Determination of -SH and -S-S- content in the process of wheat protein fiber preparation
EP
TE D
121
The concentration of free sulfhydryl groups (-SH) of the wheat protein fiber was determined using Ellman's
126
reagent (5'5-dithiobis(2-nitrobenzoic acid), DTNB). Changes in free sulfhydryl groups were measured in
127
triplicate as previously reported(Beveridge, et al, 1974). Briefly, finely grinded wheat protein fiber (42 mg) was
128
diluted to 10 mL with 4.7 g guanidine hydrochloride in Tris-glycine buffer (1.04% Tris, 0.69% glycine, 0.12%
129
EDTA (w/v), pH 8.0) and reacted for 1 h at 25°C. A solution of 1 mL of the solution, 4 mL 8 M urea solution
130
(dissolved with Tris-glycine buffer), and 0.1 mL Ellman's reagent was prepared and mixed in a 25°C‐water bath
131
for 30 min to avoid a light reaction. The solution was then subjected to centrifugation at 4500 rpm for 15 min,
AC C
125
6
ACCEPTED MANUSCRIPT
132
and the absorbance of the supernatant was measured at 412 nm using a UV-vis spectrophotometer. The concentration of total sulfhydryl groups (SH) in the wheat protein fiber was measured as follows: 1 mL
134
sample solution, 0.05 mL mercaptoethanol, 4 mL 8 M urea-5 M guanidine hydrochloride (dissolved with
135
Tris-glycine buffer) were mixed together in a 25°C‐water bath for 1 h. Then, 10 mL of 12% TCA was added
136
and the mixture continued to stay in a 25°C‐water bath for an additional 1 h. The solution was then centrifuged
137
at 4500 rpm for 15 min. The precipitate was then washed with 5 mL 12% TCA twice. The precipitate was
138
mixed with 10 mL 8 M urea solution and 0.04 mL Ellman's reagent in a 25°C water bath for 30 min to avoid a
139
light reaction. The absorbance was then measured at 412 nm using a UV-spectrophotometer. The concentration
140
of free –SH was calculated using the following equation:
141
-SH (µM/g) = 73.53 A412 × D/C, where A412 is the absorbance at 412 nm; C is wheat protein fiber concentration
142
(mg/mL) and D is the dilution factor. -S-S- content was calculated using the following formula:
143
-S-S-(µM/g) = (N2-N1)/2, where N1 is the content of sulfhydryl groups before reduction and N2 is the content of
144
sulfhydryl groups after reduction.
145
2.6. SDS-PAGE
EP
TE D
M AN U
SC
RI PT
133
Approximately 7 mg of microwaved and non-microwaved wheat gluten fibers were powdered and mixed
147
with 1 mL of reduced and non-reduced SDS-PAGE 2X sample buffer (100 mmol/L Tris-HCl (pH 6.8), 6%
148
AC C
146
m/V SDS, 0.2%
m/V Bromophenol Blue, 20%
V/V Glycerol, 300 mmol/L 2-ME or not), and left
149
standing at room temperature for 2 h. Samples were subsequently boiled for 5 min in boiling water and
150
centrifuged at 10000 rpm for 5 min. The clear top layer of each sample (10 μL) was loaded into each slot in
151
the gel. After electrophoresis, the gel was stained with Coomassie Brilliant blue G-250. After standing for one
152
night, the gel was flushed with deionized water and placed in a destaining liquid until a clear background was
7
ACCEPTED MANUSCRIPT
153
formed.
154
2.7. Secondary structure study Secondary structures of fibers were studied by Fourier transform infrared spectroscopy (FTIR). The
156
background spectrum of the ATR cell was recorded at 32 scans and a resolution of 4 cm-1. The spectra were
157
recorded under the same conditions as the background and the scanning range was 400-4000 cm-1. The spectra
158
were analyzed using Omnic and Peakfit software.
159
2.8. X-ray diffraction
160
The fiber was sheared and grinded finely. The measurements were taken using an X-ray diffraction analyzer
161
(Bruker Germany AXS Co. Ltd.) under the following conditions: a Ni-filtered Cu-Kα line was used as the X-ray
162
source, voltage of 40 kV, current of 30 mA, 2°/min in scanning speed, scanning the specimen in 2-45°.
163
2.9. Thermal performance
M AN U
SC
RI PT
155
The fibers were grinded to powders. Thermogravimetric analysis (TGA) of fiber samples (1–2 mg) was
165
carried out using a TGA/SDTA 851e T.A. instrument at 10°C/min from room temperature to 550°C under
166
nitrogen atmosphere using a flow rate of 50 mL/min. Differential scanning calorimetry (DSC) analysis of fiber
167
samples (1–2 mg) was carried out using a DSC-Q200 T.A. instrument at 10°C/min from 40 to 250°C under
168
nitrogen atmosphere using a flow rate of 50 mL/min. Before DSC analysis, the sample was mixed with distilled
169
water in a 1:4 ratio and kept balanced at 4°C in a refrigerator for 24 h.
170
2.10. SEM
171
AC C
EP
TE D
164
The morphological features, in terms of the longitudinal and cross-sectional appearance of the wheat gluten
172
fibers, were observed using a Hitachi model S-4800 scanning electron microscope.
173
2.11. Surface hydrophobicity
8
ACCEPTED MANUSCRIPT
The anionic fluorescent probe 8- anilinonaphthalene-1-sulfonic acid (ANSA) was used to measure the
175
exposed surface hydrophobicity of wheat protein and its fibers. The fibers were grinded and passed through a
176
200-mesh sieve. Protein samples were diluted to a final concentration of 0.02, 0.04, 0.08, or 0.16 mg/mL using
177
0.01 M sodium phosphate buffer at pH 7.0. An ANSA stock solution (8 mmol) was prepared in 10 mM sodium
178
phosphate buffer at pH 7.0. Aliquots of ANSA stock solution (10 µL) were titrated into 2 mL protein solutions
179
and a F-7000 fluorescence spectrometer at an excitation wavelength of 390 nm (slit 5 nm) and an emission
180
wavelength of 470 nm was used to measure the fluorescence intensity of the samples. The curve was based on
181
the protein concentration and the fluorescence intensity. The initial stage of the slope of the curve is the surface
182
hydrophobic activity of the protein samples.
183
2.12. Standard moisture regain
SC
M AN U
184
RI PT
174
The moisture regain of the fibers was determined according to the American Society for Testing and Materials (ASTM) method 2654 under standard atmospheric conditions of 65% relative humidity and 21°C.
186
3. Results and Discussion
187
3.1. Microwave Process Optimization
EP
TE D
185
The effects of different process variables including microwave power (100-500 W), time (1-5 min), and pH
189
(5-8) on the breaking strength and elongation at break of fibers were investigated using single factor
190
experiments.
AC C
188
191
As a protein modification method, microwave-modified wheat protein is made of fibers. The result shows
192
that as the microwave power increased, the breaking strength and the elongation at break initially increased and
193
then decreased, and both factors peaked at the microwave power of 300 W. Similar findings were observed in
194
the time analysis, and both the breaking strength and the elongation at break peaked at the microwave time of 2
9
ACCEPTED MANUSCRIPT
min. This may be because protein chains become unfolded with increasing power and time and therefore lower
196
power and shorter times are preferred. Previous studies have demonstrated that microwave treatment can
197
promote protein unfolding (Woerdeman & Veraverbeke, 2004), and the unfolded protein maybe more suitable
198
for spinning. As the microwave power and time increased, the protein chain crossed to a certain extent (Qasem,
199
2006) and caused an increase in the mechanical properties of the wheat protein fiber. However, further
200
increases in microwave power and time resulted in excessive crosslinking of the protein chains, and a decrease
201
in the mechanical properties.
SC
RI PT
195
Differences in pH can have varying effects on proteins. The result shows that breaking strength and the
203
elongation at break initially increased and then decreased with increasing pH, and both factors peaked at a pH
204
of 7. One possible reason for this observation could be that at lower pH (acidic atmosphere), proteins are easily
205
hydrolyzed, such that the protein chains becomes short, leading to poor mechanical properties of the spun fiber.
206
However, in neutral and weakly alkaline environments, protein chains remain in their original high molecular
207
state, thus leading to better mechanical properties of the spun fiber.
TE D
M AN U
202
Using Designexpert7.0 software, an analysis of variance test was done to generate a regression model of
209
breaking strength and elongation at break. The order of factors' effect on breaking strength is microwave time >
210
pH >microwave power. The order of factors' effect on elongation at break is pH> microwave time > microwave
211
power. Considering the two response values, the best process condition that the Designexpert7.0 software
212
showed is microwave power 20.6 W/mL, microwave time 3 min, pH 8. Preparing the fiber under this condition,
213
the fiber's breaking strength is 0.69 cN/dtex, elongation at break is 23.37%. Compared with the
214
non-microwaved fibers, the breaking strength increased by 19% and the elongation increased by 302.43%. The
215
obvious increase of elongation indicated the toughness of fiber was increased.
AC C
EP
208
10
ACCEPTED MANUSCRIPT
216
3.2. Determination of -SH and -S-S- content in the process of wheat protein fiber preparation The effect of microwave on free sulfhydryl groups and disulfide bonds during the preparation of fibers is
218
shown in Table 1. The results demonstrate that microwave treatment can promote disulfide bond rupture as
219
indicated in the comparison between fibers that were microwaved (treatment 4) and non-microwaved fibers
220
(treatment 1). This may be because the microwave through the electromagnetic field can cause material internal
221
friction between material atoms, and the heat of this friction can enhance the effect of urea and sodium sulfite.
222
The results also clearly indicate that drawing can increase the content of free sulfhydryl groups and decrease
223
the content of disulfide bond, as observed in the comparison between non-microwaved fibers that were drawn
224
(treatment 2) or not (treatment 1). This indicates that drawing can cause disulfide bond rupture(Li & Jinbo.
225
2010) and microwaved fibers show a similar result (comparison of treatments 4 and 5). Annealing was shown
226
to increase the content of disulfide bonds and decrease the content of free sulfhydryl groups, as seen in
227
non-microwaved fibers with or without annealing (treatments 2 and 3). This indicates that annealing can
228
promote free sulfhydryl groups into -S-S-( Cuq, et al, 1998) to fix the fiber. The final disulfide bond content of
229
microwaved fibers is higher than non-microwaved fibers (treatments 3 and 6). This could be due to a
230
microwave treatment induced change in the conformation of proteins as well as the location of –SH, therefore
231
making it easier to promote –S-S- formation. This higher content of -S-S- may be the reason for the better
232
mechanical properties of microwaved fibers.
233
3.3. SDS-PAGE
234
The SDS-PAGE of sample is showed in Fig.1. A comparison of the reduced and non-reduced SDS-PAGE
AC C
EP
TE D
M AN U
SC
RI PT
217
235
gels (A and B respectively) show a marked reduction in bands and band intensity in the non-reduced gel,
236
indicating that –S-S- plays an important role in the production of fibers. The observed decreases in band
11
ACCEPTED MANUSCRIPT
expression with microwave treatment compared to non-microwaved fibers (4 and 7 respectively in the
238
SDS-PAGE analysis) indicate that microwave forces the occurrence of covalent crosslinking in addition to the
239
disulfide bond. An increase in the intensity of the low molecular weight band (C) is observed in the comparison
240
of non-microwaved and microwaved fibers, with or without drawing, (5 with 4 or 8 with 7 respectively),
241
indicating that drawing can lead to a decrease in molecular weight, which may be caused by–S-S- rupture. In
242
microwaved fibers with or without annealing (9 with 8, respectively), the intensity of bands is increased. This
243
indicates that heating increases the content of –S-S-(Waqner M.et al,2011), which corresponds with the
244
findings of increased –S-S- content with annealing shown in Table 1. Therefore, non-covalent crosslinking and
245
indirect formation of disulfide bonds may play an important role in improving the mechanical properties of
246
microwave fiber.
247
3.4. Secondary structure study
M AN U
SC
RI PT
237
Fourier transform infrared spectroscopy (FTIR) is widely used for the analysis of the secondary structure of
249
proteins. Characteristic bands in the FTIR spectra of proteins mainly include amide I (1600-1700 cm-1) and
250
amide II (1500-1600 cm-1). Although amide I and amide II bands are related to the secondary structure of
251
proteins, the amide II band is not as good a predictor for quantitation of the secondary structure of
252
proteins(Kumosinski & Farrell, 1993) . Amide I was baseline corrected using Omnic software, smoothed with a
253
nine-point Savitsky-Golay function(Savitzky & Golay, 1964) to remove any possible white noise, and then
254
Savitsky-Golay 5 points were used for the second order derivative to determine whether a different secondary
255
structure corresponds to the position of the peak. The Peakfit software was then used to fit the Gauss function,
256
according to a previous report(Krimm & Bandekar,1986) in order to determine the position of the peak. There
257
are many examples using FTIR to analysis the secondary structure of proteins. For example, Sivam (A.S.
AC C
EP
TE D
248
12
ACCEPTED MANUSCRIPT
Sivam, et al, 2013) used FTIR to study the changes of conformation of bread after adding pectin and
259
polyphenols. Michael Byler (D. Michael Byler & Heino Susi)examined the secondary structure of proteins by
260
deconvolved FTIR spectra. The content of the secondary structure of wheat gluten fiber was determined based
261
on the fitting curve and is listed in Table 2.
RI PT
258
The results show that microwave treatment (treatments 1 (non-microwaved) and 4 (microwaved), Table 2)
263
can increase the random coil and α-helix content of wheat protein, which could be caused by disulfide bond
264
rupture. We also find that drawing can increase random coil and decrease α-helix, which may be caused by
265
disulfide bond rupture during drawing. Annealing caused the disappearance of random coil and increased the
266
α-helix and β-sheets content, regardless of microwave treatment. The phenomenon is however more
267
profound in microwave treated fiber, which may be the reason that the structure of microwave treated fibers‐are
268
generally more disordered and easier to change. The conformation changes in fiber may be caused by changes
269
in free sulfhydryl groups and disulfide bond content.
270
3.5. X-ray diffraction
TE D
M AN U
SC
262
Crystallinity can be used to reflect the protein structure of the fiber. The crystallinity of the fiber increases
272
with increasing molecular order of the protein. Fig. 2 shows the effects of microwave on the x-ray diffraction
273
(XRD) spectra of wheat protein fiber. The most prominent diffracting peaks are seen at about 9° and 19.5°,
274
which are the characteristic crystalline peaks of wheat gluten fiber(Reddy & Yang, 2010). There is almost no
275
difference at 9°, while the full width at half maximum (FWHM) of microwaved fibers is 9.67, which is smaller
276
than fibers without microwave treatment. Since the degree of crystallinity under the same condition is worse,
277
diffraction peaks will be broader. Therefore, microwaved fibers have a higher degree of crystallinity compared
278
with non-microwaved fibers. Furthermore, microwaved fibers add a crystalline peak at 2 θ at 32°, which further
AC C
EP
271
13
ACCEPTED MANUSCRIPT
279
increases the degree of crystallinity. Proteins with higher crystallinity require higher power to destroy the
280
ordered structure, and thus increase the mechanical properties of the fiber.
281
3.6. Thermal performance In order to investigate the effect of microwave treatment on the thermal properties of wheat gluten fiber,
283
DSC and TGA analysis were performed. Thermal profiles from DSC and TGA of wheat gluten fiber are shown
284
in Fig.3. DSC is an important method to study the thermal stability of proteins. Large Td values corresponded
285
with higher thermal stability. ∆H is the area under the endothermic peak, which is the energy required to
286
completely denature the wheat protein. The results demonstrate that the Td of microwaved fibers are higher than
287
that of non-microwaved fibers, which indicate that microwave treatment can improve the thermal stability of
288
fibers. The denaturation enthalpy of microwaved fiber is lower than that of non-microwaved fiber. This may be
289
due to protein crosslinking after microwave treatment. Protein crosslinking causes the unfolding of protein
290
chains and exposes hydrophobic residues, leading to the increase of surface hydrophobicity(Rani J.
291
Qasem,2006).TG curves show two stages of well-defined mass loss. The first stage was attributed to the
292
removal of water over the temperature interval of 30–160°C. This stage is associated with the process of
293
protein denaturation(Li-Chan & Ma, 2002). The second stage corresponds to the thermal decomposition of the
294
protein fraction after 200°C. The results clearly show that thermal decomposition of microwaved fibers is
295
higher than non-microwaved fibers, which indicate higher thermal stability. This conclusion was also consistent
296
with the findings of the DSC analysis.
297
3.7. Scanning Electron Microscopy (SEM)
298 299
AC C
EP
TE D
M AN U
SC
RI PT
282
In order to study the effect of microwave on wheat protein fibers, scanning electron microscopy (SEM) was used to observe the longitudinal appearance, as shown in Fig. 4.
14
ACCEPTED MANUSCRIPT
From the longitudinal appearance, the comparison of non-microwaved fiber with microwaved fiber (a with b,
301
respectively) shows that the surface of wheat protein fiber becomes smoother and denser with microwave
302
treatment. Enlarged images reveal granular materials on the surface. These granular materials may be coagulant
303
crystalline particles, urea crystallization, or residual starch granules. Comparison of 5K times enlarged images
304
(e with f) shows that wheat protein has better orientation after microwave treatment and the expression of the
305
fibrous protein is more obvious in microwaved fibers. The higher orientation of molecular protein may be
306
another reason for the improved mechanical properties with microwave treatment. SEM was also used to
307
observe the cross-section of wheat gluten fiber.
M AN U
SC
RI PT
300
The cross-section of non-microwaved fibers is almost circular, whereas that of microwaved fibers are flat
309
and dumbbell shaped. Furthermore, non-microwaved fibers are less dense than microwaved fibers, as observed
310
from the comparison of images c and d. Additionally, a greater number of pores were observed in the
311
non-microwaved fiber compared with the microwave treated fiber(e and f). The denser structure of microwaved
312
fiber observed may be another reason for the better mechanical properties observed with microwaved
313
treatment.
314
3.8. Surface hydrophobicity and standard moisture regain.
EP
TE D
308
The surface hydrophobicity of microwaved fiber and non-microwaved fiber was 852.5±5.8 and 660.71±9.6,
316
respectively. The surface hydrophobicity of microwaved fibers is higher than that of non- microwaved fibers.
317
This may be the result of protein denaturation caused by microwave treatment, leading to unfolding of the
318
protein chain and exposure of hydrophobic residues, thus ultimately increasing the surface hydrophobicity. The
319
standard moisture regain of microwaved fiber and non-microwaved fiber is 11.11% and 10.20%, respectively.
320
The higher surface hydrophobicity of the microwaved fiber could affect its ability to absorb water, thus
AC C
315
15
ACCEPTED MANUSCRIPT
resulting in the observed decrease in standard moisture regain. Regardless of microwave treatment, the standard
322
moisture regain of wheat protein fibers is close to the standard moisture regain of silk, indicating that the wheat
323
protein fiber has good hygroscopicity.
324
4. Conclusions
RI PT
321
In the present study, microwave-modified wheat protein fiber was produced to improve the mechanical
326
properties as well as shortened the production cycle. The best process conditions obtained from our response
327
surface analyses are a microwave power of 20.6 W/mL, microwave time of 3 min, and pH 8. Preparing the
328
fiber under these conditions results in a fiber breaking strength of 0.69 cN/dtex and an elongation at break of
329
23.37%. Compared with the non-microwaved fibers, the breaking strength increased by 19% and the elongation
330
increased by 302.43%. And microwave did play a role in the improvement of mechanical properties. To study
331
the mechanism of microwave treatment on the improvement of mechanical properties, -SH and -S-S- content in
332
the process of wheat protein fiber preparation, secondary structure study, X-ray diffraction, thermal
333
performance, SEM, surface hydrophobicity, and standard moisture regain were examined . The results show
334
that microwaved fiber had a higher content of –S-S-, α-helix, crystallinity, which may be responsible for the
335
better mechanical properties and increased thermal stability. Additionally, SEM micrographs showed that
336
microwaved fiber also had a smoother and denser structure as well as less pores. Although the surface
337
hydrophobicity and standard moisture regain were decreased, microwave fiber had good hygroscopicity, which
338
was close to that of silk.
339
Acknowledgments
AC C
EP
TE D
M AN U
SC
325
340
We thank school of textile and garment, Jiangnan University for providing us with equipment support. We
341
are also grateful for the guidance of teachers at physical platform. With their help, our experiment can run
16
ACCEPTED MANUSCRIPT
342
smoothly. At last, we also thank for the help of our fellow classmates.
343 References
345
Shewry P. R., Tatham A. S.(1997). Disulphide bonds in wheat gluten proteins. Journal of Cereal Science, 25(3),
350 351 352 353 354 355 356 357 358
SC
Day, L., Augustin, M. A., Batey, I. L., & Wrigley, C. W. (2006). Wheat-gluten uses and industry needs. Trends
M AN U
349
to breadmaking functionality. Critical reviews in food science and nutrition, 42(3), 179-208.
in Food Science & Technology, 17(2), 82-90.
Lens J. P., Mulder W. J., Kolster P.(1999). Modification of wheat gluten for nonfood applications. Cereal Foods World, 44, 5-9
Ye, P., Reitz, L., Horan, C., & Parnas, R. (2006). Manufacture and biodegradation of wheat gluten/basalt
TE D
348
Veraverbeke W. S., Delcour J. A.(2002). Wheat protein composition and properties of wheat glutenin in relation
composite material. Journal of Polymers and the Environment, 14(1), 1-7. Woerdeman, D. L., Veraverbeke, W. S., Parnas, R. S., Johnson, D., Delcour, J. A., Verpoest, I., & Plummer, C. J. (2004). Designing new materials from wheat protein. Biomacromolecules, 5(4), 1262-1269.
EP
347
207-227.
Bietz, J. A., & Lookhart, G. L. (1996). Properties and non-food potential of gluten. Cereal Foods World, 41(5),
AC C
346
RI PT
344
376-382.
359
Krull, L. H., & Inglett, G. E. (1971). Industrial uses of gluten. Cereal science today, 16, 232-261.
360
Reddy, N., & Yang, Y. (2007). Novel protein fibers from wheat gluten. Biomacromolecules, 8(2), 638-643.
361
Moncrieff, R. W. (1975). Man-made fibres. London : Newnes-Butterworths.
362
Yang, Y., Wang, L., Li, S. (1996).,Formaldehyde-free zein fiber preparation and investigation. Journal of
17
ACCEPTED MANUSCRIPT
364 365
Applied Polymer Science,59 (3), 433- 441. Farrow, B.(1956). Extensometric and Elastic Properties of Textile. Fibres. Journal of Textile Institute, 47, No. 2, T58-T101.
RI PT
363
Chiou, B. S., Jafri, H., Cao, T., Robertson, G. H., Gregorski, K. S., Imam, S. H., Greg M.& Orts, W. J. (2013).
367
Modification of wheat gluten with citric acid to produce superabsorbent materials. Journal of Applied
368
Polymer Science, 129(6), 3192-3197.
371 372 373 374
transglutaminase (TGase) regeneration gluten protein fiber. Journal of functional materials ,1(44),39-46.
M AN U
370
Jun, M.; Li, C.; Xuerong, F.; Ping, W.; Qiang, W.(2013). Study based on the preparation and properties of
Banik, S., Bandyopadhyay, S., & Ganguly, S. (2003). Bioeffects of microwave-a brief review. Bioresource technology, 87(2), 155-159.
Thostenson, E. T., & Chou, T. W. (1999). Microwave processing: fundamentals and applications. Composites Part A: Applied Science and Manufacturing, 30(9), 1055-1071.
TE D
369
SC
366
Byaruhanga, Y. B., Emmambux, M. N., Belton, P. S., Wellner, N., Ng, K. G., & Taylor, J. R. N. (2006).
376
Alteration of kafirin and kafirin film structure by heating with microwave energy and tannin complexation.
377
Journal of agricultural and food chemistry, 54(12), 4198-4207.
379 380 381 382 383
Larhed M, Hallberg A.(2001) .Microwave-assisted high speed chemistry: a new technique in drug discovery.
AC C
378
EP
375
Drug Discovery Today. 6:406-416. Beveridge, T., Toma, S. J., & Nakai, S. (1974). Determination of SH- and –S-S- groups in some food proteins using ELLMAN'S reagent. Journal of Food Science, 39(1), 49-51. Qasem, R. J. (2006). The effect of microwave thermal denaturation on release properties of bovine serum albumin and gluten matrices. AAPS PharmSciTech, 7(1), E104-E110.
18
ACCEPTED MANUSCRIPT
386 387 388 389
Journal of Tianjin Polytechnic University,29(2):19-24,44. Cuq, B., Gontard, N., Cuq, J. L., & Guilbert, S. (1998). Packaging films based on myofibrillar proteins:
RI PT
385
Li W., Jinbo Y.(2010). Fourier transform spectroscopy analysis of the secondary structure of stretch wool.
Fabrication, properties and applications. Food/Nahrung, 42(03 04), 260-263.
Waqner M., Morel MH, Bonicel J., Cuq B.(2011).Mechanisms of heat-mediated aggregation of wheat gluten protein upon pasta processing. Journal of Agricultural and Food Chemistry,59(7):3146-54.
SC
384
Kumosinski, T. F., & Farrell, H. M. (1993). Determination of the global secondary structure of proteins by
391
Fourier transform infrared (FTIR) spectroscopy. Trends in Food Science & Technology, 4(6), 169-175.
392
Savitzky, A., & Golay, M. J. (1964). Smoothing and differentiation of data by simplified least squares
396 397 398 399 400 401
proteins. Advances in protein chemistry, 38, 181-364.
TE D
395
Krimm, S., & Bandekar, J. (1986). Vibrational spectroscopy and conformation of peptides, polypeptides, and
Reddy, N., & Yang, Y. (2010). Developing water stable gliadin films without using crosslinking agents. Journal of Polymers and the Environment, 18(3), 277-283.
EP
394
procedures. Analytical chemistry, 36(8), 1627-1639.
Rani J. Qasem(2006). The Effect of Microwave Thermal Denaturation on Release Properties of Bovine Serum Albumin and Gluten Matrices, Aaps Pharmscitech, 7 (1):E1-E7
AC C
393
M AN U
390
Li-Chan, E. C. Y., & Ma, C. Y. (2002). Thermal analysis of flaxseed (Linum usitatissimum) proteins by differential scanning calorimetry. Food Chemistry, 77(4), 495-502.
402
A.S. Sivam, D. Sun-Waterhouse, C.O. Perera, G.I.N. Waterhouse(2013). Application of FT-IR and Raman
403
spectroscopy for the study of biopolymers in breads fortified with fibre and polyphenols, Food Research
404
International,50, 574-585.
19
ACCEPTED MANUSCRIPT
405 406
D. MICHAEL BYLER and HEINO SUSI(1986). Examination of the Secondary Structure of Proteins by Deconvolved FTIR Spectra, Biopolymers, 25, 469-487.
RI PT
407 408 409
SC
410 411
M AN U
412 413 414 415
419 420 421 422
EP
418
AC C
417
TE D
416
423 424 425
20
ACCEPTED MANUSCRIPT
426
Tables
427
Table 1. Effect of microwave on free sulfhydryl groups and disulfide bond of WG fibers Treatmenta-f
Content of free -SH µmol/g
2
17.08±0.09
3
13.33±0.09
4
13.83±0.1
5
18.68±0.05
6
10.17±0.11
8.61±0.12
RI PT
12.84±0.07
7.73±0.26 8.83±0.21
8.03±0.18 6.08±0.04
SC
1
Content of -S-S- µmol/g
10.27±0.15
a
1. fibers without microwave, drawing or annealing; b2. fibers without microwave and annealing but with drawing; c3. fibers
429
without microwave but with annealing and drawing; d4. microwaved fiber without drawing or annealing; e5. microwaved fiber
430
drawing but not annealing; f6. microwaved fiber drawing and annealing
431
Table 2. Secondary structure of wheat protein fiber with different treatments Treatment
M AN U
428
a-f
3 4 5 6 a
β-turns(%)
34.48±0.57
6.90±0.10
24.45±0.32
29.78±0.37
31.51±0.32
14.15±0.24
18.26±0.20
33.33±0.35
33.49±0.41
-
22.52±0.19
33.73±0.56
28.68±0.18
17.45±0.09
33.16±0.25
18.20±0.30
17.46±0.38
36.56±0.35
4.96±0.08
44.44±0.44
37.83±0.52
-
39.91±0.47
22.77±0.65
1. fibers without microwave, drawing or annealing; b2. fibers without microwave and annealing but with drawing; c3. fibers
AC C
432
α-helix(%)
EP
2
random coil(%)
TE D
1
β-sheets(%)
433
without microwave but with annealing and drawing; d4. microwaved fiber without drawing or annealing; e5. microwaved fiber
434
drawing but not annealing; f6. microwaved fiber drawing and annealing
435
Figure captions
436
Fig. 1. SDS-PAGE of the wheat protein and fibers
437
A is the reduced SDS-PAGE; B is the non-reduced SDS-PAGE. M is standard protein.1-9 and a-i represent the
438
following: 1, a) wheat gluten; 2, b) glutenin; 3, c) gliadin; 4, d) fibers without microwave, drawing or annealing; 21
ACCEPTED MANUSCRIPT
5, e) fibers without microwave and annealing but with drawing; 6, f) fibers without microwave but with
440
annealing and drawing; 7, g) microwaved fiber without drawing or annealing; 8, h) microwaved fiber with
441
drawing but not annealing; 9, i) microwaved fiber with drawing and annealing
442
Fig. 2. Effects of microwave on the XRD spectra of wheat protein fiber (1- fibers without microwave treatment,
443
2- microwaved fibers)
444
Fig.3. DSC and TG-DTG curve of wheat protein fiber
445
Fig. 4. SEM micrographs of the longitudinal appearance and cross-section of wheat protein fibers.
446
a, c, e are images of non-microwaved fiber enlarged 1K, 2K, and 5K times. b, d, f are images of microwave
447
treated fiber enlarged 1K, 2K, and 5K times.
448
Figure
449
Fig.1
451 452 453
AC C
450
EP
TE D
M AN U
SC
RI PT
439
C
A
B
454 455 456 22
ACCEPTED MANUSCRIPT
Fig.2
SC
RI PT
457
Fig.3
Non-microwaved fiber Microwaved fiber
-30 -80 -130 -180 -230 0
TE D
Heat Flow W/g
20
50
100
150
200
250
300
Temperature
EP
460 461
5.00E-04 0.00E+00 -5.00E-04 -1.00E-03 -1.50E-03 -2.00E-03 -2.50E-03 -3.00E-03
weight loss (%)
AC C
120 100 80 60 40 20 0
0
100
200
300
400
500
temperature (°C)
462 463 464 23
600
Derived weight (%/°C)
459
M AN U
458
Non-microwaved fibers Microwaved fibers
ACCEPTED MANUSCRIPT
Fig.4
RI PT
465
b
M AN U
SC
a
d
AC C
EP
TE D
c
e
466
f
467 468 469 24
RI PT
ACCEPTED MANUSCRIPT
b
M AN U
SC
a
d
470 471
AC C
EP
TE D
c
e
f
472 473
25
ACCEPTED MANUSCRIPT Highlights The breaking strength and elongation were increased by 19% and 302.43% respectively
Microwaved fiber has higher content of –S-S-, α-helices and crystallinity.
The treatment increases the thermal stability of wheat protein fibers.
The treated fiber has a smoother and denser surface and less pores.
The fiber has a good standard moisture regain and increased surface hydrophobicity.
AC C
EP
TE D
M AN U
SC
RI PT
S0733-5210(16)30080-7
DOI:
10.1016/j.jcs.2016.05.018
Reference:
YJCRS 2147
To appear in:
Journal of Cereal Science
Received Date: 14 September 2015 Revised Date:
1 April 2016
Accepted Date: 11 May 2016
Please cite this article as: Liu, C., Ma, X., Study on the mechanism of microwave modified wheat protein fiber to improve its mechanical properties, Journal of Cereal Science (2016), doi: 10.1016/ j.jcs.2016.05.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Study on the mechanism of microwave modified wheat protein fiber to
2
improve its mechanical properties
3
Cuiqing Liu, Xiaojun Ma*
4
School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu
5
Province, PR China
7
*Corresponding authors
8
Tel/fax: 13861750315
9
Email address: [email protected]
10 11
15 16 17 18 19 20 21
EP
14
AC C
13
TE D
12
M AN U
SC
6
RI PT
1
22 23 24 25 1
ACCEPTED MANUSCRIPT
26
Abstract Wheat protein is widely used in food industry. In order to expand the scope of its application on non-food
28
field, we managed to apply the wheat protein to fiber production. To improve the mechanical properties of the
29
fibers, we used the method of microwave modification. The best process conditions obtained by response
30
surface analysis were a microwave power of 20.6 W/mL, microwave time of 3 min, and pH 8. Compared to
31
non-microwaved fibers, the breaking strength was 19% higher and the elongation was 302.43% higher which
32
indicated the microwaved fiber toughness was increased. To study the mechanism underlying the effect of
33
microwave treatment on the improvement of mechanical properties, changes in the -SH and -S-S- content
34
during wheat protein fiber preparation, a secondary structure study, X-ray diffraction, thermal performance
35
analysis, SEM, surface hydrophobicity, and standard moisture regain measurement were examined. The
36
microwaved fiber had increased –S-S- content, α-helices, crystallinity, which may be responsible for the better
37
mechanical properties. DSC and TG results showed that the thermal stability of microwaved fiber was
38
increased. Additionally, SEM micrographs revealed that the structure of microwaved fibers was smoother and
39
denser, and contained less pores than non-microwaved fibers. Although the surface hydrophobicity and
40
standard moisture regain were decreased, microwaved fiber had good hygroscopicity, which was close to that
41
of silk.
42
Keywords: wheat protein, fiber, microwave, mechanical properties, mechanism
44
SC
M AN U
TE D
EP
AC C
43
RI PT
27
45 46 47
2
ACCEPTED MANUSCRIPT
48
1. Introduction Wheat gluten, the endosperm storage protein, is a typical water-insoluble protein that makes up about 85% of
50
total wheat protein. Wheat gluten is formed by glutenin and gliadin through disulfide inter– and intra–molecule
51
linkage(Shewry & Tatham, 1997). The unique properties required for forming flour dough make wheat gluten
52
very popular in food industries, and also allow the development of biodegradable biomaterials(Veraverbeke &
53
Delcour,2002). Today, gluten is extensively used in human and pet food applications, but also used in non-food
54
industries, such as natural adhesives, films for food packing, and so on (Day, et al,2006).Besides, there also
55
have reports about wheat gluten fibers which have fineness similar to that of wool (Narendra Reddy & Yiqi
56
Yang, 2007).
M AN U
SC
RI PT
49
Wheat gluten is also a cheap, abundant, and renewable source for producing protein fibers(Lens, et al,1999;
58
Ye, et al,2006; Woerdeman & Veraverbeke,2004) . In addition, it has good resistance to water and heat,
59
excellent elasticity, and easy degradability, all of which are desirable properties for fibers(Woerdeman &
60
Veraverbeke,2004; Bietz & Lookhart,1996; Krull & Inglett,1971). Therefore, the application of wheat gluten on
61
fiber can not only expand its consumption market but also increase its value addition.
EP
TE D
57
Several attempts have been made to use plant proteins such as soybeans, corn, and peanut or milk proteins
63
(casein) for fiber production. However, pure plant protein fibers are scarce because of their poor properties. For
64
example, soyprotein fibers’ breaking tenacity is about 30% of wool and the breaking elongation is less than
65
10% of wool(Moncrieff, 1975; Farrow,1956). Zein fibers’ mechanical properity is similar to soyprotein
66
fiber(Yang,et al,1996; Farrow,1956).Modification of wheat gluten by physical, chemical, and enzymatic
67
methods can change the nature of the protein, such as the spatial structure, the electric charge as well as the
68
length of peptide chain. Chiou (Chiou, et al, 2013) used citric acid to modify wheat gluten to produce
AC C
62
3
ACCEPTED MANUSCRIPT
superabsorbent materials. It was reported that a sample with a gluten:citric acid ratio of 0.5:1 and a reaction
70
temperature of 120°C‐had the largest water uptake value. Additionally, all modified gluten samples had lower
71
thermal stability than neat gluten. The reaction of transglutaminase (TGase) with wheat gluten was reported to
72
improve the physical and mechanical properties of the fiber as well as its hydrolytic stability(Jun Li, et al,
73
2013).
RI PT
69
Microwave is an electromagnetic wave whose frequency of 300 MHz-300 GHz can cause molecular
75
vibrations at the molecular level. Microwave can also induce changes in water polarity orientation with the
76
external electric field in high frequency, thus further causing molecular movement and mutual friction.
77
Therefore, microwave energy converts into heat and increases the material temperature(Banik, et al,2003;
78
Thostenson & Chou,1999) . It is reported that microwave can change the materials' structure and permanently
79
change their properties. Accordingly, Byaruhanga (Byaruhanga, Emmambux, Belton, Wellner, Ng &
80
Taylor,2006) found that the kafirin content of β-folding increased after microwave treatment.
TE D
M AN U
SC
74
Application of microwave thermal energy is considered to be a mean for producing even heating
82
throughout the sample as compared with direct oven heating, and thus could be used to create uniform
83
cross-linking throughout the sample(Larhed &Hallberg ,2001). Therefore in the current study, we prepared a
84
pure protein fiber using microwave-modified wheat protein. We investigated the effect of microwave power,
85
time, and pH on the mechanical properties of the fiber and further optimized the process using response surface
86
methodology. We hoped that the microwave can improve the mechanical properties of fiber and shorten the
87
production time. Additionally, we also hoped to find the mechanism of the improved mechanical properties
88
observed with microwave treatment.
89
2. Materials and Methods
AC C
EP
81
4
ACCEPTED MANUSCRIPT
90
2.1. Materials Commercially available wheat gluten, Whetpro 80 with about 80% protein content was purchased from
92
Chinatex HuiZe Bio-technology (De Zhou) Co. Ltd. Analytical grade urea, sodium sulfite, sulfuric acid,
93
sodium hydroxide, absolute alcohol, and sodium sulfate were purchased from commercial sources in China.
94
2.2. Separation of glutenin and gliadin
RI PT
91
Wheat gluten was mixed with 65% alcohol in a solid: liquid ratio of 1:30 and stirred at 50°C for 3 h. The
96
mixture was then centrifuged at 4500 rpm for 20 min after the extraction. Precipitation was continued using
97
65% alcohol and extracted an additional 2 times. The supernatants were pooled and the alcohol was removed
98
using a rotary evaporator at 40°C. The gliadin-rich fraction was freeze-dried. The final precipitate was then
99
mixed with water in a material: liquid ratio of 1:20. The pH of the mixture was regulated to pH 11.5 and stirred
100
at 60 °C for 3 h, and then centrifuged at 4500 rpm for 20 min after the extraction. The alcohol (65%) was added
101
to the supernatant and regulated to pH 7. The solution was then stored at 4°C for 12 h and centrifuged (4500
102
rpm, 20 min). The subsequent deposits were the glutein, which were collected and freeze-dried. The glutenin
103
and gliadin were crushed and mixed with hexane in a solid: liquid ratio of 1:2 to remove the fat, and then
104
passed through an 80 mesh stand-by.
105
2.3. Fiber Preparation
AC C
EP
TE D
M AN U
SC
95
106
Protein solutions with wheat gluten concentrations of 15% (w/w), and a glutenin: gliadin ratio of 1:1 at pH 7
107
were prepared according to the Reddy and Yang( Reddy & Yang, 2007), by dissolving wheat gluten in 8 M urea
108
solution as a swelling agent and 1% (w/w) sodium sulfite on the total weight of the bath as the reducing agent.
109
Wheat protein solutions were treated by microwave under different conditions. Fibers were extruded into a
110
coagulation bath consisting of 10% (w/w) sodium sulfate and 10% (w/w) sulfuric acid using a normal syringe
5
ACCEPTED MANUSCRIPT
and needle. The fibers formed were allowed to stay in the coagulation bath for 20 min and later rinsed with
112
water and air-dried. Then the spun fiber was drawn and annealed. The effects of microwave power (five
113
different levels: 100,200,300,400, and 500 W), exposure time (five different times: 1, 2, 3, 4, and 5 min), and
114
pH (four different pH: 5, 6, 7, and 8) on the breaking strength and breaking elongation of the fiber were
115
examined.
116
2.4. Tensile Testing
SC
RI PT
111
All of the fiber samples were conditioned before testing in a standard testing atmosphere of 21°C and 65%
118
relative humidity for 24 h. Fibers were tested for their tensile properties using a texture analyzer (TA). A gauge
119
length of 40 mm and an extension speed of 2 mm/min were used for tensile testing. Approximately 15 fibers
120
were tested for each condition and the average values were obtained.
M AN U
117
To determine the optimal conditions for microwave technology, we chose microwave power, time, and pH as
122
single factors. Based on this experiment, we chose the breaking strength and elongation at break as the
123
evaluation index and determined the response surface experiment with these three factors at three levels.
124
2.5. Determination of -SH and -S-S- content in the process of wheat protein fiber preparation
EP
TE D
121
The concentration of free sulfhydryl groups (-SH) of the wheat protein fiber was determined using Ellman's
126
reagent (5'5-dithiobis(2-nitrobenzoic acid), DTNB). Changes in free sulfhydryl groups were measured in
127
triplicate as previously reported(Beveridge, et al, 1974). Briefly, finely grinded wheat protein fiber (42 mg) was
128
diluted to 10 mL with 4.7 g guanidine hydrochloride in Tris-glycine buffer (1.04% Tris, 0.69% glycine, 0.12%
129
EDTA (w/v), pH 8.0) and reacted for 1 h at 25°C. A solution of 1 mL of the solution, 4 mL 8 M urea solution
130
(dissolved with Tris-glycine buffer), and 0.1 mL Ellman's reagent was prepared and mixed in a 25°C‐water bath
131
for 30 min to avoid a light reaction. The solution was then subjected to centrifugation at 4500 rpm for 15 min,
AC C
125
6
ACCEPTED MANUSCRIPT
132
and the absorbance of the supernatant was measured at 412 nm using a UV-vis spectrophotometer. The concentration of total sulfhydryl groups (SH) in the wheat protein fiber was measured as follows: 1 mL
134
sample solution, 0.05 mL mercaptoethanol, 4 mL 8 M urea-5 M guanidine hydrochloride (dissolved with
135
Tris-glycine buffer) were mixed together in a 25°C‐water bath for 1 h. Then, 10 mL of 12% TCA was added
136
and the mixture continued to stay in a 25°C‐water bath for an additional 1 h. The solution was then centrifuged
137
at 4500 rpm for 15 min. The precipitate was then washed with 5 mL 12% TCA twice. The precipitate was
138
mixed with 10 mL 8 M urea solution and 0.04 mL Ellman's reagent in a 25°C water bath for 30 min to avoid a
139
light reaction. The absorbance was then measured at 412 nm using a UV-spectrophotometer. The concentration
140
of free –SH was calculated using the following equation:
141
-SH (µM/g) = 73.53 A412 × D/C, where A412 is the absorbance at 412 nm; C is wheat protein fiber concentration
142
(mg/mL) and D is the dilution factor. -S-S- content was calculated using the following formula:
143
-S-S-(µM/g) = (N2-N1)/2, where N1 is the content of sulfhydryl groups before reduction and N2 is the content of
144
sulfhydryl groups after reduction.
145
2.6. SDS-PAGE
EP
TE D
M AN U
SC
RI PT
133
Approximately 7 mg of microwaved and non-microwaved wheat gluten fibers were powdered and mixed
147
with 1 mL of reduced and non-reduced SDS-PAGE 2X sample buffer (100 mmol/L Tris-HCl (pH 6.8), 6%
148
AC C
146
m/V SDS, 0.2%
m/V Bromophenol Blue, 20%
V/V Glycerol, 300 mmol/L 2-ME or not), and left
149
standing at room temperature for 2 h. Samples were subsequently boiled for 5 min in boiling water and
150
centrifuged at 10000 rpm for 5 min. The clear top layer of each sample (10 μL) was loaded into each slot in
151
the gel. After electrophoresis, the gel was stained with Coomassie Brilliant blue G-250. After standing for one
152
night, the gel was flushed with deionized water and placed in a destaining liquid until a clear background was
7
ACCEPTED MANUSCRIPT
153
formed.
154
2.7. Secondary structure study Secondary structures of fibers were studied by Fourier transform infrared spectroscopy (FTIR). The
156
background spectrum of the ATR cell was recorded at 32 scans and a resolution of 4 cm-1. The spectra were
157
recorded under the same conditions as the background and the scanning range was 400-4000 cm-1. The spectra
158
were analyzed using Omnic and Peakfit software.
159
2.8. X-ray diffraction
160
The fiber was sheared and grinded finely. The measurements were taken using an X-ray diffraction analyzer
161
(Bruker Germany AXS Co. Ltd.) under the following conditions: a Ni-filtered Cu-Kα line was used as the X-ray
162
source, voltage of 40 kV, current of 30 mA, 2°/min in scanning speed, scanning the specimen in 2-45°.
163
2.9. Thermal performance
M AN U
SC
RI PT
155
The fibers were grinded to powders. Thermogravimetric analysis (TGA) of fiber samples (1–2 mg) was
165
carried out using a TGA/SDTA 851e T.A. instrument at 10°C/min from room temperature to 550°C under
166
nitrogen atmosphere using a flow rate of 50 mL/min. Differential scanning calorimetry (DSC) analysis of fiber
167
samples (1–2 mg) was carried out using a DSC-Q200 T.A. instrument at 10°C/min from 40 to 250°C under
168
nitrogen atmosphere using a flow rate of 50 mL/min. Before DSC analysis, the sample was mixed with distilled
169
water in a 1:4 ratio and kept balanced at 4°C in a refrigerator for 24 h.
170
2.10. SEM
171
AC C
EP
TE D
164
The morphological features, in terms of the longitudinal and cross-sectional appearance of the wheat gluten
172
fibers, were observed using a Hitachi model S-4800 scanning electron microscope.
173
2.11. Surface hydrophobicity
8
ACCEPTED MANUSCRIPT
The anionic fluorescent probe 8- anilinonaphthalene-1-sulfonic acid (ANSA) was used to measure the
175
exposed surface hydrophobicity of wheat protein and its fibers. The fibers were grinded and passed through a
176
200-mesh sieve. Protein samples were diluted to a final concentration of 0.02, 0.04, 0.08, or 0.16 mg/mL using
177
0.01 M sodium phosphate buffer at pH 7.0. An ANSA stock solution (8 mmol) was prepared in 10 mM sodium
178
phosphate buffer at pH 7.0. Aliquots of ANSA stock solution (10 µL) were titrated into 2 mL protein solutions
179
and a F-7000 fluorescence spectrometer at an excitation wavelength of 390 nm (slit 5 nm) and an emission
180
wavelength of 470 nm was used to measure the fluorescence intensity of the samples. The curve was based on
181
the protein concentration and the fluorescence intensity. The initial stage of the slope of the curve is the surface
182
hydrophobic activity of the protein samples.
183
2.12. Standard moisture regain
SC
M AN U
184
RI PT
174
The moisture regain of the fibers was determined according to the American Society for Testing and Materials (ASTM) method 2654 under standard atmospheric conditions of 65% relative humidity and 21°C.
186
3. Results and Discussion
187
3.1. Microwave Process Optimization
EP
TE D
185
The effects of different process variables including microwave power (100-500 W), time (1-5 min), and pH
189
(5-8) on the breaking strength and elongation at break of fibers were investigated using single factor
190
experiments.
AC C
188
191
As a protein modification method, microwave-modified wheat protein is made of fibers. The result shows
192
that as the microwave power increased, the breaking strength and the elongation at break initially increased and
193
then decreased, and both factors peaked at the microwave power of 300 W. Similar findings were observed in
194
the time analysis, and both the breaking strength and the elongation at break peaked at the microwave time of 2
9
ACCEPTED MANUSCRIPT
min. This may be because protein chains become unfolded with increasing power and time and therefore lower
196
power and shorter times are preferred. Previous studies have demonstrated that microwave treatment can
197
promote protein unfolding (Woerdeman & Veraverbeke, 2004), and the unfolded protein maybe more suitable
198
for spinning. As the microwave power and time increased, the protein chain crossed to a certain extent (Qasem,
199
2006) and caused an increase in the mechanical properties of the wheat protein fiber. However, further
200
increases in microwave power and time resulted in excessive crosslinking of the protein chains, and a decrease
201
in the mechanical properties.
SC
RI PT
195
Differences in pH can have varying effects on proteins. The result shows that breaking strength and the
203
elongation at break initially increased and then decreased with increasing pH, and both factors peaked at a pH
204
of 7. One possible reason for this observation could be that at lower pH (acidic atmosphere), proteins are easily
205
hydrolyzed, such that the protein chains becomes short, leading to poor mechanical properties of the spun fiber.
206
However, in neutral and weakly alkaline environments, protein chains remain in their original high molecular
207
state, thus leading to better mechanical properties of the spun fiber.
TE D
M AN U
202
Using Designexpert7.0 software, an analysis of variance test was done to generate a regression model of
209
breaking strength and elongation at break. The order of factors' effect on breaking strength is microwave time >
210
pH >microwave power. The order of factors' effect on elongation at break is pH> microwave time > microwave
211
power. Considering the two response values, the best process condition that the Designexpert7.0 software
212
showed is microwave power 20.6 W/mL, microwave time 3 min, pH 8. Preparing the fiber under this condition,
213
the fiber's breaking strength is 0.69 cN/dtex, elongation at break is 23.37%. Compared with the
214
non-microwaved fibers, the breaking strength increased by 19% and the elongation increased by 302.43%. The
215
obvious increase of elongation indicated the toughness of fiber was increased.
AC C
EP
208
10
ACCEPTED MANUSCRIPT
216
3.2. Determination of -SH and -S-S- content in the process of wheat protein fiber preparation The effect of microwave on free sulfhydryl groups and disulfide bonds during the preparation of fibers is
218
shown in Table 1. The results demonstrate that microwave treatment can promote disulfide bond rupture as
219
indicated in the comparison between fibers that were microwaved (treatment 4) and non-microwaved fibers
220
(treatment 1). This may be because the microwave through the electromagnetic field can cause material internal
221
friction between material atoms, and the heat of this friction can enhance the effect of urea and sodium sulfite.
222
The results also clearly indicate that drawing can increase the content of free sulfhydryl groups and decrease
223
the content of disulfide bond, as observed in the comparison between non-microwaved fibers that were drawn
224
(treatment 2) or not (treatment 1). This indicates that drawing can cause disulfide bond rupture(Li & Jinbo.
225
2010) and microwaved fibers show a similar result (comparison of treatments 4 and 5). Annealing was shown
226
to increase the content of disulfide bonds and decrease the content of free sulfhydryl groups, as seen in
227
non-microwaved fibers with or without annealing (treatments 2 and 3). This indicates that annealing can
228
promote free sulfhydryl groups into -S-S-( Cuq, et al, 1998) to fix the fiber. The final disulfide bond content of
229
microwaved fibers is higher than non-microwaved fibers (treatments 3 and 6). This could be due to a
230
microwave treatment induced change in the conformation of proteins as well as the location of –SH, therefore
231
making it easier to promote –S-S- formation. This higher content of -S-S- may be the reason for the better
232
mechanical properties of microwaved fibers.
233
3.3. SDS-PAGE
234
The SDS-PAGE of sample is showed in Fig.1. A comparison of the reduced and non-reduced SDS-PAGE
AC C
EP
TE D
M AN U
SC
RI PT
217
235
gels (A and B respectively) show a marked reduction in bands and band intensity in the non-reduced gel,
236
indicating that –S-S- plays an important role in the production of fibers. The observed decreases in band
11
ACCEPTED MANUSCRIPT
expression with microwave treatment compared to non-microwaved fibers (4 and 7 respectively in the
238
SDS-PAGE analysis) indicate that microwave forces the occurrence of covalent crosslinking in addition to the
239
disulfide bond. An increase in the intensity of the low molecular weight band (C) is observed in the comparison
240
of non-microwaved and microwaved fibers, with or without drawing, (5 with 4 or 8 with 7 respectively),
241
indicating that drawing can lead to a decrease in molecular weight, which may be caused by–S-S- rupture. In
242
microwaved fibers with or without annealing (9 with 8, respectively), the intensity of bands is increased. This
243
indicates that heating increases the content of –S-S-(Waqner M.et al,2011), which corresponds with the
244
findings of increased –S-S- content with annealing shown in Table 1. Therefore, non-covalent crosslinking and
245
indirect formation of disulfide bonds may play an important role in improving the mechanical properties of
246
microwave fiber.
247
3.4. Secondary structure study
M AN U
SC
RI PT
237
Fourier transform infrared spectroscopy (FTIR) is widely used for the analysis of the secondary structure of
249
proteins. Characteristic bands in the FTIR spectra of proteins mainly include amide I (1600-1700 cm-1) and
250
amide II (1500-1600 cm-1). Although amide I and amide II bands are related to the secondary structure of
251
proteins, the amide II band is not as good a predictor for quantitation of the secondary structure of
252
proteins(Kumosinski & Farrell, 1993) . Amide I was baseline corrected using Omnic software, smoothed with a
253
nine-point Savitsky-Golay function(Savitzky & Golay, 1964) to remove any possible white noise, and then
254
Savitsky-Golay 5 points were used for the second order derivative to determine whether a different secondary
255
structure corresponds to the position of the peak. The Peakfit software was then used to fit the Gauss function,
256
according to a previous report(Krimm & Bandekar,1986) in order to determine the position of the peak. There
257
are many examples using FTIR to analysis the secondary structure of proteins. For example, Sivam (A.S.
AC C
EP
TE D
248
12
ACCEPTED MANUSCRIPT
Sivam, et al, 2013) used FTIR to study the changes of conformation of bread after adding pectin and
259
polyphenols. Michael Byler (D. Michael Byler & Heino Susi)examined the secondary structure of proteins by
260
deconvolved FTIR spectra. The content of the secondary structure of wheat gluten fiber was determined based
261
on the fitting curve and is listed in Table 2.
RI PT
258
The results show that microwave treatment (treatments 1 (non-microwaved) and 4 (microwaved), Table 2)
263
can increase the random coil and α-helix content of wheat protein, which could be caused by disulfide bond
264
rupture. We also find that drawing can increase random coil and decrease α-helix, which may be caused by
265
disulfide bond rupture during drawing. Annealing caused the disappearance of random coil and increased the
266
α-helix and β-sheets content, regardless of microwave treatment. The phenomenon is however more
267
profound in microwave treated fiber, which may be the reason that the structure of microwave treated fibers‐are
268
generally more disordered and easier to change. The conformation changes in fiber may be caused by changes
269
in free sulfhydryl groups and disulfide bond content.
270
3.5. X-ray diffraction
TE D
M AN U
SC
262
Crystallinity can be used to reflect the protein structure of the fiber. The crystallinity of the fiber increases
272
with increasing molecular order of the protein. Fig. 2 shows the effects of microwave on the x-ray diffraction
273
(XRD) spectra of wheat protein fiber. The most prominent diffracting peaks are seen at about 9° and 19.5°,
274
which are the characteristic crystalline peaks of wheat gluten fiber(Reddy & Yang, 2010). There is almost no
275
difference at 9°, while the full width at half maximum (FWHM) of microwaved fibers is 9.67, which is smaller
276
than fibers without microwave treatment. Since the degree of crystallinity under the same condition is worse,
277
diffraction peaks will be broader. Therefore, microwaved fibers have a higher degree of crystallinity compared
278
with non-microwaved fibers. Furthermore, microwaved fibers add a crystalline peak at 2 θ at 32°, which further
AC C
EP
271
13
ACCEPTED MANUSCRIPT
279
increases the degree of crystallinity. Proteins with higher crystallinity require higher power to destroy the
280
ordered structure, and thus increase the mechanical properties of the fiber.
281
3.6. Thermal performance In order to investigate the effect of microwave treatment on the thermal properties of wheat gluten fiber,
283
DSC and TGA analysis were performed. Thermal profiles from DSC and TGA of wheat gluten fiber are shown
284
in Fig.3. DSC is an important method to study the thermal stability of proteins. Large Td values corresponded
285
with higher thermal stability. ∆H is the area under the endothermic peak, which is the energy required to
286
completely denature the wheat protein. The results demonstrate that the Td of microwaved fibers are higher than
287
that of non-microwaved fibers, which indicate that microwave treatment can improve the thermal stability of
288
fibers. The denaturation enthalpy of microwaved fiber is lower than that of non-microwaved fiber. This may be
289
due to protein crosslinking after microwave treatment. Protein crosslinking causes the unfolding of protein
290
chains and exposes hydrophobic residues, leading to the increase of surface hydrophobicity(Rani J.
291
Qasem,2006).TG curves show two stages of well-defined mass loss. The first stage was attributed to the
292
removal of water over the temperature interval of 30–160°C. This stage is associated with the process of
293
protein denaturation(Li-Chan & Ma, 2002). The second stage corresponds to the thermal decomposition of the
294
protein fraction after 200°C. The results clearly show that thermal decomposition of microwaved fibers is
295
higher than non-microwaved fibers, which indicate higher thermal stability. This conclusion was also consistent
296
with the findings of the DSC analysis.
297
3.7. Scanning Electron Microscopy (SEM)
298 299
AC C
EP
TE D
M AN U
SC
RI PT
282
In order to study the effect of microwave on wheat protein fibers, scanning electron microscopy (SEM) was used to observe the longitudinal appearance, as shown in Fig. 4.
14
ACCEPTED MANUSCRIPT
From the longitudinal appearance, the comparison of non-microwaved fiber with microwaved fiber (a with b,
301
respectively) shows that the surface of wheat protein fiber becomes smoother and denser with microwave
302
treatment. Enlarged images reveal granular materials on the surface. These granular materials may be coagulant
303
crystalline particles, urea crystallization, or residual starch granules. Comparison of 5K times enlarged images
304
(e with f) shows that wheat protein has better orientation after microwave treatment and the expression of the
305
fibrous protein is more obvious in microwaved fibers. The higher orientation of molecular protein may be
306
another reason for the improved mechanical properties with microwave treatment. SEM was also used to
307
observe the cross-section of wheat gluten fiber.
M AN U
SC
RI PT
300
The cross-section of non-microwaved fibers is almost circular, whereas that of microwaved fibers are flat
309
and dumbbell shaped. Furthermore, non-microwaved fibers are less dense than microwaved fibers, as observed
310
from the comparison of images c and d. Additionally, a greater number of pores were observed in the
311
non-microwaved fiber compared with the microwave treated fiber(e and f). The denser structure of microwaved
312
fiber observed may be another reason for the better mechanical properties observed with microwaved
313
treatment.
314
3.8. Surface hydrophobicity and standard moisture regain.
EP
TE D
308
The surface hydrophobicity of microwaved fiber and non-microwaved fiber was 852.5±5.8 and 660.71±9.6,
316
respectively. The surface hydrophobicity of microwaved fibers is higher than that of non- microwaved fibers.
317
This may be the result of protein denaturation caused by microwave treatment, leading to unfolding of the
318
protein chain and exposure of hydrophobic residues, thus ultimately increasing the surface hydrophobicity. The
319
standard moisture regain of microwaved fiber and non-microwaved fiber is 11.11% and 10.20%, respectively.
320
The higher surface hydrophobicity of the microwaved fiber could affect its ability to absorb water, thus
AC C
315
15
ACCEPTED MANUSCRIPT
resulting in the observed decrease in standard moisture regain. Regardless of microwave treatment, the standard
322
moisture regain of wheat protein fibers is close to the standard moisture regain of silk, indicating that the wheat
323
protein fiber has good hygroscopicity.
324
4. Conclusions
RI PT
321
In the present study, microwave-modified wheat protein fiber was produced to improve the mechanical
326
properties as well as shortened the production cycle. The best process conditions obtained from our response
327
surface analyses are a microwave power of 20.6 W/mL, microwave time of 3 min, and pH 8. Preparing the
328
fiber under these conditions results in a fiber breaking strength of 0.69 cN/dtex and an elongation at break of
329
23.37%. Compared with the non-microwaved fibers, the breaking strength increased by 19% and the elongation
330
increased by 302.43%. And microwave did play a role in the improvement of mechanical properties. To study
331
the mechanism of microwave treatment on the improvement of mechanical properties, -SH and -S-S- content in
332
the process of wheat protein fiber preparation, secondary structure study, X-ray diffraction, thermal
333
performance, SEM, surface hydrophobicity, and standard moisture regain were examined . The results show
334
that microwaved fiber had a higher content of –S-S-, α-helix, crystallinity, which may be responsible for the
335
better mechanical properties and increased thermal stability. Additionally, SEM micrographs showed that
336
microwaved fiber also had a smoother and denser structure as well as less pores. Although the surface
337
hydrophobicity and standard moisture regain were decreased, microwave fiber had good hygroscopicity, which
338
was close to that of silk.
339
Acknowledgments
AC C
EP
TE D
M AN U
SC
325
340
We thank school of textile and garment, Jiangnan University for providing us with equipment support. We
341
are also grateful for the guidance of teachers at physical platform. With their help, our experiment can run
16
ACCEPTED MANUSCRIPT
342
smoothly. At last, we also thank for the help of our fellow classmates.
343 References
345
Shewry P. R., Tatham A. S.(1997). Disulphide bonds in wheat gluten proteins. Journal of Cereal Science, 25(3),
350 351 352 353 354 355 356 357 358
SC
Day, L., Augustin, M. A., Batey, I. L., & Wrigley, C. W. (2006). Wheat-gluten uses and industry needs. Trends
M AN U
349
to breadmaking functionality. Critical reviews in food science and nutrition, 42(3), 179-208.
in Food Science & Technology, 17(2), 82-90.
Lens J. P., Mulder W. J., Kolster P.(1999). Modification of wheat gluten for nonfood applications. Cereal Foods World, 44, 5-9
Ye, P., Reitz, L., Horan, C., & Parnas, R. (2006). Manufacture and biodegradation of wheat gluten/basalt
TE D
348
Veraverbeke W. S., Delcour J. A.(2002). Wheat protein composition and properties of wheat glutenin in relation
composite material. Journal of Polymers and the Environment, 14(1), 1-7. Woerdeman, D. L., Veraverbeke, W. S., Parnas, R. S., Johnson, D., Delcour, J. A., Verpoest, I., & Plummer, C. J. (2004). Designing new materials from wheat protein. Biomacromolecules, 5(4), 1262-1269.
EP
347
207-227.
Bietz, J. A., & Lookhart, G. L. (1996). Properties and non-food potential of gluten. Cereal Foods World, 41(5),
AC C
346
RI PT
344
376-382.
359
Krull, L. H., & Inglett, G. E. (1971). Industrial uses of gluten. Cereal science today, 16, 232-261.
360
Reddy, N., & Yang, Y. (2007). Novel protein fibers from wheat gluten. Biomacromolecules, 8(2), 638-643.
361
Moncrieff, R. W. (1975). Man-made fibres. London : Newnes-Butterworths.
362
Yang, Y., Wang, L., Li, S. (1996).,Formaldehyde-free zein fiber preparation and investigation. Journal of
17
ACCEPTED MANUSCRIPT
364 365
Applied Polymer Science,59 (3), 433- 441. Farrow, B.(1956). Extensometric and Elastic Properties of Textile. Fibres. Journal of Textile Institute, 47, No. 2, T58-T101.
RI PT
363
Chiou, B. S., Jafri, H., Cao, T., Robertson, G. H., Gregorski, K. S., Imam, S. H., Greg M.& Orts, W. J. (2013).
367
Modification of wheat gluten with citric acid to produce superabsorbent materials. Journal of Applied
368
Polymer Science, 129(6), 3192-3197.
371 372 373 374
transglutaminase (TGase) regeneration gluten protein fiber. Journal of functional materials ,1(44),39-46.
M AN U
370
Jun, M.; Li, C.; Xuerong, F.; Ping, W.; Qiang, W.(2013). Study based on the preparation and properties of
Banik, S., Bandyopadhyay, S., & Ganguly, S. (2003). Bioeffects of microwave-a brief review. Bioresource technology, 87(2), 155-159.
Thostenson, E. T., & Chou, T. W. (1999). Microwave processing: fundamentals and applications. Composites Part A: Applied Science and Manufacturing, 30(9), 1055-1071.
TE D
369
SC
366
Byaruhanga, Y. B., Emmambux, M. N., Belton, P. S., Wellner, N., Ng, K. G., & Taylor, J. R. N. (2006).
376
Alteration of kafirin and kafirin film structure by heating with microwave energy and tannin complexation.
377
Journal of agricultural and food chemistry, 54(12), 4198-4207.
379 380 381 382 383
Larhed M, Hallberg A.(2001) .Microwave-assisted high speed chemistry: a new technique in drug discovery.
AC C
378
EP
375
Drug Discovery Today. 6:406-416. Beveridge, T., Toma, S. J., & Nakai, S. (1974). Determination of SH- and –S-S- groups in some food proteins using ELLMAN'S reagent. Journal of Food Science, 39(1), 49-51. Qasem, R. J. (2006). The effect of microwave thermal denaturation on release properties of bovine serum albumin and gluten matrices. AAPS PharmSciTech, 7(1), E104-E110.
18
ACCEPTED MANUSCRIPT
386 387 388 389
Journal of Tianjin Polytechnic University,29(2):19-24,44. Cuq, B., Gontard, N., Cuq, J. L., & Guilbert, S. (1998). Packaging films based on myofibrillar proteins:
RI PT
385
Li W., Jinbo Y.(2010). Fourier transform spectroscopy analysis of the secondary structure of stretch wool.
Fabrication, properties and applications. Food/Nahrung, 42(03 04), 260-263.
Waqner M., Morel MH, Bonicel J., Cuq B.(2011).Mechanisms of heat-mediated aggregation of wheat gluten protein upon pasta processing. Journal of Agricultural and Food Chemistry,59(7):3146-54.
SC
384
Kumosinski, T. F., & Farrell, H. M. (1993). Determination of the global secondary structure of proteins by
391
Fourier transform infrared (FTIR) spectroscopy. Trends in Food Science & Technology, 4(6), 169-175.
392
Savitzky, A., & Golay, M. J. (1964). Smoothing and differentiation of data by simplified least squares
396 397 398 399 400 401
proteins. Advances in protein chemistry, 38, 181-364.
TE D
395
Krimm, S., & Bandekar, J. (1986). Vibrational spectroscopy and conformation of peptides, polypeptides, and
Reddy, N., & Yang, Y. (2010). Developing water stable gliadin films without using crosslinking agents. Journal of Polymers and the Environment, 18(3), 277-283.
EP
394
procedures. Analytical chemistry, 36(8), 1627-1639.
Rani J. Qasem(2006). The Effect of Microwave Thermal Denaturation on Release Properties of Bovine Serum Albumin and Gluten Matrices, Aaps Pharmscitech, 7 (1):E1-E7
AC C
393
M AN U
390
Li-Chan, E. C. Y., & Ma, C. Y. (2002). Thermal analysis of flaxseed (Linum usitatissimum) proteins by differential scanning calorimetry. Food Chemistry, 77(4), 495-502.
402
A.S. Sivam, D. Sun-Waterhouse, C.O. Perera, G.I.N. Waterhouse(2013). Application of FT-IR and Raman
403
spectroscopy for the study of biopolymers in breads fortified with fibre and polyphenols, Food Research
404
International,50, 574-585.
19
ACCEPTED MANUSCRIPT
405 406
D. MICHAEL BYLER and HEINO SUSI(1986). Examination of the Secondary Structure of Proteins by Deconvolved FTIR Spectra, Biopolymers, 25, 469-487.
RI PT
407 408 409
SC
410 411
M AN U
412 413 414 415
419 420 421 422
EP
418
AC C
417
TE D
416
423 424 425
20
ACCEPTED MANUSCRIPT
426
Tables
427
Table 1. Effect of microwave on free sulfhydryl groups and disulfide bond of WG fibers Treatmenta-f
Content of free -SH µmol/g
2
17.08±0.09
3
13.33±0.09
4
13.83±0.1
5
18.68±0.05
6
10.17±0.11
8.61±0.12
RI PT
12.84±0.07
7.73±0.26 8.83±0.21
8.03±0.18 6.08±0.04
SC
1
Content of -S-S- µmol/g
10.27±0.15
a
1. fibers without microwave, drawing or annealing; b2. fibers without microwave and annealing but with drawing; c3. fibers
429
without microwave but with annealing and drawing; d4. microwaved fiber without drawing or annealing; e5. microwaved fiber
430
drawing but not annealing; f6. microwaved fiber drawing and annealing
431
Table 2. Secondary structure of wheat protein fiber with different treatments Treatment
M AN U
428
a-f
3 4 5 6 a
β-turns(%)
34.48±0.57
6.90±0.10
24.45±0.32
29.78±0.37
31.51±0.32
14.15±0.24
18.26±0.20
33.33±0.35
33.49±0.41
-
22.52±0.19
33.73±0.56
28.68±0.18
17.45±0.09
33.16±0.25
18.20±0.30
17.46±0.38
36.56±0.35
4.96±0.08
44.44±0.44
37.83±0.52
-
39.91±0.47
22.77±0.65
1. fibers without microwave, drawing or annealing; b2. fibers without microwave and annealing but with drawing; c3. fibers
AC C
432
α-helix(%)
EP
2
random coil(%)
TE D
1
β-sheets(%)
433
without microwave but with annealing and drawing; d4. microwaved fiber without drawing or annealing; e5. microwaved fiber
434
drawing but not annealing; f6. microwaved fiber drawing and annealing
435
Figure captions
436
Fig. 1. SDS-PAGE of the wheat protein and fibers
437
A is the reduced SDS-PAGE; B is the non-reduced SDS-PAGE. M is standard protein.1-9 and a-i represent the
438
following: 1, a) wheat gluten; 2, b) glutenin; 3, c) gliadin; 4, d) fibers without microwave, drawing or annealing; 21
ACCEPTED MANUSCRIPT
5, e) fibers without microwave and annealing but with drawing; 6, f) fibers without microwave but with
440
annealing and drawing; 7, g) microwaved fiber without drawing or annealing; 8, h) microwaved fiber with
441
drawing but not annealing; 9, i) microwaved fiber with drawing and annealing
442
Fig. 2. Effects of microwave on the XRD spectra of wheat protein fiber (1- fibers without microwave treatment,
443
2- microwaved fibers)
444
Fig.3. DSC and TG-DTG curve of wheat protein fiber
445
Fig. 4. SEM micrographs of the longitudinal appearance and cross-section of wheat protein fibers.
446
a, c, e are images of non-microwaved fiber enlarged 1K, 2K, and 5K times. b, d, f are images of microwave
447
treated fiber enlarged 1K, 2K, and 5K times.
448
Figure
449
Fig.1
451 452 453
AC C
450
EP
TE D
M AN U
SC
RI PT
439
C
A
B
454 455 456 22
ACCEPTED MANUSCRIPT
Fig.2
SC
RI PT
457
Fig.3
Non-microwaved fiber Microwaved fiber
-30 -80 -130 -180 -230 0
TE D
Heat Flow W/g
20
50
100
150
200
250
300
Temperature
EP
460 461
5.00E-04 0.00E+00 -5.00E-04 -1.00E-03 -1.50E-03 -2.00E-03 -2.50E-03 -3.00E-03
weight loss (%)
AC C
120 100 80 60 40 20 0
0
100
200
300
400
500
temperature (°C)
462 463 464 23
600
Derived weight (%/°C)
459
M AN U
458
Non-microwaved fibers Microwaved fibers
ACCEPTED MANUSCRIPT
Fig.4
RI PT
465
b
M AN U
SC
a
d
AC C
EP
TE D
c
e
466
f
467 468 469 24
RI PT
ACCEPTED MANUSCRIPT
b
M AN U
SC
a
d
470 471
AC C
EP
TE D
c
e
f
472 473
25
ACCEPTED MANUSCRIPT Highlights The breaking strength and elongation were increased by 19% and 302.43% respectively
Microwaved fiber has higher content of –S-S-, α-helices and crystallinity.
The treatment increases the thermal stability of wheat protein fibers.
The treated fiber has a smoother and denser surface and less pores.
The fiber has a good standard moisture regain and increased surface hydrophobicity.
AC C
EP
TE D
M AN U
SC
RI PT