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Effects of rice bran rancidity on the oxidation and structural characteristics of rice bran protein
Journal Pre-proof Effects of rice bran rancidity on the oxidation and structural characteristics of rice bran protein Xiaojuan Wu, Fang Li, Wei Wu PII:
S0023-6438(19)31285-X
DOI:
https://doi.org/10.1016/j.lwt.2019.108943
Reference:
YFSTL 108943
To appear in:
LWT - Food Science and Technology
Received Date: 3 October 2019 Revised Date:
8 December 2019
Accepted Date: 10 December 2019
Please cite this article as: Wu, X., Li, F., Wu, W., Effects of rice bran rancidity on the oxidation and structural characteristics of rice bran protein, LWT - Food Science and Technology (2020), doi: https:// doi.org/10.1016/j.lwt.2019.108943. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
Effects of rice bran rancidity on the oxidation and structural characteristics of rice bran protein Xiaojuan Wu#, Fang Li#, & Wei Wu* National Engineering Laboratory for Rice and By-product Deep Processing, College of Food Science and Engineering, Central South University of Forestry and Technology, Changsha, Hunan 410004, China *Corresponding author Tel.: +86-731-85658893; Fax: +86-731-85658893. E-mail address: [email protected] (Wei Wu) #These
authors contributed to the work equally and should be regarded as
co-first authors.
↑ Acid value Hydrolytic rancidity
↑ Carbonyl ↑ Dityrosine
Rice bran protein oxidation
Rice bran storage
Oxidative rancidity
↑ Peroxide ↑ TBA
Protein aggregation
↓ Sulfhydryl -SH → –S-S Protein cross-link
1
Effects of rice bran rancidity on the oxidation and structural characteristics of
2
rice bran protein
3
Xiaojuan Wu#, Fang Li#, & Wei Wu*
4
National Engineering Laboratory for Rice and By-product Deep Processing, College
5
of Food Science and Engineering, Central South University of Forestry and
6
Technology, Changsha, Hunan 410004, China
7
*Corresponding author Tel.: +86-731-85658893; Fax: +86-731-85658893.
8
E-mail address: [email protected] (Wei Wu)
9
#
10
These authors contributed to the work equally and should be regarded as co-first
authors.
11
Page 1 of 23
12
Abstract
13
Rice bran rancidity may affect rice bran protein through protein oxidation. However,
14
little is known about the relationship between rice bran rancidity and rice bran protein
15
oxidation. The effects of rice bran rancidity on the oxidation extent and structural
16
characteristics of rice bran protein were investigated. As storage time of rice bran
17
increased, the acid value, peroxide value, and value of thiobarbituric acid reactive
18
substances in crude rice bran oil increased from 4.31 mg KOH/g, 2.84 Meq/kg, and
19
6.22 µg MDA/g to 38.72 mg KOH/g, 15.58 Meq/kg, and 28.99 µg MDA/g,
20
respectively, which indicated that hydrolytic rancidity and oxidative rancidity of rice
21
bran occurred simultaneously. The gradual increase in protein carbonyl and dityrosine
22
content from 2.12 nmol/mg and 88.61 A.U. to 13.8 nmol/mg and 159.37 A.U. was
23
accompanied by a steady decrease in free sulfhydryl content of rice bran protein from
24
22.6 to 9.6 nmol/mg, which implied that the products of rice bran rancidity induced
25
rice bran protein oxidation. The rice bran protein oxidation subsequently resulted in a
26
loss of the ordered state of secondary structure and the formation of aggregates as
27
well as cross-link, when both disulfide bonds and non-disulfide covalent bonds
28
participated in cross-link formation.
29
Keywords: rice bran rancidity; rice bran protein; protein oxidation; aggregation;
30
cross-link
31
1. Introduction
32
Rice (Oryza sativa L.) is one of the important staple food crops for more than half
33
of the global population, and also consumed as a staple food for over 65% of the Page 2 of 23
34
population in China (Tong, Gao, Luo, Liu, & Bao, 2019). China is the largest
35
producer and consumer of rice in the world, which harvested over 200 million tons
36
and processed over 100 million tons of rice annually (International Grains Council，
37
2019), resulting in the production of at least 10 million tons of rice bran ever year. As
38
a major by-product of rice milling, rice bran is a rich source of oil, protein, fiber, and
39
other functional compounds, which can be used as an important functional food
40
ingredient (Sharif, Butt, Anjum, & Khan, 2014). Despite its excellent nutritional
41
benefits and outstanding application prospects, rice bran is underutilized. At present,
42
the majority of rice bran is used to produce cattle feed or non-edible oil, and only a
43
small percentage of rice bran is processed into edible oil (Burlando & Cornara, 2014).
44
Comprehensive utilization of rice bran is severely restricted due to the rapid
45
rancidity that starts with the rice milling process (Sharif et al., 2014). A large number
46
of unsaturated lipids and the presence of potent hydrolytic and oxidative enzymes
47
such as lipase and lipoxygenase result in drastic quality deterioration of rice bran
48
(Chen, Bergman, & McClung, 2019). The active endogenous lipase can hydrolyze the
49
triglycerides of rice bran into glycerol and free fatty acids, resulting in hydrolytic
50
rancidity which leads to an increase in acidity and the formation of off-flavor (Chen et
51
al., 2019). Free fatty acids are vulnerable to the endogenous lipoxygenase of rice bran
52
in the presence of oxygen, and the primary products of fatty acids hydroperoxides can
53
be further decomposed into off-flavor volatile compounds and free radicals,
54
facilitating oxidative rancidity, and further inducing the formation of off-flavor along
55
with the potential harmfulness to human health (Rodchuajeen, Niamnuy, Charunuch, Page 3 of 23
56
Soponronnarit, & Devahastin, 2016). Therefore, the fresh rice bran must be stabilized
57
immediately after the rice milling to inhibit rancidity, and various stabilization
58
methods of rice bran have been investigated in recent years (Liu, Strappe, Zhou, &
59
Blanchard, 2019). However, lipolytic activity in rice bran begins as soon as bran
60
layers are removed from endosperm during the rice milling process. Besides, owing to
61
the distance and transportation between rice milling plant and rice bran oil processing
62
factory, stabilization of rice bran soon after the milling process is not practical for the
63
commercial scale, which implies that a certain degree of hydrolytic rancidity and
64
oxidative rancidity has already occurred before stabilization of rice bran (Thanonkaew,
65
Wongyai, McClements, & Decker, 2012).
66
Reducing the negative effects of hydrolytic rancidity and oxidative rancidity on the
67
quality of rice bran derived products is mainly reflected in the deacidification and
68
deodorization methods of rice bran oil. As a major by-product of rice bran oil,
69
defatted rice bran contains about 18% protein. Rice bran protein has unique nutritional
70
and hypoallergenic properties, which can be utilized as a suitable ingredient for
71
nutraceutical and functional food formulation (Fabian & Ju, 2011). However, rice
72
bran protein is not commercially useful because of its unpleasant characteristic odor
73
(Arsa, Theerakulkait, & Cadwallader, 2019). As a primary product of rice bran
74
oxidative rancidity, fatty acids hydroperoxides are also strong prooxidants due to their
75
ability to decompose into low molecular weight aldehydes, ketones, and alkoxy
76
radicals (Bui, Hsu, & Hankinson, 2009). The active aldehydes, ketones and alkoxy
77
radicals can trigger protein oxidation in rice bran (Davies, 2016). Oxidative Page 4 of 23
78
modification of protein during food processing and storage could alter the structure,
79
functional properties, and nutritional characteristics of protein, and might pose
80
potential threat to human health, which ultimately affects the development and
81
application of protein in food industry (Estevez & Luna, 2017). However, the effects
82
of rice bran rancidity on the structure and properties of rice bran protein have not been
83
reported yet. Therefore, the purpose of this study was to identify the relationship
84
between rice bran rancidity and rice bran protein oxidation. Moreover, in order to
85
simulate the varying degrees of rancidity of most rice bran in China, the fresh rice
86
bran was stored at room temperature for different periods.
87
2. Materials and methods
88
2.1. Materials
89
Freshly milled rice bran from indica rice (Oryza sativa L., Variety Zhong Jia Zao
90
17) was supplied by Hunan Grain Group Co., Ltd. (Changsha, Hunan Province,
91
China). The total dietary fiber, crude protein, fat, ash, and moisture of rice bran were
92
282 g/kg, 148 g/kg, 168 g/kg, 75 g/kg, and 120 g/kg, respectively. Blue plus protein
93
markers with molecular weight from 14 to 100 kDa for protein electrophoresis were
94
purchased from TransGen Biotech (Beijing, China). 1-anilino-8-naphthalene-sulfonate
95
(ANS),
96
5,5'-dithiobis(2-nitrobenzoic acid), acrylamide, and N, N’-methylenebisacrylamide
97
were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals
98
were of analytical grade.
99
2.2. Preparation of defatted rice bran from rice bran with different degree of rancidity
1,1,3,3-tetramethoxypropane,
Page 5 of 23
2,
4-dinitrophenylhydrazine,
100
Freshly milled rice bran was passed through 60 meshes (size of 0.25 mm). After
101
stored at 25 °C and relative humidity of 85% for 0, 1, 3, 5, and 10 days, the rice bran
102
with different degrees of rancidity were stabilized by a twin-screw extruder (FMHE
103
36-24, FUMACH, Changsha, Hunan Province, China). The feed moisture was
104
adjusted to 16 g/100 g. The rice bran was fed into the extruder at a constant speed of
105
15 kg/h, and the screw speed was 150 rpm. The extruder barrel temperatures from the
106
first zone (feeding zone) to the fifth zone were set at 70, 120, 120, 70 and 60 °C,
107
respectively. After stabilization, the rice bran was immediately dried at 45 °C for 12 h,
108
and then smashed to pass through 80 meshes (size of 0.18 mm). The stabilized rice
109
bran powder was defatted using hexane with a ratio of 1:4 (w/v) at 25 °C for three
110
times. After vacuum filtering, the filter cake was vacuum dried at 25 °C. The dried
111
defatted rice bran was ground to pass through 80 meshes and stored at 4 °C. The
112
filtrate was evaporated to obtain crude rice bran oil and recovered hexane.
113
2.3. Preparation of rice bran protein
114
The defatted rice bran was mixed with distilled water at a ratio of 1:10 (w/v), and
115
the pH of the solution was adjusted to 9.0 with 2 mol/L NaOH. After stirring for 4 h at
116
40 °C, the suspension was centrifuged (Lynx 6000, Thermo Fisher Scientific,
117
Germany) at 8,000 g for 20 min at 4 °C to recover the supernatant. Rice bran protein
118
was precipitated by adjusting pH to 4.0 with 2 mol/L HCl and hold for 30 minutes,
119
and then centrifuged at 8,000 g for 15 min at 4 °C. After washed with distilled water,
120
the protein precipitate was resuspended in distilled water at a ratio of 1:5 (w/v), and
121
neutralized to pH 7.0 with 2 mol/L NaOH. Finally, the solution was dialyzed with Page 6 of 23
122
distilled water at 4 °C for 24 h, freeze-dried and stored at 4 °C. The wet base purity of
123
rice bran protein prepared by rice bran which was stored for 0, 1, 3, 5, 10 days was
124
74.65%, 74.38%, 73.46%, 74.55%, 74.35%, respectively, and the purity was obtained
125
by the method of micro-Kjeldahl with the nitrogen conversion factor of 5.95.
126
2.4. Determination of acid value (AV), peroxide value (PV) and thiobarbituric acid
127
reactive substances (TBARS) assay result of crude rice bran oil
128
Determination of PV and AV of crude rice bran oil were performed according to
129
the standard methods of AOCS Cd 3d-63 and AOCS Cd 8-53. TBARS assay result is
130
usually expressed as the malondialdehyde (MDA) concentration, and MDA
131
concentration was determined by reaction with 2-thiobarbituric acid reagent according
132
to the method of Papastergiadis, Mubiru, Van Langenhove, and De Meulenaer (2012).
133
2.5. Determination of rice bran protein carbonyl content
134
Protein
carbonylation
was
determined
by
reaction
with
2,
135
4-dinitrophenylhydrazine according to the method described by Wu, Zhang, Kong,
136
and Hua (2009). The results were expressed as nmoles of carbonyl groups per
137
milligram of soluble protein with a molar extinction coefficient of 22,000 (mol/L)−1
138
cm−1. The concentration of soluble rice bran protein was determined by the method of
139
Bradford using bovine serum albumin as the standard (Smith et al., 1985).
140
2.6. Measurement of rice bran protein sulfhydryl and disulfide content
141
Contents of free sulfhydryl and total disulfide/sulfhydryl groups in rice bran
142
protein were determined using Ellman’s procedure modified by Wu et al (2009). The
143
content of sulfhydryl groups was calculated by using the extinction coefficient of Page 7 of 23
144
13,600 (mol/L)−1 cm−1. Disulfide groups of rice bran protein were estimated by
145
subtracting the free sulfhydryl from the total content of sulfhydryl. The concentration
146
of soluble rice bran protein was measured by the method of Bradford using bovine
147
serum albumin as the standard (Smith et al., 1985).
148
2.7. Measurement of dityrosine content
149
The dityrosine content of rice bran protein was estimated by the method of Cui,
150
Xiong, Kong, Zhao, and Liu (2012). The rice bran protein samples about 50 mg were
151
dissolved in 50 mL 0.02 mol/L pH 6.0 phosphate buffer. The concentration of soluble
152
rice bran protein was measured by the determined by the method of Bradford (Smith
153
et al., 1985). With the excitation wavelength of 325 nm and emission wavelength at
154
395 nm (band width of 5 nm), the dityrosine content was measured as the ratio of
155
fluorescence
156
spectrophotometer (Shimadzu, Kyoto, Japan).
157
2.8. Measurement of Fourier transform infrared spectra
intensity
to
protein
concentration
by
F7000
fluorescence
158
According to the method of Sun, Zhou, Sun, and Zhao (2013), IRTracer-100
159
spectrometer (Shimadzu, Kyoto, Japan) was used to measure the Fourier transform
160
infrared spectra of rice bran protein from 4000 to 400 cm-1 with a resolution of 4 cm-1
161
and accumulation of 128 scans. The bands in the amide region I (1600 to 1700 cm-1)
162
were used to assign secondary structural components and calculate the percentage of
163
secondary structure. Baseline correction, smoothing and curve fitting of infrared
164
spectra were conducted by the PeakFit Version 4.12 software (SPSS Inc., Chicago, IL,
165
USA) Page 8 of 23
166
2.9. Measurement of surface hydrophobicity
167
The surface hydrophobicity of rice bran protein was determined using ANS
168
according to the method of Wu et al (2009). Rice bran protein was dissolved in 0.05
169
mol/L phosphate buffer (pH 8.0) and stirred at 25 °C for 2 h, and then centrifuged
170
(10,000 g, 30 min). The concentration of soluble protein was determined by the
171
method of Bradford using bovine serum albumin as the standard (Smith et al., 1985).
172
The rice bran protein solution was diluted to the concentration of 0.005, 0.01, 0.05,
173
0.2, 0.4, 0.5 mg/mL protein. Then, 4 mL protein solution was mixed with 50 µL 8
174
mmol/L ANS. The fluorescence intensity of the rice bran protein was measured by
175
F-7000 fluorescence spectrometer (Shimadzu, Kyoto, Japan), at 390 nm (excitation
176
wavelength) and 470 nm (emission wavelength). The surface hydrophobicity was
177
calculated by the initial slope of the fluorescence intensity versus the rice bran protein
178
concentration.
179
2.10. Measurement of intrinsic fluorescence spectra
180
Intrinsic fluorescence spectra of rice bran protein was determined according to the
181
method of Wu et al (2009). Rice bran protein samples were dissolved in phosphate
182
buffer (0.01 mol/L, pH 7.0), and the concentration of soluble rice bran protein was
183
adjusted to 0.1 mg/mL. F-7000 fluorescence spectrometer (Shimadzu, Kyoto, Japan)
184
was used to measure intrinsic fluorescence spectra of rice bran protein at excitation
185
wavelength of 295 nm, emission wavelength in the range of 300-500 nm and scanning
186
speed of 10 nm/s.
187
2.11. Measurement of molecular weight distribution Page 9 of 23
188
LC-20A liquid chromatogram (Shimadzu, Kyoto, Japan) and the column of
189
TSK-Gel G4000 SWXL (7.8 mm×300 mm, Tosoh Biosep, Japan) were used to
190
measure molecular weight distribution of rice bran protein. The rice bran protein
191
samples about 1.00 g were dissolved in 100 mL 0.05 mol/L pH 7.2 phosphate buffer
192
(containing 0.05 mol/L NaCl) and centrifuged at 10,000 g for 15 min. After filtered
193
through a cellulose acetate membrane with a pore size of 0.45 µm and degassed, 10
194
µL supernatant was injected into the column. The flow rate was 1 mL/min using
195
phosphate buffer (0.05 mol/L, pH 7.2, and containing 0.05 mol/L NaCl) as the mobile
196
phase, and the eluent was monitored at 280 nm.
197
2.12. Reducing and non-reducing sodium dodecyl sulfate-polyacrylamide gel
198
electrophoresis
199
The reducing (with 0.05 g/mL β-mercaptoethanol in sample buffer) and
200
non-reducing (without β-mercaptoethanol in protein sample buffer) sodium dodecyl
201
sulfate-polyacrylamide gel electrophoresis of rice bran protein samples were
202
performed according to the method of Wu et al. using 125 g/L separation gel and 40
203
g/L stacking gel (Wu et al., 2009). The protein samples were loaded at 10 µL/channel
204
and run at 25 mA constant current.
205
2.13. Statistical analysis
206
Statistical calculations were performed using the statistical package SPSS 18.0
207
(SPSS Inc., Chicago, IL, USA) for data analysis (ANOVA, Duncan’s multiple range
208
tests). Statistical significance was set at P<0.05. The results are presented as mean ±
209
standard deviation of three independent experiments. Page 10 of 23
210
3. Results and discussion
211
3.1. The effect of rice bran rancidity on the rancidity indicators of crude rice bran oil
212
As shown in Table 1, the AV of crude rice bran oil prepared by rice bran with
213
storage time of 0, 1, 3, 5, 10 days were 4.31, 16.28, 26.55, 33.63, and 38.72 mg
214
KOH/g, which was close to the AV of the majority of crude rice bran oil in China
215
(15-40 mg KOH/g) (You, Huang, Wu, & Wu, 2019). The results of AV indicated that
216
the different rancidity extent of rice bran obtained by storing fresh rice bran at room
217
temperature for 0, 1, 3, 5, and 10 days could represent the rancidity extent of the
218
majority of rice bran in China. As storage time of rice bran increased, the AV, PV, and
219
TBARS of crude rice bran oil increased significantly (P<0.05), which implied that
220
hydrolytic rancidity and oxidative rancidity simultaneously occurred during rice bran
221
storage. The lipase in the rice bran specifically hydrolyzes the 1,3-site of
222
triacylglyerol right after rice milling, resulting in rapid formation of free fatty acids
223
(Rodchuajeen et al., 2016; Thanonkaew et al., 2012). The endogenous lipoxygenase
224
could cause deleterious effect of oxidative rancidity on rice bran, leading to the
225
formation of fatty acids hydroperoxides and a wide range of low molecular weight
226
carbonyl compounds, which contributed to the increase in PV and TBARS value
227
(Loypimai, Moongngarm, & Chottanom, 2015).
228
3.2. The effect of rice bran rancidity on oxidation extent of rice bran protein
229
Protein oxidation is usually accompanied by protein carbonylation. As shown in
230
Table 1, the protein carbonyl content of rice bran protein increased significantly
231
(P<0.05) as storage time of rice bran increased. The free radicals which derived from Page 11 of 23
232
rice bran rancidity could simultaneously attack protein backbone and side chains to
233
form protein radicals which could convert to protein peroxyl radicals in the presence
234
of oxygen. The further reactions of protein peroxyl radicals located at side chains of
235
arginine, lysine, proline, and threonine residues could form carbonyl groups (Huang,
236
Hua, & Qiu, 2006). The subsequent oxidation of protein peroxyl radicals which
237
located in the backbone led to C-terminal decarboxylation and backbone
238
fragmentation, which resulted in protein carbonylation (Wu et al., 2009). Apart from
239
the free radicals, oxidative modification by the secondary degradation products of rice
240
bran rancidity such as MDA could also introduce carbonyl groups into protein (Davies,
241
2016).
242
As shown in Table 1, as storage time of rice bran increased, free sulfhydryl
243
content of rice bran protein gradually decreased, while disulfide groups first increased
244
and then decreased, reaching the maximum as storage time of rice bran was 5 days.
245
Sulfhydryl groups were vulnerable to oxidation (Wu et al., 2009). Oxidative
246
modification could alter the equilibrium constant of sulfhydryl-disulfide interchange
247
reaction, which led to the conversion of protein sulfhydryl groups to disulfide groups
248
and non-disulfide groups (Wu, Wu, & Hua, 2010). The simultaneous decrease in free
249
sulfhydryl and disulfide as storage time of rice bran was 10 days could be attributed to
250
the formation of sulfur-containing oxidation products other than disulfide bonds. The
251
rice bran oxidative rancidity-derived free radicals could react with sulfhydryl groups
252
to form sulfhydryl peroxyl radicals in the presence of oxygen, which led to a decrease
253
of sulfhydryl groups (Wu et al., 2009). In addition, the secondary degradation Page 12 of 23
254
products of rice bran rancidity such as MDA could form stable adducts with protein
255
sulfhydryl groups (Davies, 2016; Estevez & Luna, 2017).
256
As shown in Table 1, the dityrosine content of rice bran protein increased steadily
257
as rice bran storage time increased. The tyrosine residues were susceptible to free
258
radicals induced oxidation. The free radicals which derived from rice bran rancidity
259
could attack the side chains of tyrosine residues to form tyrosyl radicals. As two
260
tyrosyl radicals were close to each other, dityrosine adduct easily formed due to the
261
high reactivity of free radicals, resulting in protein cross-linking (Duan et al., 2018).
262
3.3. Relationship between lipid rancidity of crude rice bran oil and oxidation extent of
263
rice bran protein
264
Correlation analysis between lipid rancidity of crude rice bran oil and oxidation
265
extent of rice bran protein was performed to establish potential linkage between rice
266
bran rancidity and rice bran protein oxidation. As shown in Table 2, the carbonyl
267
content and dityrosine content of rice bran protein were significantly positively
268
correlated with the AV of crude rice bran oil (P<0.05), and significantly positively
269
correlated with the PV and TBARS of crude rice bran oil (P<0.01). The free
270
sulfhydryl groups of rice bran protein were significantly negatively correlated with the
271
PV and TBARS of crude rice bran oil (P<0.01). The results confirmed a high
272
correlation between rice bran rancidity and rice bran protein oxidation.
273
3.4. The effect of rice bran rancidity on the secondary structure of rice bran protein
274
As shown in Fig. 1A, ten major bands corresponded to rice bran protein secondary
275
structure were observed in the stacked deconvoluted infrared amide I spectra of rice Page 13 of 23
276
bran protein. The band at 1655 cm-1 could be attributed to C=O stretching vibration in
277
protein α-helix structure (Liu et al., 2011). The bands at 1618 cm-1 could be assigned
278
to protein intermolecular β-sheet components, and the bands from 1630 to 1638 cm-1
279
as well as bands from 1676 to 1693 cm-1 could be attributed to antiparallel β-sheet
280
structure of the protein (Bocker, Ofstad, Bertram, Egelandsdal, & Kohler, 2006). The
281
band at 1646 cm-1 could be assigned to the random coil structure, while the bands at
282
1662 and 1668 cm-1 could be attributed to the β-turn structure (Singh & Sogi, 2018).
283
The secondary structural percentage of rice bran protein calculated by the
284
wavenumber assignment was shown in Fig. 1B, and the results showed that secondary
285
structure of rice bran protein contained relatively high content of β-sheet (about 40%)
286
and relatively low content of α-helix (about 20%), which was agreed with the report
287
of Singh and Sogi (2018). In addition, as storage time of rice bran increased, the
288
simultaneous decrease in α-helix and β-sheet structure was accompanied by an
289
increase in random coil and β-turn structure of rice bran protein, which indicated that
290
rice bran rancidity resulted in the conversion of rice bran protein secondary structure
291
from ordered state to disordered state.
292
3.5. The effect of rice bran rancidity on the intrinsic fluorescence spectra and surface
293
hydrophobicity of rice bran protein
294
As shown in Fig. 2A, as excitation wavelength was set at 295 nm, the fluorescence
295
emission spectra of rice bran protein were typical fluorescence emission spectra of
296
tryptophan residues in protein. With increasing storage time of rice bran, a gradual
297
blue shift of the intrinsic fluorescence spectral peak position (from 360 to 357 nm) Page 14 of 23
298
was observed along with a continuous decrease in intrinsic fluorescence intensity of
299
rice bran protein. Tryptophan residue was vulnerable to oxidative modification, and
300
quenching of tryptophan fluorescence was an accompanying phenomenon of protein
301
oxidation (Wu et al., 2009). The products which derived from rice bran oxidative
302
rancidity could lead to oxidation of tryptophan residues, which resulted in the loss of
303
tryptophan residues and the decrease in tryptophan fluorescence intensity (Wu et al.,
304
2009; Wang, Zhang, Fang, & Bhandari, 2016). In addition, as storage time of rice bran
305
increased, blue shifts of the maximum emission wavelength of the rice bran protein
306
indicated that the microenvironment of tryptophan residues transformed from polar to
307
non-polar, resulting from protein cross-link by oxidative modification (Wu et al.,
308
2009).
309
As shown in Fig. 2B, with the storage time of rice bran increased from 0 to 10
310
days, surface hydrophobicity of rice bran protein decreased significantly (P<0.05). A
311
similar observation of the effect of fatty acid peroxidation-derived peroxy radicals and
312
acrolein modification on the surface hydrophobicity in soy protein was reported (Wu
313
et al., 2009; Wu et al., 2010). The free radicals and active aldehydes with low
314
molecular weight derived from rice bran oxidative rancidity could simultaneously
315
modify backbone and side chains of protein, and resulted in unfolding as well as
316
aggregation of protein (Cao, True, Chen, & Xiong, 2016). The observed decrease in
317
surface hydrophobicity might be attributed to the formation of aggregates and
318
cross-link caused by the increased protein-protein interaction, such as disulfide，
319
dityrosine, and hydrophobic interaction, leading to the shielding of hydrophobicity Page 15 of 23
320
site in rice bran protein, which was consistent with the analysis of rice bran protein
321
intrinsic fluorescence.
322
3.6. The effect of rice bran rancidity on the molecular weight distribution of rice bran
323
protein
324
As shown in Fig. 3A and Fig. 3B, the elution pattern of rice bran protein exhibited
325
a polydisperse distribution, and four eluting peaks were observed which were not
326
completely separated with the retention time of 5.60, 11.18, 12.12, and 12.05 min,
327
respectively. The retention time of 5.60 min peak corresponded to the molecular
328
weight distribution from 200 to 2000 kDa, which was assigned to the rice bran protein
329
aggregates. Relatively high molecular weight aggregates which were cross-linked by
330
disulfide bonds existed in the nature state of rice bran protein, and heat stabilization of
331
rice bran usually resulted in protein denaturation and aggregation (Xia et al., 2012).
332
The peaks with retention time of 11.18, 12.12, and 12.05 min corresponded to the
333
molecular weight distribution from 33 to 200 kDa, from 18 to 33 kDa, and from 3 to
334
18 kDa, respectively. By the method of size exclusion chromatography, Hamada
335
(1997) found that molecular weight of rice bran albumin, globulin, prolamin, and
336
acid-soluble glutelin were 10-100 kDa, 10-150 kDa, 33-150 kDa, and 25-100 kDa,
337
respectively. Adebiyi, Adebiyi, Hasegawa, Ogawa, and Muramoto (2009) measured
338
the molecular weight of rice bran protein by MALDI-TOF mass spectrometry, and
339
found that the molecular weight of rice bran albumin, globulin, and glutelin
340
distributed from 11 to 76 kDa, from 13 to 127 kDa, and from 11 to 52 kDa,
341
respectively, and the molecular weight of rice bran prolamin was about 19 kDa. Page 16 of 23
342
Therefore, the retention time of 11.18, 12.12, and 12.05 min was assigned to the
343
mixture of rice bran albumin, globulin, glutelin, and prolamin. As storage time of rice
344
bran increased, the area percentage of peaks with the retention time of 5.60, 12.12,
345
and 12.30 min gradually increased, and peak area percentage with the retention time
346
of 11.18 min decreased steadily. The phenomena implied that rice bran rancidity
347
induced rice bran protein aggregation, which was consistent with the analysis of rice
348
bran protein surface hydrophobicity.
349
3.7. The effect of rice bran rancidity on the sodium dodecyl sulfate-polyacrylamide
350
gel electrophoresis of rice bran protein
351
As shown in Fig. 4A and Fig. 4B, about 10 bands were observed in the reducing
352
and non-reducing electrophoresis patterns of rice bran protein, and their molecular
353
weight ranged from 14 to 70 kDa, which was in agreement with the report of Tang,
354
Hettiarachchy, Horax, and Eswaranandam (2003). According to the reports of Ling,
355
Ouyang, and Wang (2019) and Xia, et al. (2012), the electrophoresis bands of rice
356
bran protein could be separated into three regions, 40-70 kDa, 30-40 kDa, and 14-25
357
kDa, respectively. Band corresponded to the high molecular weight aggregates of rice
358
bran protein which was located at the top of the separating gels in the reducing and
359
non-reducing electrophoresis patterns were observed. As storage time of rice bran
360
increased, the intensity of protein aggregates bands both in the reducing and
361
non-reducing electrophoresis patterns gradually increased. Moreover, when rice bran
362
was stored for the same period, the intensity of protein aggregates bands in reducing
363
electrophoresis pattern was much darker than the corresponded rice bran protein in the Page 17 of 23
364
non-reducing electrophoresis pattern. In this experiment, disulfide bonds in rice bran
365
protein were cleaved by β-mercaptoethanol in reducing electrophoresis, and the
366
β-mercaptoethanol was absent in the non-reducing electrophoresis, where disulfide
367
bonds were reserved. Therefore, the results of electrophoresis indicated that rice bran
368
rancidity caused rice bran protein cross-link via disulfide bonds as well as
369
non-disulfide covalent bonds, and compared to the non-disulfide covalent bonds, the
370
formation of rice bran protein crosslink mainly depended on disulfide bonds.
371
4. Conclusions
372
Storage of rice bran at room temperature resulted in the hydrolytic rancidity and
373
oxidative rancidity of rice bran. The oxidative rancidity products of rice bran induced
374
continuous oxidation of rice bran protein, which led to the conversion of the
375
secondary structure of rice bran protein from ordered state to disordered state, and
376
was accompanied by the formation of aggregates and cross-link. This study revealed
377
the relationship between rice bran rancidity and rice bran protein oxidation, and
378
provided a new important factor which could affect structural characteristics,
379
functional and nutritional properties of rice bran protein in the actual production and
380
processing of rice bran.
381
Conflict of interest
382 383 384 385
The authors declare that they have no conflict of interest. Acknowledgments This work was financed by the National Natural Science Foundation of China (No. 31771918). Page 18 of 23
386
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Page 23 of 23
1
Table 1
2
The rancidity indicators of crude rice bran oil and oxidation markers of rice bran
3
protein prepared by rice bran which was stored at room temperature for 0, 1, 3, 5, 10
4
days. crude rice bran oil
rice bran protein
storage
dityrosine protein
time
AV
(days)
(mg KOH/g)
PV (Meq/kg)
free
disulfide (expressed as
TBARS carbonyl
sulfhydryl
groups
(nmol/mg)
(nmol/mg)
(nmol/mg)
(µg MDA /g)
fluorescence) (A.U.)
0
4.31±0.08a
2.84±0.14a
6.22±0.38a
2.12±0.19a
22.66±0.45e
11.50±0.12b
86.11±0.69a
1
16.28±0.09b
3.60±0.16b
7.98±0.39b
3.39±0.21b
21.65±0.42d
12.01±0.12c
104.24±1.15b
3
26.55±0.09c
4.24±0.22c
11.09±0.34c
5.51±0.24c
18.80±0.36c
12.60±0.15d
110.52±1.01c
5
33.63±0.11d
6.54±0.24d
15.58±0.43d
7.80±0.31d
18.10±0.43b
12.80±0.13e
122.69±0.78d
10
38.72±0.12e
15.58±0.40e
28.99±0.45e
13.80±0.38e
9.60±0.41a
9.00±0.14a
159.37±1.22e
5
The different letters in column indicate significantly differences at P<0.05.
6
AV, acid value; PV, peroxide value; TBARS, thiobarbituric acid reactive substances.
7
Table 2
8
Correlation coefficients (r) between rancidity indicators of crude rice bran oil and
9
oxidation markers of rice bran protein.
10
protein carbonyl
free sulfhydryl
disulfide groups
AV
0.895*
-0.845
-0.300
0.907*
PV
0.972**
-0.982**
-0.832
0.962**
TBARS
0.996**
-0.992**
-0.747
0.984**
*P<0.05, **P<0.01.
dityrosine
1
Figure captions
2
Fig. 1. Deconvoluted FTIR spectra (A) and secondary structure distribution (B) of
3
rice bran protein prepared by rice bran which was stored at room temperature for 0, 1,
4
3, 5, 10 days. Different letters in Fig. 1B indicate a significant difference (P<0.05) of
5
the secondary structure percentage of rice bran protein prepared by rice bran which
6
was stored at room temperature for 0, 1, 3, 5, 10 days.
7
Fig. 2. Intrinsic fluorescence spectra (A) and surface hydrophobicity (B) of rice bran
8
protein prepared by rice bran which was stored at room temperature for 0, 1, 3, 5, 10
9
days. Columns with different letters are significantly different (P<0.05).
10
Fig. 3. High-performance size-exclusion chromatogram (A) and peak area percentage
11
in molecular weight distribution (B) of rice bran protein prepared by rice bran which
12
was stored at room temperature for 0, 1, 3, 5, 10 days. Different letters indicate a
13
significant difference (P<0.05) among rice bran protein prepared by rice bran with
14
different storage time for the same retention time.
15
Fig. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern of rice bran
16
protein prepared by rice bran which was stored at room temperature for 0, 1, 3, 5, 10
17
days with (A) and without (B) 0.05 g/mL β-mercaptoethanol. (Lane 1, molecular
18
weight markers; Lane 2-lane 6 corresponded to the rice bran protein prepared by rice
19
bran which was stored at room temperature for 0, 1, 3, 5, 10 days, respectively).
20
21
Fig. 1.
A
22
B
23
24
Fig. 2. A
25
B
26
27
Fig. 3.
A
28
B
29
30
Fig. 4. marker
0d
1d
3d
5d
10 d
A
aggregates 100 kDa 70 kDa 50 kDa 40 kDa 30 kDa 25 kDa
14 kDa 31
marker 0 d
1d
3d
5d
10 d
B
aggregates 100 kDa 70 kDa 50 kDa 40 kDa 30 kDa 25 kDa
14 kDa 32
Highlights 1. Hydrolytic and oxidative rancidity of rice bran simultaneously occurred during storage. 2. The products of rice bran rancidity induced rice bran protein oxidation. 3. Rice bran rancidity resulted in rice bran protein aggregation and cross-linking.
S0023-6438(19)31285-X
DOI:
https://doi.org/10.1016/j.lwt.2019.108943
Reference:
YFSTL 108943
To appear in:
LWT - Food Science and Technology
Received Date: 3 October 2019 Revised Date:
8 December 2019
Accepted Date: 10 December 2019
Please cite this article as: Wu, X., Li, F., Wu, W., Effects of rice bran rancidity on the oxidation and structural characteristics of rice bran protein, LWT - Food Science and Technology (2020), doi: https:// doi.org/10.1016/j.lwt.2019.108943. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.
Effects of rice bran rancidity on the oxidation and structural characteristics of rice bran protein Xiaojuan Wu#, Fang Li#, & Wei Wu* National Engineering Laboratory for Rice and By-product Deep Processing, College of Food Science and Engineering, Central South University of Forestry and Technology, Changsha, Hunan 410004, China *Corresponding author Tel.: +86-731-85658893; Fax: +86-731-85658893. E-mail address: [email protected] (Wei Wu) #These
authors contributed to the work equally and should be regarded as
co-first authors.
↑ Acid value Hydrolytic rancidity
↑ Carbonyl ↑ Dityrosine
Rice bran protein oxidation
Rice bran storage
Oxidative rancidity
↑ Peroxide ↑ TBA
Protein aggregation
↓ Sulfhydryl -SH → –S-S Protein cross-link
1
Effects of rice bran rancidity on the oxidation and structural characteristics of
2
rice bran protein
3
Xiaojuan Wu#, Fang Li#, & Wei Wu*
4
National Engineering Laboratory for Rice and By-product Deep Processing, College
5
of Food Science and Engineering, Central South University of Forestry and
6
Technology, Changsha, Hunan 410004, China
7
*Corresponding author Tel.: +86-731-85658893; Fax: +86-731-85658893.
8
E-mail address: [email protected] (Wei Wu)
9
#
10
These authors contributed to the work equally and should be regarded as co-first
authors.
11
Page 1 of 23
12
Abstract
13
Rice bran rancidity may affect rice bran protein through protein oxidation. However,
14
little is known about the relationship between rice bran rancidity and rice bran protein
15
oxidation. The effects of rice bran rancidity on the oxidation extent and structural
16
characteristics of rice bran protein were investigated. As storage time of rice bran
17
increased, the acid value, peroxide value, and value of thiobarbituric acid reactive
18
substances in crude rice bran oil increased from 4.31 mg KOH/g, 2.84 Meq/kg, and
19
6.22 µg MDA/g to 38.72 mg KOH/g, 15.58 Meq/kg, and 28.99 µg MDA/g,
20
respectively, which indicated that hydrolytic rancidity and oxidative rancidity of rice
21
bran occurred simultaneously. The gradual increase in protein carbonyl and dityrosine
22
content from 2.12 nmol/mg and 88.61 A.U. to 13.8 nmol/mg and 159.37 A.U. was
23
accompanied by a steady decrease in free sulfhydryl content of rice bran protein from
24
22.6 to 9.6 nmol/mg, which implied that the products of rice bran rancidity induced
25
rice bran protein oxidation. The rice bran protein oxidation subsequently resulted in a
26
loss of the ordered state of secondary structure and the formation of aggregates as
27
well as cross-link, when both disulfide bonds and non-disulfide covalent bonds
28
participated in cross-link formation.
29
Keywords: rice bran rancidity; rice bran protein; protein oxidation; aggregation;
30
cross-link
31
1. Introduction
32
Rice (Oryza sativa L.) is one of the important staple food crops for more than half
33
of the global population, and also consumed as a staple food for over 65% of the Page 2 of 23
34
population in China (Tong, Gao, Luo, Liu, & Bao, 2019). China is the largest
35
producer and consumer of rice in the world, which harvested over 200 million tons
36
and processed over 100 million tons of rice annually (International Grains Council，
37
2019), resulting in the production of at least 10 million tons of rice bran ever year. As
38
a major by-product of rice milling, rice bran is a rich source of oil, protein, fiber, and
39
other functional compounds, which can be used as an important functional food
40
ingredient (Sharif, Butt, Anjum, & Khan, 2014). Despite its excellent nutritional
41
benefits and outstanding application prospects, rice bran is underutilized. At present,
42
the majority of rice bran is used to produce cattle feed or non-edible oil, and only a
43
small percentage of rice bran is processed into edible oil (Burlando & Cornara, 2014).
44
Comprehensive utilization of rice bran is severely restricted due to the rapid
45
rancidity that starts with the rice milling process (Sharif et al., 2014). A large number
46
of unsaturated lipids and the presence of potent hydrolytic and oxidative enzymes
47
such as lipase and lipoxygenase result in drastic quality deterioration of rice bran
48
(Chen, Bergman, & McClung, 2019). The active endogenous lipase can hydrolyze the
49
triglycerides of rice bran into glycerol and free fatty acids, resulting in hydrolytic
50
rancidity which leads to an increase in acidity and the formation of off-flavor (Chen et
51
al., 2019). Free fatty acids are vulnerable to the endogenous lipoxygenase of rice bran
52
in the presence of oxygen, and the primary products of fatty acids hydroperoxides can
53
be further decomposed into off-flavor volatile compounds and free radicals,
54
facilitating oxidative rancidity, and further inducing the formation of off-flavor along
55
with the potential harmfulness to human health (Rodchuajeen, Niamnuy, Charunuch, Page 3 of 23
56
Soponronnarit, & Devahastin, 2016). Therefore, the fresh rice bran must be stabilized
57
immediately after the rice milling to inhibit rancidity, and various stabilization
58
methods of rice bran have been investigated in recent years (Liu, Strappe, Zhou, &
59
Blanchard, 2019). However, lipolytic activity in rice bran begins as soon as bran
60
layers are removed from endosperm during the rice milling process. Besides, owing to
61
the distance and transportation between rice milling plant and rice bran oil processing
62
factory, stabilization of rice bran soon after the milling process is not practical for the
63
commercial scale, which implies that a certain degree of hydrolytic rancidity and
64
oxidative rancidity has already occurred before stabilization of rice bran (Thanonkaew,
65
Wongyai, McClements, & Decker, 2012).
66
Reducing the negative effects of hydrolytic rancidity and oxidative rancidity on the
67
quality of rice bran derived products is mainly reflected in the deacidification and
68
deodorization methods of rice bran oil. As a major by-product of rice bran oil,
69
defatted rice bran contains about 18% protein. Rice bran protein has unique nutritional
70
and hypoallergenic properties, which can be utilized as a suitable ingredient for
71
nutraceutical and functional food formulation (Fabian & Ju, 2011). However, rice
72
bran protein is not commercially useful because of its unpleasant characteristic odor
73
(Arsa, Theerakulkait, & Cadwallader, 2019). As a primary product of rice bran
74
oxidative rancidity, fatty acids hydroperoxides are also strong prooxidants due to their
75
ability to decompose into low molecular weight aldehydes, ketones, and alkoxy
76
radicals (Bui, Hsu, & Hankinson, 2009). The active aldehydes, ketones and alkoxy
77
radicals can trigger protein oxidation in rice bran (Davies, 2016). Oxidative Page 4 of 23
78
modification of protein during food processing and storage could alter the structure,
79
functional properties, and nutritional characteristics of protein, and might pose
80
potential threat to human health, which ultimately affects the development and
81
application of protein in food industry (Estevez & Luna, 2017). However, the effects
82
of rice bran rancidity on the structure and properties of rice bran protein have not been
83
reported yet. Therefore, the purpose of this study was to identify the relationship
84
between rice bran rancidity and rice bran protein oxidation. Moreover, in order to
85
simulate the varying degrees of rancidity of most rice bran in China, the fresh rice
86
bran was stored at room temperature for different periods.
87
2. Materials and methods
88
2.1. Materials
89
Freshly milled rice bran from indica rice (Oryza sativa L., Variety Zhong Jia Zao
90
17) was supplied by Hunan Grain Group Co., Ltd. (Changsha, Hunan Province,
91
China). The total dietary fiber, crude protein, fat, ash, and moisture of rice bran were
92
282 g/kg, 148 g/kg, 168 g/kg, 75 g/kg, and 120 g/kg, respectively. Blue plus protein
93
markers with molecular weight from 14 to 100 kDa for protein electrophoresis were
94
purchased from TransGen Biotech (Beijing, China). 1-anilino-8-naphthalene-sulfonate
95
(ANS),
96
5,5'-dithiobis(2-nitrobenzoic acid), acrylamide, and N, N’-methylenebisacrylamide
97
were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals
98
were of analytical grade.
99
2.2. Preparation of defatted rice bran from rice bran with different degree of rancidity
1,1,3,3-tetramethoxypropane,
Page 5 of 23
2,
4-dinitrophenylhydrazine,
100
Freshly milled rice bran was passed through 60 meshes (size of 0.25 mm). After
101
stored at 25 °C and relative humidity of 85% for 0, 1, 3, 5, and 10 days, the rice bran
102
with different degrees of rancidity were stabilized by a twin-screw extruder (FMHE
103
36-24, FUMACH, Changsha, Hunan Province, China). The feed moisture was
104
adjusted to 16 g/100 g. The rice bran was fed into the extruder at a constant speed of
105
15 kg/h, and the screw speed was 150 rpm. The extruder barrel temperatures from the
106
first zone (feeding zone) to the fifth zone were set at 70, 120, 120, 70 and 60 °C,
107
respectively. After stabilization, the rice bran was immediately dried at 45 °C for 12 h,
108
and then smashed to pass through 80 meshes (size of 0.18 mm). The stabilized rice
109
bran powder was defatted using hexane with a ratio of 1:4 (w/v) at 25 °C for three
110
times. After vacuum filtering, the filter cake was vacuum dried at 25 °C. The dried
111
defatted rice bran was ground to pass through 80 meshes and stored at 4 °C. The
112
filtrate was evaporated to obtain crude rice bran oil and recovered hexane.
113
2.3. Preparation of rice bran protein
114
The defatted rice bran was mixed with distilled water at a ratio of 1:10 (w/v), and
115
the pH of the solution was adjusted to 9.0 with 2 mol/L NaOH. After stirring for 4 h at
116
40 °C, the suspension was centrifuged (Lynx 6000, Thermo Fisher Scientific,
117
Germany) at 8,000 g for 20 min at 4 °C to recover the supernatant. Rice bran protein
118
was precipitated by adjusting pH to 4.0 with 2 mol/L HCl and hold for 30 minutes,
119
and then centrifuged at 8,000 g for 15 min at 4 °C. After washed with distilled water,
120
the protein precipitate was resuspended in distilled water at a ratio of 1:5 (w/v), and
121
neutralized to pH 7.0 with 2 mol/L NaOH. Finally, the solution was dialyzed with Page 6 of 23
122
distilled water at 4 °C for 24 h, freeze-dried and stored at 4 °C. The wet base purity of
123
rice bran protein prepared by rice bran which was stored for 0, 1, 3, 5, 10 days was
124
74.65%, 74.38%, 73.46%, 74.55%, 74.35%, respectively, and the purity was obtained
125
by the method of micro-Kjeldahl with the nitrogen conversion factor of 5.95.
126
2.4. Determination of acid value (AV), peroxide value (PV) and thiobarbituric acid
127
reactive substances (TBARS) assay result of crude rice bran oil
128
Determination of PV and AV of crude rice bran oil were performed according to
129
the standard methods of AOCS Cd 3d-63 and AOCS Cd 8-53. TBARS assay result is
130
usually expressed as the malondialdehyde (MDA) concentration, and MDA
131
concentration was determined by reaction with 2-thiobarbituric acid reagent according
132
to the method of Papastergiadis, Mubiru, Van Langenhove, and De Meulenaer (2012).
133
2.5. Determination of rice bran protein carbonyl content
134
Protein
carbonylation
was
determined
by
reaction
with
2,
135
4-dinitrophenylhydrazine according to the method described by Wu, Zhang, Kong,
136
and Hua (2009). The results were expressed as nmoles of carbonyl groups per
137
milligram of soluble protein with a molar extinction coefficient of 22,000 (mol/L)−1
138
cm−1. The concentration of soluble rice bran protein was determined by the method of
139
Bradford using bovine serum albumin as the standard (Smith et al., 1985).
140
2.6. Measurement of rice bran protein sulfhydryl and disulfide content
141
Contents of free sulfhydryl and total disulfide/sulfhydryl groups in rice bran
142
protein were determined using Ellman’s procedure modified by Wu et al (2009). The
143
content of sulfhydryl groups was calculated by using the extinction coefficient of Page 7 of 23
144
13,600 (mol/L)−1 cm−1. Disulfide groups of rice bran protein were estimated by
145
subtracting the free sulfhydryl from the total content of sulfhydryl. The concentration
146
of soluble rice bran protein was measured by the method of Bradford using bovine
147
serum albumin as the standard (Smith et al., 1985).
148
2.7. Measurement of dityrosine content
149
The dityrosine content of rice bran protein was estimated by the method of Cui,
150
Xiong, Kong, Zhao, and Liu (2012). The rice bran protein samples about 50 mg were
151
dissolved in 50 mL 0.02 mol/L pH 6.0 phosphate buffer. The concentration of soluble
152
rice bran protein was measured by the determined by the method of Bradford (Smith
153
et al., 1985). With the excitation wavelength of 325 nm and emission wavelength at
154
395 nm (band width of 5 nm), the dityrosine content was measured as the ratio of
155
fluorescence
156
spectrophotometer (Shimadzu, Kyoto, Japan).
157
2.8. Measurement of Fourier transform infrared spectra
intensity
to
protein
concentration
by
F7000
fluorescence
158
According to the method of Sun, Zhou, Sun, and Zhao (2013), IRTracer-100
159
spectrometer (Shimadzu, Kyoto, Japan) was used to measure the Fourier transform
160
infrared spectra of rice bran protein from 4000 to 400 cm-1 with a resolution of 4 cm-1
161
and accumulation of 128 scans. The bands in the amide region I (1600 to 1700 cm-1)
162
were used to assign secondary structural components and calculate the percentage of
163
secondary structure. Baseline correction, smoothing and curve fitting of infrared
164
spectra were conducted by the PeakFit Version 4.12 software (SPSS Inc., Chicago, IL,
165
USA) Page 8 of 23
166
2.9. Measurement of surface hydrophobicity
167
The surface hydrophobicity of rice bran protein was determined using ANS
168
according to the method of Wu et al (2009). Rice bran protein was dissolved in 0.05
169
mol/L phosphate buffer (pH 8.0) and stirred at 25 °C for 2 h, and then centrifuged
170
(10,000 g, 30 min). The concentration of soluble protein was determined by the
171
method of Bradford using bovine serum albumin as the standard (Smith et al., 1985).
172
The rice bran protein solution was diluted to the concentration of 0.005, 0.01, 0.05,
173
0.2, 0.4, 0.5 mg/mL protein. Then, 4 mL protein solution was mixed with 50 µL 8
174
mmol/L ANS. The fluorescence intensity of the rice bran protein was measured by
175
F-7000 fluorescence spectrometer (Shimadzu, Kyoto, Japan), at 390 nm (excitation
176
wavelength) and 470 nm (emission wavelength). The surface hydrophobicity was
177
calculated by the initial slope of the fluorescence intensity versus the rice bran protein
178
concentration.
179
2.10. Measurement of intrinsic fluorescence spectra
180
Intrinsic fluorescence spectra of rice bran protein was determined according to the
181
method of Wu et al (2009). Rice bran protein samples were dissolved in phosphate
182
buffer (0.01 mol/L, pH 7.0), and the concentration of soluble rice bran protein was
183
adjusted to 0.1 mg/mL. F-7000 fluorescence spectrometer (Shimadzu, Kyoto, Japan)
184
was used to measure intrinsic fluorescence spectra of rice bran protein at excitation
185
wavelength of 295 nm, emission wavelength in the range of 300-500 nm and scanning
186
speed of 10 nm/s.
187
2.11. Measurement of molecular weight distribution Page 9 of 23
188
LC-20A liquid chromatogram (Shimadzu, Kyoto, Japan) and the column of
189
TSK-Gel G4000 SWXL (7.8 mm×300 mm, Tosoh Biosep, Japan) were used to
190
measure molecular weight distribution of rice bran protein. The rice bran protein
191
samples about 1.00 g were dissolved in 100 mL 0.05 mol/L pH 7.2 phosphate buffer
192
(containing 0.05 mol/L NaCl) and centrifuged at 10,000 g for 15 min. After filtered
193
through a cellulose acetate membrane with a pore size of 0.45 µm and degassed, 10
194
µL supernatant was injected into the column. The flow rate was 1 mL/min using
195
phosphate buffer (0.05 mol/L, pH 7.2, and containing 0.05 mol/L NaCl) as the mobile
196
phase, and the eluent was monitored at 280 nm.
197
2.12. Reducing and non-reducing sodium dodecyl sulfate-polyacrylamide gel
198
electrophoresis
199
The reducing (with 0.05 g/mL β-mercaptoethanol in sample buffer) and
200
non-reducing (without β-mercaptoethanol in protein sample buffer) sodium dodecyl
201
sulfate-polyacrylamide gel electrophoresis of rice bran protein samples were
202
performed according to the method of Wu et al. using 125 g/L separation gel and 40
203
g/L stacking gel (Wu et al., 2009). The protein samples were loaded at 10 µL/channel
204
and run at 25 mA constant current.
205
2.13. Statistical analysis
206
Statistical calculations were performed using the statistical package SPSS 18.0
207
(SPSS Inc., Chicago, IL, USA) for data analysis (ANOVA, Duncan’s multiple range
208
tests). Statistical significance was set at P<0.05. The results are presented as mean ±
209
standard deviation of three independent experiments. Page 10 of 23
210
3. Results and discussion
211
3.1. The effect of rice bran rancidity on the rancidity indicators of crude rice bran oil
212
As shown in Table 1, the AV of crude rice bran oil prepared by rice bran with
213
storage time of 0, 1, 3, 5, 10 days were 4.31, 16.28, 26.55, 33.63, and 38.72 mg
214
KOH/g, which was close to the AV of the majority of crude rice bran oil in China
215
(15-40 mg KOH/g) (You, Huang, Wu, & Wu, 2019). The results of AV indicated that
216
the different rancidity extent of rice bran obtained by storing fresh rice bran at room
217
temperature for 0, 1, 3, 5, and 10 days could represent the rancidity extent of the
218
majority of rice bran in China. As storage time of rice bran increased, the AV, PV, and
219
TBARS of crude rice bran oil increased significantly (P<0.05), which implied that
220
hydrolytic rancidity and oxidative rancidity simultaneously occurred during rice bran
221
storage. The lipase in the rice bran specifically hydrolyzes the 1,3-site of
222
triacylglyerol right after rice milling, resulting in rapid formation of free fatty acids
223
(Rodchuajeen et al., 2016; Thanonkaew et al., 2012). The endogenous lipoxygenase
224
could cause deleterious effect of oxidative rancidity on rice bran, leading to the
225
formation of fatty acids hydroperoxides and a wide range of low molecular weight
226
carbonyl compounds, which contributed to the increase in PV and TBARS value
227
(Loypimai, Moongngarm, & Chottanom, 2015).
228
3.2. The effect of rice bran rancidity on oxidation extent of rice bran protein
229
Protein oxidation is usually accompanied by protein carbonylation. As shown in
230
Table 1, the protein carbonyl content of rice bran protein increased significantly
231
(P<0.05) as storage time of rice bran increased. The free radicals which derived from Page 11 of 23
232
rice bran rancidity could simultaneously attack protein backbone and side chains to
233
form protein radicals which could convert to protein peroxyl radicals in the presence
234
of oxygen. The further reactions of protein peroxyl radicals located at side chains of
235
arginine, lysine, proline, and threonine residues could form carbonyl groups (Huang,
236
Hua, & Qiu, 2006). The subsequent oxidation of protein peroxyl radicals which
237
located in the backbone led to C-terminal decarboxylation and backbone
238
fragmentation, which resulted in protein carbonylation (Wu et al., 2009). Apart from
239
the free radicals, oxidative modification by the secondary degradation products of rice
240
bran rancidity such as MDA could also introduce carbonyl groups into protein (Davies,
241
2016).
242
As shown in Table 1, as storage time of rice bran increased, free sulfhydryl
243
content of rice bran protein gradually decreased, while disulfide groups first increased
244
and then decreased, reaching the maximum as storage time of rice bran was 5 days.
245
Sulfhydryl groups were vulnerable to oxidation (Wu et al., 2009). Oxidative
246
modification could alter the equilibrium constant of sulfhydryl-disulfide interchange
247
reaction, which led to the conversion of protein sulfhydryl groups to disulfide groups
248
and non-disulfide groups (Wu, Wu, & Hua, 2010). The simultaneous decrease in free
249
sulfhydryl and disulfide as storage time of rice bran was 10 days could be attributed to
250
the formation of sulfur-containing oxidation products other than disulfide bonds. The
251
rice bran oxidative rancidity-derived free radicals could react with sulfhydryl groups
252
to form sulfhydryl peroxyl radicals in the presence of oxygen, which led to a decrease
253
of sulfhydryl groups (Wu et al., 2009). In addition, the secondary degradation Page 12 of 23
254
products of rice bran rancidity such as MDA could form stable adducts with protein
255
sulfhydryl groups (Davies, 2016; Estevez & Luna, 2017).
256
As shown in Table 1, the dityrosine content of rice bran protein increased steadily
257
as rice bran storage time increased. The tyrosine residues were susceptible to free
258
radicals induced oxidation. The free radicals which derived from rice bran rancidity
259
could attack the side chains of tyrosine residues to form tyrosyl radicals. As two
260
tyrosyl radicals were close to each other, dityrosine adduct easily formed due to the
261
high reactivity of free radicals, resulting in protein cross-linking (Duan et al., 2018).
262
3.3. Relationship between lipid rancidity of crude rice bran oil and oxidation extent of
263
rice bran protein
264
Correlation analysis between lipid rancidity of crude rice bran oil and oxidation
265
extent of rice bran protein was performed to establish potential linkage between rice
266
bran rancidity and rice bran protein oxidation. As shown in Table 2, the carbonyl
267
content and dityrosine content of rice bran protein were significantly positively
268
correlated with the AV of crude rice bran oil (P<0.05), and significantly positively
269
correlated with the PV and TBARS of crude rice bran oil (P<0.01). The free
270
sulfhydryl groups of rice bran protein were significantly negatively correlated with the
271
PV and TBARS of crude rice bran oil (P<0.01). The results confirmed a high
272
correlation between rice bran rancidity and rice bran protein oxidation.
273
3.4. The effect of rice bran rancidity on the secondary structure of rice bran protein
274
As shown in Fig. 1A, ten major bands corresponded to rice bran protein secondary
275
structure were observed in the stacked deconvoluted infrared amide I spectra of rice Page 13 of 23
276
bran protein. The band at 1655 cm-1 could be attributed to C=O stretching vibration in
277
protein α-helix structure (Liu et al., 2011). The bands at 1618 cm-1 could be assigned
278
to protein intermolecular β-sheet components, and the bands from 1630 to 1638 cm-1
279
as well as bands from 1676 to 1693 cm-1 could be attributed to antiparallel β-sheet
280
structure of the protein (Bocker, Ofstad, Bertram, Egelandsdal, & Kohler, 2006). The
281
band at 1646 cm-1 could be assigned to the random coil structure, while the bands at
282
1662 and 1668 cm-1 could be attributed to the β-turn structure (Singh & Sogi, 2018).
283
The secondary structural percentage of rice bran protein calculated by the
284
wavenumber assignment was shown in Fig. 1B, and the results showed that secondary
285
structure of rice bran protein contained relatively high content of β-sheet (about 40%)
286
and relatively low content of α-helix (about 20%), which was agreed with the report
287
of Singh and Sogi (2018). In addition, as storage time of rice bran increased, the
288
simultaneous decrease in α-helix and β-sheet structure was accompanied by an
289
increase in random coil and β-turn structure of rice bran protein, which indicated that
290
rice bran rancidity resulted in the conversion of rice bran protein secondary structure
291
from ordered state to disordered state.
292
3.5. The effect of rice bran rancidity on the intrinsic fluorescence spectra and surface
293
hydrophobicity of rice bran protein
294
As shown in Fig. 2A, as excitation wavelength was set at 295 nm, the fluorescence
295
emission spectra of rice bran protein were typical fluorescence emission spectra of
296
tryptophan residues in protein. With increasing storage time of rice bran, a gradual
297
blue shift of the intrinsic fluorescence spectral peak position (from 360 to 357 nm) Page 14 of 23
298
was observed along with a continuous decrease in intrinsic fluorescence intensity of
299
rice bran protein. Tryptophan residue was vulnerable to oxidative modification, and
300
quenching of tryptophan fluorescence was an accompanying phenomenon of protein
301
oxidation (Wu et al., 2009). The products which derived from rice bran oxidative
302
rancidity could lead to oxidation of tryptophan residues, which resulted in the loss of
303
tryptophan residues and the decrease in tryptophan fluorescence intensity (Wu et al.,
304
2009; Wang, Zhang, Fang, & Bhandari, 2016). In addition, as storage time of rice bran
305
increased, blue shifts of the maximum emission wavelength of the rice bran protein
306
indicated that the microenvironment of tryptophan residues transformed from polar to
307
non-polar, resulting from protein cross-link by oxidative modification (Wu et al.,
308
2009).
309
As shown in Fig. 2B, with the storage time of rice bran increased from 0 to 10
310
days, surface hydrophobicity of rice bran protein decreased significantly (P<0.05). A
311
similar observation of the effect of fatty acid peroxidation-derived peroxy radicals and
312
acrolein modification on the surface hydrophobicity in soy protein was reported (Wu
313
et al., 2009; Wu et al., 2010). The free radicals and active aldehydes with low
314
molecular weight derived from rice bran oxidative rancidity could simultaneously
315
modify backbone and side chains of protein, and resulted in unfolding as well as
316
aggregation of protein (Cao, True, Chen, & Xiong, 2016). The observed decrease in
317
surface hydrophobicity might be attributed to the formation of aggregates and
318
cross-link caused by the increased protein-protein interaction, such as disulfide，
319
dityrosine, and hydrophobic interaction, leading to the shielding of hydrophobicity Page 15 of 23
320
site in rice bran protein, which was consistent with the analysis of rice bran protein
321
intrinsic fluorescence.
322
3.6. The effect of rice bran rancidity on the molecular weight distribution of rice bran
323
protein
324
As shown in Fig. 3A and Fig. 3B, the elution pattern of rice bran protein exhibited
325
a polydisperse distribution, and four eluting peaks were observed which were not
326
completely separated with the retention time of 5.60, 11.18, 12.12, and 12.05 min,
327
respectively. The retention time of 5.60 min peak corresponded to the molecular
328
weight distribution from 200 to 2000 kDa, which was assigned to the rice bran protein
329
aggregates. Relatively high molecular weight aggregates which were cross-linked by
330
disulfide bonds existed in the nature state of rice bran protein, and heat stabilization of
331
rice bran usually resulted in protein denaturation and aggregation (Xia et al., 2012).
332
The peaks with retention time of 11.18, 12.12, and 12.05 min corresponded to the
333
molecular weight distribution from 33 to 200 kDa, from 18 to 33 kDa, and from 3 to
334
18 kDa, respectively. By the method of size exclusion chromatography, Hamada
335
(1997) found that molecular weight of rice bran albumin, globulin, prolamin, and
336
acid-soluble glutelin were 10-100 kDa, 10-150 kDa, 33-150 kDa, and 25-100 kDa,
337
respectively. Adebiyi, Adebiyi, Hasegawa, Ogawa, and Muramoto (2009) measured
338
the molecular weight of rice bran protein by MALDI-TOF mass spectrometry, and
339
found that the molecular weight of rice bran albumin, globulin, and glutelin
340
distributed from 11 to 76 kDa, from 13 to 127 kDa, and from 11 to 52 kDa,
341
respectively, and the molecular weight of rice bran prolamin was about 19 kDa. Page 16 of 23
342
Therefore, the retention time of 11.18, 12.12, and 12.05 min was assigned to the
343
mixture of rice bran albumin, globulin, glutelin, and prolamin. As storage time of rice
344
bran increased, the area percentage of peaks with the retention time of 5.60, 12.12,
345
and 12.30 min gradually increased, and peak area percentage with the retention time
346
of 11.18 min decreased steadily. The phenomena implied that rice bran rancidity
347
induced rice bran protein aggregation, which was consistent with the analysis of rice
348
bran protein surface hydrophobicity.
349
3.7. The effect of rice bran rancidity on the sodium dodecyl sulfate-polyacrylamide
350
gel electrophoresis of rice bran protein
351
As shown in Fig. 4A and Fig. 4B, about 10 bands were observed in the reducing
352
and non-reducing electrophoresis patterns of rice bran protein, and their molecular
353
weight ranged from 14 to 70 kDa, which was in agreement with the report of Tang,
354
Hettiarachchy, Horax, and Eswaranandam (2003). According to the reports of Ling,
355
Ouyang, and Wang (2019) and Xia, et al. (2012), the electrophoresis bands of rice
356
bran protein could be separated into three regions, 40-70 kDa, 30-40 kDa, and 14-25
357
kDa, respectively. Band corresponded to the high molecular weight aggregates of rice
358
bran protein which was located at the top of the separating gels in the reducing and
359
non-reducing electrophoresis patterns were observed. As storage time of rice bran
360
increased, the intensity of protein aggregates bands both in the reducing and
361
non-reducing electrophoresis patterns gradually increased. Moreover, when rice bran
362
was stored for the same period, the intensity of protein aggregates bands in reducing
363
electrophoresis pattern was much darker than the corresponded rice bran protein in the Page 17 of 23
364
non-reducing electrophoresis pattern. In this experiment, disulfide bonds in rice bran
365
protein were cleaved by β-mercaptoethanol in reducing electrophoresis, and the
366
β-mercaptoethanol was absent in the non-reducing electrophoresis, where disulfide
367
bonds were reserved. Therefore, the results of electrophoresis indicated that rice bran
368
rancidity caused rice bran protein cross-link via disulfide bonds as well as
369
non-disulfide covalent bonds, and compared to the non-disulfide covalent bonds, the
370
formation of rice bran protein crosslink mainly depended on disulfide bonds.
371
4. Conclusions
372
Storage of rice bran at room temperature resulted in the hydrolytic rancidity and
373
oxidative rancidity of rice bran. The oxidative rancidity products of rice bran induced
374
continuous oxidation of rice bran protein, which led to the conversion of the
375
secondary structure of rice bran protein from ordered state to disordered state, and
376
was accompanied by the formation of aggregates and cross-link. This study revealed
377
the relationship between rice bran rancidity and rice bran protein oxidation, and
378
provided a new important factor which could affect structural characteristics,
379
functional and nutritional properties of rice bran protein in the actual production and
380
processing of rice bran.
381
Conflict of interest
382 383 384 385
The authors declare that they have no conflict of interest. Acknowledgments This work was financed by the National Natural Science Foundation of China (No. 31771918). Page 18 of 23
386
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1
Table 1
2
The rancidity indicators of crude rice bran oil and oxidation markers of rice bran
3
protein prepared by rice bran which was stored at room temperature for 0, 1, 3, 5, 10
4
days. crude rice bran oil
rice bran protein
storage
dityrosine protein
time
AV
(days)
(mg KOH/g)
PV (Meq/kg)
free
disulfide (expressed as
TBARS carbonyl
sulfhydryl
groups
(nmol/mg)
(nmol/mg)
(nmol/mg)
(µg MDA /g)
fluorescence) (A.U.)
0
4.31±0.08a
2.84±0.14a
6.22±0.38a
2.12±0.19a
22.66±0.45e
11.50±0.12b
86.11±0.69a
1
16.28±0.09b
3.60±0.16b
7.98±0.39b
3.39±0.21b
21.65±0.42d
12.01±0.12c
104.24±1.15b
3
26.55±0.09c
4.24±0.22c
11.09±0.34c
5.51±0.24c
18.80±0.36c
12.60±0.15d
110.52±1.01c
5
33.63±0.11d
6.54±0.24d
15.58±0.43d
7.80±0.31d
18.10±0.43b
12.80±0.13e
122.69±0.78d
10
38.72±0.12e
15.58±0.40e
28.99±0.45e
13.80±0.38e
9.60±0.41a
9.00±0.14a
159.37±1.22e
5
The different letters in column indicate significantly differences at P<0.05.
6
AV, acid value; PV, peroxide value; TBARS, thiobarbituric acid reactive substances.
7
Table 2
8
Correlation coefficients (r) between rancidity indicators of crude rice bran oil and
9
oxidation markers of rice bran protein.
10
protein carbonyl
free sulfhydryl
disulfide groups
AV
0.895*
-0.845
-0.300
0.907*
PV
0.972**
-0.982**
-0.832
0.962**
TBARS
0.996**
-0.992**
-0.747
0.984**
*P<0.05, **P<0.01.
dityrosine
1
Figure captions
2
Fig. 1. Deconvoluted FTIR spectra (A) and secondary structure distribution (B) of
3
rice bran protein prepared by rice bran which was stored at room temperature for 0, 1,
4
3, 5, 10 days. Different letters in Fig. 1B indicate a significant difference (P<0.05) of
5
the secondary structure percentage of rice bran protein prepared by rice bran which
6
was stored at room temperature for 0, 1, 3, 5, 10 days.
7
Fig. 2. Intrinsic fluorescence spectra (A) and surface hydrophobicity (B) of rice bran
8
protein prepared by rice bran which was stored at room temperature for 0, 1, 3, 5, 10
9
days. Columns with different letters are significantly different (P<0.05).
10
Fig. 3. High-performance size-exclusion chromatogram (A) and peak area percentage
11
in molecular weight distribution (B) of rice bran protein prepared by rice bran which
12
was stored at room temperature for 0, 1, 3, 5, 10 days. Different letters indicate a
13
significant difference (P<0.05) among rice bran protein prepared by rice bran with
14
different storage time for the same retention time.
15
Fig. 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern of rice bran
16
protein prepared by rice bran which was stored at room temperature for 0, 1, 3, 5, 10
17
days with (A) and without (B) 0.05 g/mL β-mercaptoethanol. (Lane 1, molecular
18
weight markers; Lane 2-lane 6 corresponded to the rice bran protein prepared by rice
19
bran which was stored at room temperature for 0, 1, 3, 5, 10 days, respectively).
20
21
Fig. 1.
A
22
B
23
24
Fig. 2. A
25
B
26
27
Fig. 3.
A
28
B
29
30
Fig. 4. marker
0d
1d
3d
5d
10 d
A
aggregates 100 kDa 70 kDa 50 kDa 40 kDa 30 kDa 25 kDa
14 kDa 31
marker 0 d
1d
3d
5d
10 d
B
aggregates 100 kDa 70 kDa 50 kDa 40 kDa 30 kDa 25 kDa
14 kDa 32
Highlights 1. Hydrolytic and oxidative rancidity of rice bran simultaneously occurred during storage. 2. The products of rice bran rancidity induced rice bran protein oxidation. 3. Rice bran rancidity resulted in rice bran protein aggregation and cross-linking.