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Study on the antioxidant activity and emulsifying properties of flaxseed gum-whey protein isolate conjugates prepared by Maillard reaction
Journal Pre-proofs Study on the antioxidant activitiy and emulsifying properties of flaxseed gumwhey protein isolate conjugates prepared by Maillard reaction Xuyan Dong, Shanshan Du, Qianchun Deng, Hu Tang, Chen Yang, Fang Wei, Hong Chen, Siew Young Quek, Aijun Zhou, Liang Liu PII: DOI: Reference:
S0141-8130(19)36928-4 https://doi.org/10.1016/j.ijbiomac.2019.10.245 BIOMAC 13746
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
28 August 2019 25 October 2019 26 October 2019
Please cite this article as: X. Dong, S. Du, Q. Deng, H. Tang, C. Yang, F. Wei, H. Chen, S. Young Quek, A. Zhou, L. Liu, Study on the antioxidant activitiy and emulsifying properties of flaxseed gum-whey protein isolate conjugates prepared by Maillard reaction, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.245
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1
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Study on the antioxidant activitiy and emulsifying properties of flaxseed
3
gum-whey protein isolate conjugates prepared by Maillard reaction
4
Xuyan Donga,b, Shanshan Dub,c, Qianchun Dengb, Hu Tangb, Chen Yangb, Fang Weib,
5
Hong Chenb, Siew Young Quekd,e, Aijun Zhouc, Liang Liua,*
6 7 8
a
9
China
College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, P.R.
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b
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Oilseeds processing ， Ministry of Agriculture and Rural Area - Hubei Key Laboratory of Lipid
12
Chemistry and Nutrition, Wuhan, Hubei 430062, P.R. China
13
c
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430074, P. R. China
15
d
School of Chemical Sciences, The University of Auckland, Auckland 1142, New Zealand
16
e
Riddet Institute, Palmerston North, New Zealand.
Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Key Laboratory of
College of Materials and Science and Engineering, Wuhan Institute of Technology, Wuhan, Hubei
17 18 19 20 21 22 * Corresponding author Tel: +86-532-86080771; Fax: +86-532-86080771; Email: [email protected] 1
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ABSTRACT: The antioxidant and emulsifying properties of flaxseed gum-whey protein
28
isolate (FSG-WPI) conjugates prepared by Maillard reaction via controlled dry-heating
29
were investigated. The reaction was carried out using a ratio of FSG to WPI of 1:3 at
30
60C and 79% relative humidity for different incubation times. The reaction was
31
confirmed by analysis of the browning index, free amino content and soluble sulfhydryl
32
content, as well as by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
33
ultraviolet-visible spectroscopy, and Fourier transform infrared spectroscopy. We found
34
that nearly 35% of the protein participated in conjugation with FSG after 48 h of
35
incubation. The antioxidant activity of the conjugates improved markedly after 48 and
36
72 h incubation time. Differential scanning calorimetry results indicated that the
37
denaturation temperature of the conjugates increased. The FSG-WPI conjugate prepared
38
by 72 h incubation had the best emulsifying properties in stabilizing an oil-in-water
39
emulsion. This research provides significant knowledge for the potential applications of
40
FSG in food industry.
41
Keywords: Maillard reaction; Conjugate; Flaxseed gum; Whey protein isolate;
42
Emulsifying property
43 44 45
2
46
Highlights
47 48
The FSG-WPI conjugate was successfully prepared by controlled dry heating.
49
Nearly 35% of the protein participated in conjugation with FSG after 48 h of incubation.
50 51
conjugates.
52 53 54
The Maillard reaction between FSG and WPI improved the antioxidant activity of
The FSG-WPI conjugate prepared by 72 h incubation exhibited the best emulsifying properties.
55
3
56
1. Introduction
57
Whey protein isolate (WPI) is extensively used as the key nutitional and functional
58
ingredients in a variety of food products, which is the excellent source of high quality
59
protein obtained from cheese-making process and contained all the essential amino
60
acids(Qi and Xiao et al., 2016). Moreover, WPI presents various physico-chemical and
61
structural properties such as gelation, emulsification, foaming and flavor binding, which
62
impart food products with satisfied appearance, taste, texture, and rheological behavior.
63
However, the industrial application of WPI is limited due to reduced solubility,
64
decreased emulsifying stability, and even coagulation in certain processing conditions as
65
high ionic strength, pH and/or temperature(Chen and Lv et al., 2019).
66
To overcome these limits, many efforts have been made to improve or alter the
67
performance of WPI by using physical, chemical and/or enzymatic approaches(Sutariya
68
and Patel, 2017). However, the potential health risks associated with reagents used in the
69
above processes remain a major concern. Recently, conjugation of the ε-amino groups
70
of the amino acid or protein with reducing sugar via Maillard reaction without using any
71
other chemicals has received great attention(Oliveira and Coimbra et al., 2016). The most
72
notable fact is that this reaction is capable of improving emulsifying properties, thermal
73
stability, and antioxidant effect by forming the covalently conjugated products(Cui and
74
Steve et al., 2013). Monosaccharides like glucose and disaccharides like lactose and
75
maltose are often used to study the glycosylation of proteins(Zhao and Zhou et al., 2016;
76
Liu and Wang et al., 2019; Wang and Li et al., 2019). The increasing evidence showed that the
77
functional properties obtained by glycation of protein with polysaccharide were far
78
superior to those of obtained with mono- or disaccharides(Chen and Lv et al., 2019).
79
Flaxseed gum (FSG), an anionic hydrophilic colloid, are naturally-occurring plant
80
polysaccharide, accounts for 8%-10% of the weight of flaxseed. The annual production
81
of flaxseed in China is about 450 kilotons, accounting for 20% of total world production.
82
There are more than 165 kilotons flaxseed oil and 20 kilotons flaxseed gum every
83
year(Yousuf and Srivastava, 2017). In the past few decades, the extraction, characterization, 4
84
chemical composition and physiochemical properties of FSG has been completely
85
revealed. FSG can be extracted from flaxseed, flaxseed hull, or flaxseed cake(Liu and
86
Shim et al., 2018; Rashid and Ahmed et al., 2019) . After extraction of oil from flaxseed, a
87
large amount of flaxseed cake is generated and it is generally used as fertilizer, apart
88
from its limited use for livestock and poultry feed. Its potential value is far from being
89
developed, and FSG obtained from flaxseed cake has not been fully utilized. Moreover,
90
the uncontrolled hydration rate of FSG restricts its wide application(Liu and Shen et al.,
91
2016).
92
Recently, the renewed interest in FSG as food source due to its health benefits
93
attributed to reduction of blood glucose, cholesterol, and antioxidtive activity(Thakur and
94
Mitra et al., 2009; Nounou and Deif et al., 2012) . In addition to its thickening and
95
water-holding capacities, FSG also plays an important
96
emulsions(Wang and Feng et al., 2017). It is found that FSG exhibited much lower
97
viscosity at a concentration of 0.3%(w/v) than locust bean gum, guar gum, and xanthan
98
gum(Qian and Cui et al., 2012). FSG can effectively improved the texture properties of
99
lotus root starch gels, making starch gels softer and have a better mouthfeel, which is
100
suitable to produce gelatinised food(Liu and Xu, 2019). Therefore, it is supposed that FSG
101
can potentially replace gum Arabic in food emulsion(Kaushik and Dowling et al., 2017) and
102
is expected to be more widely used in the food industry because of its sustainable,
103
biodegradable functional properties(Rashid and Ahmed et al., 2019) and unique nutritional
104
value(Xu and Qi et al., 2016).
role
in stabilizing
105
Hitherto, many attempts have been made to improve the performance of WPI by
106
addition of FSG. It has been revealed that the interaction and attachment of FSG onto
107
the surface of WPI-stabilized emulsion through electrostatic interaction had a significant
108
impact on the microrheological and physicochemical properties of WPI-stabilized
109
emulsion(Khalloufi and Corredig et al., 2009; Xu and Qi et al., 2016) . Liu’s research
110
demonstrated that FSG-WPI mixtures in aqueous solution favored electroneutral
111
coacervate formation with a more compact structure than charged coacervates, which
112
attributed to improved rheological properties (Liu and Shim et al., 2017). Zhang found that 5
113
the addition of FSG increased the viscosity of FSG-WPI solutions and the strength of
114
the mixed gels(Zhang and Li et al., 2013). However, up-to-date the covalent conjugates
115
between FSG and WPI produced via Maillard reaction and their functional properties
116
for application in food industry has not been systematically investigated. Thus, the
117
present study aimed to fill the above research gap by investigating the conjugation of
118
FSG and WPI via controlled dry-heating Maillard reaction at various incubation/
119
reaction time and to evaluate the functional properties of the conjugates produced
120
including antioxidant activity, thermal stability and emulsifying properties.
121
122
2. Materials and methods
123
2.1. Materials
124
FSG (90%) with viscosity of 12,250 mPa·s was purchased from Li Shi De Bio
125
Technology Co. Ltd. (Xinjiang, China). The average molecular weight of the FSP was
126
1.7×104 - 5.7×106 Da, consisting of mannose, galactose, glucose, arabinose, glucuronic
127
acid, xylose, rhamnose, ribose, galacturonic acid. The natural carbohydrate, uronic acid,
128
and protein contents were 55.20±3.07%, 37.78±1.26%, and 2.93±0.06%, respectively.
129
The WPI (90.46%) was purchased from Fonterra Co-operative Group Ltd. (Auckland,
130
New Zealand). Medium-chain triglyceride (MCT) was provided by Houman Biological
131
Technology Co. Ltd. (Shanghai, China). All other chemicals were of analytical grade.
132 133
2.2. Preparation of FSG-WPI conjugates
134
The conjugation process was carried out according to the method described by previous
135
researchers(Yang and Cui et al., 2015; Tamnak and Mirhosseini et al., 2016) , with minor
136
modification. FSG and WPI were dispersed in 0.01 M phosphate buffer solution (PBS，
137
pH=7.2~7.4) at mass ratio of 1:3 with stirring at room temperature (25 ± 1C) for 2 h,
138
and then stored at 4C overnight for complete hydration. The resultant solution was
139
successively freeze-dried, ground, and sieved (300 µm) to obtain uniform particles. The 6
140
powder was incubated (LHS-80HC-I; Yiheng, Shanghai, China) at 60C in relative
141
humidity (RH) of 79%. At 22h, 48h, and 72h, the generalted FSG-WPI conjugates were
142
taken out and stored at 4C for further analysis.
143 144
2.3. Determination of the browning intensity
145
Browning intensity(BI) of FSG-WPI conjugates collected at selected intervals were
146
measured to determine the progress of the reaction(Zhao and Zhou et al., 2016). Briefly,
147
FSG-WPI conjugates dispersions (5 mg/mL) were prepared using Milli-Q water, and the
148
absorbance was measured at 420 nm on a SpectraMax M2 (Molecular Devices, Silicon
149
Valley, USA). The experiments were performed in triplicate.
150 151
2.4. Determination of free amino group content
152
To determine free amino group (FAG) content in the FSG-WPI conjugates, 1mL of 5
153
mg/mL FSG-WPI conjugate solution was mixed with 1 mL of 4% NaHCO 3 (pH 8.5)
154
solution and 1 mL of 0.1% (w/w) 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) solution,
155
and then stored at 40C for 2 h. Subsequently, 1 mL of 10% sodium dodecyl sulfate
156
solution and 0.5 mL of 1 M HCl solution were added to terminate the reaction. The
157
absorbance of the resultant solution was measured at 340 nm. The FAG concentration
158
was calculated through the following equation:
159
FAG(%) = 𝐴 𝑡 × 100%
160
where A0 is the absorbance of the FSG-WPI mixture, At are the absorbances of the
161
conjugate solutions with different incubation times. The FAG concentration was
162
measured in triplicate.
𝐴
(1)
0
163 164
2.5. Determination of sulfhydryl content on the protein surface
7
165
Total surface sulfhydryl content on the protein surface was analyzed according to a
166
modified method(Qi and Xiao et al., 2017). The samples were dissolved in the buffer
167
solution (PBS containing 1.0 mM EDTA, pH 8.0) to a concentration of 20 mg/mL. The
168
sample solution (500 µL) and buffer solution (2.25 mL) were mixed with 50 µL of 1.0
169
mM 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman reagent) solution. Afterward,
170
the reaction solution was kept in the dark for 20 min at 25C. The absorbance was
171
measured at 412 nm against a blank, which is 500 µL of reaction buffer solution. All
172
experiments were performed in triplicate. The sulfhydryl group content, [SH], was
173
calculated through the following formula:
174
[SH] (µmol/g) =
175
where A412 is the absorbance at 412 nm, 13,600 is the extinction coefficient, C is the
176
protein concentration (mg/mL), and D is the dilution factor of the sample solution.
106 ×A412 ×D
(2)
13,600×C
177 178
2.6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
179
SDS-PAGE using 15% (w/v) acrylamide separating gel was performed according to a
180
modified
181
Radioimmunoprecipitation (RIPA) lysis buffer (strong) and with 10% (w/v) SDS were
182
prepared. The conjugate solution (20 μL) was mixed with 5 μL of 5× loading buffer, and
183
the mixture was denatured at 100C for 5 min. Electrophoresis was carried out at 90 V
184
for 20 min and then at a voltage of 150 V for 70 min. Subsequently, the gel was stained
185
using Coomassie brilliant blue R-250. The electropherograms were obtained using a
186
Bio-Rad GS-800 gel scanner (California, USA).
method
described
by
Laemmli(Laemmli,
1970).
Samples
in
187 188
2.7. Fourier transform infrared (FT-IR) spectroscopy
189
For FT-IR spectroscopy, samples were freeze-dried for 24 h and then stored in a
190
decicator. The dry powder samples were mixed with potassium bromide (KBr) at a ratio 8
191
of 1:100 and then fully ground and compressed under 20 MPa for 2-3 min to form
192
uniform slices. All samples were scanned from 4000-400 cm−1 at a resolution of 4 cm-1
193
using a Perkin Elmer FT-IR 1600 instrument (Norwalk, USA).
194 195
2.8. Ultraviolet-visible (UV-VIS) spectroscopy
196
Sample dispersions at a concentration of 0.5 mg/mL in PBS (pH = 7.2-7.4) were
197
prepared. Absorbance spectra were obtained on a SpectraMax M2 (Molecular Devices,
198
Silicon Valley, USA), and the wavelengths were recorded from 220 to 400 nm.
199 200
2.9. Antioxidant activity
201
The scavenging efficiency of FSG-WPI conjugates for DPPH• radical was
202
evaluated through the method(Cheng and Yu et al., 2019), with slight modifications. In
203
brief, 0.1 mM of DPPH• in ethanol solution and FSG-WPI conjugate solutions of
204
different concentrations subjected to reactions for different durations were prepared.
205
The butylated hydroxytoluene (BHT) in ethanol solution (0.5 mg/mL) was used as
206
control. Afterward, 1 mL of FSG-WPI conjugate solution was mixed with 1 mL of
207
DPPH• of solution and shaken well. The reaction system was placed away from light for
208
30 min at room temperature. At was measured at 517 nm on a SpectraMax M2. Milli-Q
209
water (1 mL) was used instead of sample solution in the same operation, and the reading
210
was recorded as Ac. In addition, the absorbance of a 1 mL sample solution was mixed
211
with 1 mL of absolute alcohol, and the reading was recorded as Ab. Experiments were
212
conducted in triplicate. The scavenging effect of FSG-WPI conjugates on DPPH•
213
radical was calculated as follows:
214
Scavenging Efficiency (%) =
215
Where At is the absorbance of sample solution with DPPH• solution, Ac is the
216
absorbance of Milli-Q water with DPPH• solution, and Ab is the absorbance of sample
217
solution with absolute alcohol.
𝐴𝑐 −(𝐴𝑡 −𝐴𝑏 ) 𝐴𝑐
× 100
9
(3)
218 219
2.10. Thermal properties
220
The thermal properties of conjugates were characterized by differential scanning
221
calorimetry (DSC) using a TA Instruments Q2000 (New Castle, USA). About 6 mg of
222
conjugate powder was loaded in a sample pan in a nitrogen atmosphere, and an empty
223
sample pan was used as reference. The temperature was raised from 30 oC to 250 oC at a
224
rate of 5 oC/min. The variations of heat flow with temperature were recorded.
225 226
2.11 Emulsifying activity index (EAI) and emulsion stability index (ESI)
227
EAI and ESI were determined by the turbidimetric method of Pearce and Kinsella
228
(Pearce and Kinsella, 1978) with minor modifications. For emulsion formation, 30 mL of
229
0.5 % (w/v) protein solutions in 100 mM PBS (pH 7.0) and 10 mL of soybean oil were
230
homogenized in Ultra-Turrax T25 homogenizer (IKA Werke GmbH & Co. KG, Staufen,
231
Germany) at 10,000 rpm for 1 min. One hundred microliters of the resultant emulsion
232
was withdrawn at 0 min and 10 min, diluted (1:100, v/v) with 0.1 % (w/v) SDS solution.
233
After shaking in a vortex mixer for 5 s, the absorbance of dilute emulsions was
234
determined at 500 nm using a UV2300 spectrophotometer (Techcomp, Shanghai, China)
235
immediately against a blank (0.1 % (w/v) SDS solution instead of the emulsion). The
236
EAI was determined from the absorbance measured immediately after the emulsion had
237
formed (0 min). The EAI and ESI were calculated using the following equations:
2 2.303 A0 N m2 ) g c L 10000
238
EAI (
239
ESI (min)
240
where, N was the dilution factor, c was the protein concentration (g/m3), Φ is the
241
oil volume fraction (v/v) in the emulsion, L is the optical path (1 cm), and A0 and A10
242
are the absorbance of diluted emulsions at 0 and 10 min, respectively; t is 10 min.
A0 t A0 A10
10
243
Measurements were performed in triplicate.
244 245
2.12. Emulsion preparation and analysis
246
The samples were dissolved in Milli-Q water to a concentration of 0.5% (w/w), and
247
then MCT was added slowly to the solutions to a concentration of 5%. The emulsions
248
were homogenized using an IKA T25 digital Ultra-Turrax (Staufen, Germany) at 15,000
249
rpm for 3 min in an ice bath. The particle size of the emulsions was measured on a
250
Mastersizer 2000 (Malvern, UK) and the zeta potential was determined on a Malvern
251
Zetasizer Nano-ZS90 (Malvern, UK). The refractive indexes of the oil and aqueous
252
phases were set at 1.54 and 1.33, respectively.
253
2.13. Evaluation of emulsion stability
254
The stability of the emulsion samples was evaluated as described previously (Bi and Yang
255
et al., 2017), with slight modification. The emulsions were stored at 4 oC for 13 days.
256
Then observation of macroscopic features and measurement of particle size distribution
257
were conducted (section 2.12). All experiments were performed in triplicate.
258 259
2.14 Statistical analysis
260
All the samples were measured in triplicate and results were expressed as mean ±
261
standard deviation (SD).
262
263
3. Results and discussion
264
3.1. Formation of the FSG-WPI conjugates
265
3.1.1. BI and FAG content
266
Maillard reaction involves a series of complex non-enzymatic browning reactions. The
267
extent of the Maillard reaction can be indicated by the decrease in the protein or 11
268
carbohydrates involved in the reaction and the degree of browning. Therefore, the BI
269
and FAG content of the protein and polysaccharide conjugates have been commonly
270
used to characterize the progress of the Maillard reaction(Chen and Xue et al., 2014;
271
Setiowati and Vermeir et al., 2016; Han and Yi et al., 2017) . As shown in Fig. 1 (a), elongated
272
heating led to increased BI and decreased FAG content, indicating the Maillard reaction
273
was occurred during dry heating process(Guan and Qiu et al., 2006; Jafar and Fadia, 2010).
274
The FAG content decreased rapidly from time zero to 22 h, and then became nearly
275
constant at 65%. These observations were supported by Bi et al.(Bi and Yang et al., 2017),
276
who studied the conjugation of β-lactoglobulin (β-LG) with gum from Acacia Seyal.
277 278
3.1.2. Sulfhydryl content of the protein surface
279
As shown in Fig. 1 (b), a reduction of sulfhydryl content of the WPI was observed after
280
48 h of heating in comparison with the native WPI, which was mainly due to protein
281
denaturation and aggregation. However, the surface sulfhydryl content increased in the
282
FSG-WPI system during dry heating process, indicating the intramolecular sulfhydryl
283
groups could be exposed to the surface due to the changes of protein structure during
284
Maillard reaction. The broken disulfide bond induced by heating also contributes to the
285
increase of sulfhydryl groups(Creamer and Bienvenue et al., 2004). In addition, the
286
sulfhydryl activity on the molecule surface was enhanced during the reaction(Schmidt
287
and Illingworth et al., 1979) due to thermal denaturation, which causes its exposure and
288
participation in sulfhydryl/disulfide linkages interchange reaction, involving both β-LG
289
and α-LG(Wang and Ismail, 2012). It is therefore not surprising that the result led to an
290
increasing trend of sulfhydryl content on the protein surface, albeit it was less than that
291
on the native WPI.
292 293
3.1.3. SDS-PAGE
294
SDS-PAGE was used to confirm the covalent cross-linking between FSG and WPI due 12
295
to the formation of high-molecular-weight conjugates(Schmidt and Pietsch et al., 2016). As
296
shown in Fig. 2, the FSG-WPI mixture without dry-heating treatment (lane 1) produced
297
prominent bands for α-lactalbumin, β-LG, β-LG dimer, and bovine serum albumin
298
consistent with literature(Schmidt and Pietsch et al., 2016; Setiowati and Vermeir et al., 2016) ..
299
The molecular weights of all protein-containing substances were below 130 kDa.
300
Low-molecular-weight bands in lanes 2-4 seemed to be gradually fading.
301
Simultaneously, high-molecular-weight bands appeared at the boundary between the
302
separating gel and stacking gel. These phenomena became more obvious with prolonged
303
incubation time. Therefore, we could conclude that there was covalent conjugation of
304
FSG and WPI mixtures by dry heating in agreement with previous studies (Liu and Ma et
305
al., 2016; Mengibar and Miralles et al., 2017). Similar SDS-PAGE patterns for egg
306
white-pectin conjugates were reported by Al-Hakkak(Jafar and Fadia, 2010). The
307
SDS-PAGE profiles are consistent with the reduction in FAG content of the FSG-WPI
308
conjugates (Fig. 1(a)). Based on these results, it was clear that conjugation of FSG and
309
WPI had occurred where the carbonyl group of FSG was bonded to the amino groups of
310
WPI through the Maillard reaction.
311 312
3.1.4. FT-IR spectroscopy
313
FT-IR spectroscopy can be useful to investigate the molecular structure and interactions
314
of protein polysaccharide systems(Liu and Shen et al., 2016). The characteristic absorption
315
peaks of FSG located at wavenumbers 3438, 2928, and 1639 cm−1 (Fig. 3) were
316
attributed to O–H, C–H, and C=O stretching vibrations respectively(Zhou and Hu et al.,
317
2016). An absorption peak for WPI at 2928 m−1 is due to anti-symmetric stretching of –
318
CH2. The most distinctive absorption features for WPI were observed at 1647 cm−1
319
(C=O stretching) and 1541 cm−1 (N–H bending)(Gu and Jin et al., 2010). In the spectra of
320
the FSG-WPI mixture, the intensity of the band at 2928 cm−1 was markedly reduced.
321
This may be due to the intermolecular interactions resulting in the reduction of
322
methylene vibrations. After incubation of the FSG-WPI mixture for 48 h, the spectra
323
showed apparent differences in intermolecular interaction between FSG and WPI. It can 13
324
be observed that the characteristic band of O–H at 3456 cm−1 became flatter, indicating
325
the formation of hydrogen bonds. Additionally, the characteristic band of FSG at 1639
326
cm−1 (C=O stretching) disappeared, and the intensity of the band for WPI at 1541 cm−1
327
(N–H bending) decreased remarkably while a new band appeared at 1637 cm−1 (amide
328
bond). These changes were ascribed to the Maillard reaction between FSG and
329
WPI(Golkar and Nasirpour et al., 2016).
330 331
3.1.5. UV-VIS spectroscopy
332
To further explore the interaction between FSG and WPI, the UV-VIS absorption
333
spectra of FSG after 48h of heating (FSG-48h), WPI after 48h heating (WPI-48h),
334
FSG-WPI mixture, and conjugates were studied (Fig. 4). The main absorption peaks of
335
FSG-48h and WPI-48h were observed at 260 and 280 nm, respectively. The absorbance
336
intensity of the FSG-WPI conjugates increased with the reaction time, and the UV-VIS
337
absorption maximum showed a characteristic red shift with the incubation period. This
338
red shift may be explained by a difference in the Schiff base environments, which led to
339
formation of Schiff base products. Meanwhile, it was also noticed that the sample
340
solutions gradually turned into yellow color with prolong incubation time from 22 to 72
341
hours. These changes further indicate that FSG and WPI had interacted to produce
342
Maillard products. Similar results were published by Zhu et al.(Dan and Srinivasan et al.,
343
2008), in which they reported a red shift of the UV absorption maximum of WPI-dextran
344
conjugates.
345 346
3.2. DPPH• scavenging activity
347
Results show that FSG-WPI conjugates had the ability to scavenge DPPH• (Fig. 5). The
348
scavenging capacity of the conjugates increased with the incubation time and
349
concentration of conjugates. A dramatic increase in the antioxidant activity of the
350
conjugates was observed during incubation for 22 to 48 h, especially for higher 14
351
concentration of conjugates. After 48 hours of incubation, the scavenging capacity of
352
the 6 mg/mL conjugate reached 91.17%. To give a better comparison, we had conducted
353
DPPH assay for the commercial antioxidant BHT and observed its scavenging capacity
354
as 87.12% at 0.5 mg/mL. Current results also show that the antioxidant activity of
355
conjugates at a concentration of 2 mg/mL was limited, and the DPPH• scavenging
356
ability did not apparently improve with longer incubation period. In a previous study,
357
ovalbumin-glucose conjugates and ovalbumin-maltose conjugates formed by Maillard
358
reaction had shown to exhibit a drastic increase in DPPH• scavenging activity under
359
heat/moisture treatment at 120 C for 20 min(Huang and Tu et al., 2012).
360 361
3.3. Thermal properties
362
It has been reported that Maillard reaction between proteins and polysaccharides can
363
improve the thermal stability of proteins and their mixtures(Liu and Ma et al., 2016). DSC
364
can detect alterations of the sample in the heat flow during temperature changes, and it
365
can provide very useful information related to thermal stability of the sample.
366
From the DSC characteristics of the FSG-WPI mixture and the conjugates at different
367
incubation times (Fig. 6), we observed that the denaturation temperatures of the
368
conjugates were markedly higher than that of the mixture. The denaturation temperature
369
of the conjugate after 22 h of incubation increased from 129 ºC to 165 ºC. Higher
370
denaturation temperature means better thermal stability. The result indicates that the
371
thermal stability of the FSG-WPI mixture was improved markedly through the Maillard
372
reaction. This phenomenon may be explained by the increase in the steric repulsion
373
forces between WPI molecules due to glycation(Liu and Ma et al., 2016). The onset
374
temperature of FSG and WPI was 108 ºC while the FSG-WPI conjugates increased with
375
incubation time, from 140 ºC to 150 ºC. Previous studies had reported that the glycation
376
could enhance the heat stability of protein, thus our results were in agreement with the
377
others(Huang and Tu et al., 2012).
378 15
379
3.4. Emulsifying properties
380
3.4.1 Emulsifying activity index (EAI) and emulsion stability index (ESI)
381
The EAI and ESI of WPI and FSG-WPI mixture were investigated. As can be observed
382
from Fig.7(a), with the increase of reaction time, the EAI of FSG-WPI mixture
383
increased gradually, and then descended slightly. The same result has also been reported
384
in a previous literature(Li and Wang et al., 2015; Chen and Lv et al., 2019; Xu and Huang et al.,
385
2019). The slight decline of EAI might be attributed to the developing of polymerization
386
products, which could decrease the molecules mobility and make the conjugates
387
absorbed in the oil/water interface much slower. The ESI of FSG-WPI mixture dropped
388
sharply, and then had a slight increase. Significant increase in EAI of FSG-WPI
389
conjugates could be mainly attributed to the conformation ability of protein to expose
390
the hydrophobic groups and lysyl residues buried in the interior which react easily with
391
the reducing-end carbonyl group in polysaccharides. A new balance value of
392
hydrophobic and hydrophilic groups consents to be achieved and favor of emulsion
393
formation. Additionally, it can also be attributed to conjugating WPI with highly soluble
394
and charged saccharides, proteins in the emulsions were able to provide a better
395
potentiality for the adsorption at the oil-water interface, resulting in enhancement of
396
emulsion properties. After Millard reaction, there was a slight increase trend of ESI of
397
FSG-WPI mixture as a function of reaction time. Millard reaction facilitated the
398
emulsion stability of FSG-WPI mixture.
399
3.4.2 Emulsion stabilized by FSG-WPI mixture
400
The partical size of FSG-WPI-0h mixture and FSG-WPI conjugates storaged at 4 oC for
401
2, 6, and 13 days are shown in Fig. 7(b). The droplet size distributions of the fresh
402
emulsions in Fig. 7(c) show substantial differences in particle size distribution of the
403
emulsions stabilized by the FSG-WPI mixture and the conjugates incubated for different
404
durations. The emulsions stabilized by the FSG-WPI conjugates after Maillard reaction
405
(especially those conjugated for 48 and 72 h) had fewer large droplets as compared with
406
that stabilized by the FSG-WPI mixture. The results clearly indicate that the 16
407
emulsifying activity of the FSG-WPI conjugates improved with the progress of Maillard
408
reaction. The droplet size distribution of the emulsions stored for 13 days (Fig. 7(d))
409
produced with FSG-WPI-48h and FSG-WPI-72h conjugates did not change noticeably
410
when compared with that of the fresh sample (Fig. 7(c)). However, the particle size
411
distribution of the FSG-WPI mixture had a notable change because of droplets
412
aggregation. These findings indicate that the stability of the emulsions is improved by
413
the FSG-WPI conjugates produced via Maillard reaction, especially with a reaction time
414
of 72 h. Visual observation of macroscopic appearance show that the emulsions
415
stabilized by the conjugates (after 48 and 72 h of incubation) exhibited uniform
416
behavior after storage at 4°C and for 13 days. However, creaming was observed in the
417
emulsions stabilized by the native WPI and FSG-WPI mixture and the conjugates
418
produced by 22 h of incubation, indicating lesser emulsion stability, which conformed to
419
the results of ESI.
420
The changes in zeta potential of the emulsions stabilized by WPI, the FSG-WPI
421
mixture, and the FSG-WPI conjugates is as shown in Table 1. The zeta potential values
422
of the FSG-WPI conjugates after 48 and 72 h of incubation were −40.1 mV and −41.9
423
mV, respectively. These values were higher than those of WPI and the FSG-WPI
424
mixture, indicating better emulsion stability of the FSG-WPI conjugates than the
425
formers. These phenomena, once again, illustrating that Maillard reaction has improved
426
the emulsifying properties of the FSG-WPI conjugates and the incubation time has an
427
obvious impact on this properties.
428
Our results are in agreement with the work reported by Kato et al. (Kato and Minaki et
429
al., 1993). Their study showed that the conjugate of egg white protein and galactomannan
430
formed by controlled dry-heating reaction had dramatically enhanced the emulsifying
431
activity and stability as compared with their mixture. In addition, they also observed
432
that the conjugate had better emulsifying properties than some commercial emulsifiers
433
including sucrose-fatty acid ester and decaglyceryl monostearate. The emulsifying
434
properties of conjugates obtained via reaction between β-LG and gum from Acacia
435
Seyal were found to improve with the extent of Maillard reaction(Bi and Yang et al., 2017), 17
436
consistent
with
our
findings.
The
improvement
of
emulsion
stability
of
437
protein-polysaccharide conjugates by Maillard reaction may be mainly due to the
438
presence of hydrophilic polysaccharide, which provides sufficient steric repulsion and
439
electrostatic repulsion around the oil droplets, this protecting the oil droplets and
440
preventing their aggregation(Zhou and Wang et al., 2016). Similar results were also
441
reported by Xu et al.(Xu and Qi et al., 2017), who studied the effect of FSG on WPI
442
stabilized β-carotene emulsions, and they proved that FSG at a concentration of 0.1 wt%
443
exhibited a remarkable increase in physical stability.
444
4. Conclusions
445
Current study was conducted to investigate the functional properties of FSG-WPI
446
conjugates produced via Maillard reaction in an attempt to explore the potential
447
application of FSG in food industry. Our results show that BI and surface sulfhydryl
448
content increased with increasing incubation time, while the FAG content first
449
decreased rapidlybefore approaching a constant value. Both SDS-PAGE and FT-IR
450
spectroscopy confirmed the formation of large-molecular-weight fragments and the
451
binding of carbonyl groups and amino groups and thus the occurrence of Maillard
452
reaction. The conjugates showed substantially improved antioxidant activity and
453
thermal stability, especially that obtained after 72 h of incubation time. Furthermore,
454
this conjugate also showed the best emulsifying activity after 22 h and stability as
455
reflected by emulsion particle size distribution and zeta potential measurements. The
456
conjugate, thus, appears to have promising applications for nutrient delivery and for
457
effective emulsifiers because of their improved functional performance.
458 459
Acknowledgements
460
This work was supported by the National Natural Science Foundation of China
461
(Grant No 31701643 and 31171661), National High Technology Research and
462
Development Program of China (863 Program: 2017YFD0400200). 18
463 464
Conflict of interest
465
We declares that he has no conflict of interest.
466
19
467 468
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583 584 585
23
586
Table 1
587
Zeta potentials of freshly prepared emulsions with whey protein isolate (WPI) (0 h), FSG–WPI
588
mixture (0 h), and FSG–WPI Maillard reaction products (22, 48, and 72 h) at 25ºC.
Aqueous phase
Z-potential (mV)
WPI-0h
−36.6 ± 0.56
FSG-WPI-0h
−36.7 ± 0.21
FSG-WPI-22h
−35.6 ± 1.35
FSG-WPI-48h
−40.1 ± 1.81
FSG-WPI-72h
−41.9 ± 2.02
589
24
590
Figures captions
591
Fig. 1 Effect of incubation time on the formation of the FSG-WPI conjugates. a: Change in free
592
amino group (FAG) content and browning intensity (BI) of FSG-WPI conjugates during dry heating.
593
Samples include a mixture at 0 h incubation time, as well as the conjugates after 22, 48, and 72 h of
594
incubation time; b: Soluble sulfhydryl content ([SH]) of WPI, WPI at 48 h, FSG-WPI mixture, and
595
conjugates as determined using Ellman reagent.
596
Fig. 2 SDS-PAGE pattern of FSG-WPI conjugates at different incubation times. Lane 1 contains a
597
mixture of FSG and WPI. Lanes 2, 3, and 4 contain the conjugates of FSG-WPI subjected to dry
598
heating for 22, 48, and 72 h, respectively. MW contains the protein markers that indicate molecular
599
weights (kDa).
600
Fig. 3. FT-IR spectra of FSG, WPI, FSG-WPI-0h mixture, and FSG-WPI-48h conjugate.
601
Fig. 4. UV-VIS spectra of FSG-48h, WPI-48h, FSG-WPI mixture, and conjugates. The concentration
602
of all samples was 0.5 mg/mL.
603
Fig. 5. Changes in DPPH• scavenging efficiency of the FSG-WPI conjugates at concentrations of 2,
604
4, and 6 mg/mL and at different incubation times.
605
Fig. 6. DSC analysis of FSG-WPI-0h mixture and FSG-WPI conjugates incubated for 22, 48, and 72
606
h.
607
Fig. 7. Emulsifying properties of FSG-WPI mixture and FSG-WPI conjugates. The EAI and ESI of
608
FSG-WPI mixture and FSG-WPI conjugates (a); Partical size of FSG-WPI-0h mixture and
609
FSG-WPI conjugates after storage at 4 oC for 2, 6, and 13 days (b); Particle size distribution of the
610
emulsions stabilized by the FSG-WPI mixture and the conjugates incubated for different durations (c)
611
after 13 days (d).
612
Table 1 Zeta potentials of freshly prepared emulsions with whey protein isolate (WPI) (0 h),
613
FSG-WPI mixture (0 h), and FSG-WPI Maillard reaction products (22, 48, and 72 h) at 25ºC.
614 25
615 616
26
617 618
27
619 620
28
621 622
29
623 624
30
625 626
31
627
32
S0141-8130(19)36928-4 https://doi.org/10.1016/j.ijbiomac.2019.10.245 BIOMAC 13746
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International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
28 August 2019 25 October 2019 26 October 2019
Please cite this article as: X. Dong, S. Du, Q. Deng, H. Tang, C. Yang, F. Wei, H. Chen, S. Young Quek, A. Zhou, L. Liu, Study on the antioxidant activitiy and emulsifying properties of flaxseed gum-whey protein isolate conjugates prepared by Maillard reaction, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.245
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1
2
Study on the antioxidant activitiy and emulsifying properties of flaxseed
3
gum-whey protein isolate conjugates prepared by Maillard reaction
4
Xuyan Donga,b, Shanshan Dub,c, Qianchun Dengb, Hu Tangb, Chen Yangb, Fang Weib,
5
Hong Chenb, Siew Young Quekd,e, Aijun Zhouc, Liang Liua,*
6 7 8
a
9
China
College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, P.R.
10
b
11
Oilseeds processing ， Ministry of Agriculture and Rural Area - Hubei Key Laboratory of Lipid
12
Chemistry and Nutrition, Wuhan, Hubei 430062, P.R. China
13
c
14
430074, P. R. China
15
d
School of Chemical Sciences, The University of Auckland, Auckland 1142, New Zealand
16
e
Riddet Institute, Palmerston North, New Zealand.
Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Key Laboratory of
College of Materials and Science and Engineering, Wuhan Institute of Technology, Wuhan, Hubei
17 18 19 20 21 22 * Corresponding author Tel: +86-532-86080771; Fax: +86-532-86080771; Email: [email protected] 1
23 24 25 26 27
ABSTRACT: The antioxidant and emulsifying properties of flaxseed gum-whey protein
28
isolate (FSG-WPI) conjugates prepared by Maillard reaction via controlled dry-heating
29
were investigated. The reaction was carried out using a ratio of FSG to WPI of 1:3 at
30
60C and 79% relative humidity for different incubation times. The reaction was
31
confirmed by analysis of the browning index, free amino content and soluble sulfhydryl
32
content, as well as by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
33
ultraviolet-visible spectroscopy, and Fourier transform infrared spectroscopy. We found
34
that nearly 35% of the protein participated in conjugation with FSG after 48 h of
35
incubation. The antioxidant activity of the conjugates improved markedly after 48 and
36
72 h incubation time. Differential scanning calorimetry results indicated that the
37
denaturation temperature of the conjugates increased. The FSG-WPI conjugate prepared
38
by 72 h incubation had the best emulsifying properties in stabilizing an oil-in-water
39
emulsion. This research provides significant knowledge for the potential applications of
40
FSG in food industry.
41
Keywords: Maillard reaction; Conjugate; Flaxseed gum; Whey protein isolate;
42
Emulsifying property
43 44 45
2
46
Highlights
47 48
The FSG-WPI conjugate was successfully prepared by controlled dry heating.
49
Nearly 35% of the protein participated in conjugation with FSG after 48 h of incubation.
50 51
conjugates.
52 53 54
The Maillard reaction between FSG and WPI improved the antioxidant activity of
The FSG-WPI conjugate prepared by 72 h incubation exhibited the best emulsifying properties.
55
3
56
1. Introduction
57
Whey protein isolate (WPI) is extensively used as the key nutitional and functional
58
ingredients in a variety of food products, which is the excellent source of high quality
59
protein obtained from cheese-making process and contained all the essential amino
60
acids(Qi and Xiao et al., 2016). Moreover, WPI presents various physico-chemical and
61
structural properties such as gelation, emulsification, foaming and flavor binding, which
62
impart food products with satisfied appearance, taste, texture, and rheological behavior.
63
However, the industrial application of WPI is limited due to reduced solubility,
64
decreased emulsifying stability, and even coagulation in certain processing conditions as
65
high ionic strength, pH and/or temperature(Chen and Lv et al., 2019).
66
To overcome these limits, many efforts have been made to improve or alter the
67
performance of WPI by using physical, chemical and/or enzymatic approaches(Sutariya
68
and Patel, 2017). However, the potential health risks associated with reagents used in the
69
above processes remain a major concern. Recently, conjugation of the ε-amino groups
70
of the amino acid or protein with reducing sugar via Maillard reaction without using any
71
other chemicals has received great attention(Oliveira and Coimbra et al., 2016). The most
72
notable fact is that this reaction is capable of improving emulsifying properties, thermal
73
stability, and antioxidant effect by forming the covalently conjugated products(Cui and
74
Steve et al., 2013). Monosaccharides like glucose and disaccharides like lactose and
75
maltose are often used to study the glycosylation of proteins(Zhao and Zhou et al., 2016;
76
Liu and Wang et al., 2019; Wang and Li et al., 2019). The increasing evidence showed that the
77
functional properties obtained by glycation of protein with polysaccharide were far
78
superior to those of obtained with mono- or disaccharides(Chen and Lv et al., 2019).
79
Flaxseed gum (FSG), an anionic hydrophilic colloid, are naturally-occurring plant
80
polysaccharide, accounts for 8%-10% of the weight of flaxseed. The annual production
81
of flaxseed in China is about 450 kilotons, accounting for 20% of total world production.
82
There are more than 165 kilotons flaxseed oil and 20 kilotons flaxseed gum every
83
year(Yousuf and Srivastava, 2017). In the past few decades, the extraction, characterization, 4
84
chemical composition and physiochemical properties of FSG has been completely
85
revealed. FSG can be extracted from flaxseed, flaxseed hull, or flaxseed cake(Liu and
86
Shim et al., 2018; Rashid and Ahmed et al., 2019) . After extraction of oil from flaxseed, a
87
large amount of flaxseed cake is generated and it is generally used as fertilizer, apart
88
from its limited use for livestock and poultry feed. Its potential value is far from being
89
developed, and FSG obtained from flaxseed cake has not been fully utilized. Moreover,
90
the uncontrolled hydration rate of FSG restricts its wide application(Liu and Shen et al.,
91
2016).
92
Recently, the renewed interest in FSG as food source due to its health benefits
93
attributed to reduction of blood glucose, cholesterol, and antioxidtive activity(Thakur and
94
Mitra et al., 2009; Nounou and Deif et al., 2012) . In addition to its thickening and
95
water-holding capacities, FSG also plays an important
96
emulsions(Wang and Feng et al., 2017). It is found that FSG exhibited much lower
97
viscosity at a concentration of 0.3%(w/v) than locust bean gum, guar gum, and xanthan
98
gum(Qian and Cui et al., 2012). FSG can effectively improved the texture properties of
99
lotus root starch gels, making starch gels softer and have a better mouthfeel, which is
100
suitable to produce gelatinised food(Liu and Xu, 2019). Therefore, it is supposed that FSG
101
can potentially replace gum Arabic in food emulsion(Kaushik and Dowling et al., 2017) and
102
is expected to be more widely used in the food industry because of its sustainable,
103
biodegradable functional properties(Rashid and Ahmed et al., 2019) and unique nutritional
104
value(Xu and Qi et al., 2016).
role
in stabilizing
105
Hitherto, many attempts have been made to improve the performance of WPI by
106
addition of FSG. It has been revealed that the interaction and attachment of FSG onto
107
the surface of WPI-stabilized emulsion through electrostatic interaction had a significant
108
impact on the microrheological and physicochemical properties of WPI-stabilized
109
emulsion(Khalloufi and Corredig et al., 2009; Xu and Qi et al., 2016) . Liu’s research
110
demonstrated that FSG-WPI mixtures in aqueous solution favored electroneutral
111
coacervate formation with a more compact structure than charged coacervates, which
112
attributed to improved rheological properties (Liu and Shim et al., 2017). Zhang found that 5
113
the addition of FSG increased the viscosity of FSG-WPI solutions and the strength of
114
the mixed gels(Zhang and Li et al., 2013). However, up-to-date the covalent conjugates
115
between FSG and WPI produced via Maillard reaction and their functional properties
116
for application in food industry has not been systematically investigated. Thus, the
117
present study aimed to fill the above research gap by investigating the conjugation of
118
FSG and WPI via controlled dry-heating Maillard reaction at various incubation/
119
reaction time and to evaluate the functional properties of the conjugates produced
120
including antioxidant activity, thermal stability and emulsifying properties.
121
122
2. Materials and methods
123
2.1. Materials
124
FSG (90%) with viscosity of 12,250 mPa·s was purchased from Li Shi De Bio
125
Technology Co. Ltd. (Xinjiang, China). The average molecular weight of the FSP was
126
1.7×104 - 5.7×106 Da, consisting of mannose, galactose, glucose, arabinose, glucuronic
127
acid, xylose, rhamnose, ribose, galacturonic acid. The natural carbohydrate, uronic acid,
128
and protein contents were 55.20±3.07%, 37.78±1.26%, and 2.93±0.06%, respectively.
129
The WPI (90.46%) was purchased from Fonterra Co-operative Group Ltd. (Auckland,
130
New Zealand). Medium-chain triglyceride (MCT) was provided by Houman Biological
131
Technology Co. Ltd. (Shanghai, China). All other chemicals were of analytical grade.
132 133
2.2. Preparation of FSG-WPI conjugates
134
The conjugation process was carried out according to the method described by previous
135
researchers(Yang and Cui et al., 2015; Tamnak and Mirhosseini et al., 2016) , with minor
136
modification. FSG and WPI were dispersed in 0.01 M phosphate buffer solution (PBS，
137
pH=7.2~7.4) at mass ratio of 1:3 with stirring at room temperature (25 ± 1C) for 2 h,
138
and then stored at 4C overnight for complete hydration. The resultant solution was
139
successively freeze-dried, ground, and sieved (300 µm) to obtain uniform particles. The 6
140
powder was incubated (LHS-80HC-I; Yiheng, Shanghai, China) at 60C in relative
141
humidity (RH) of 79%. At 22h, 48h, and 72h, the generalted FSG-WPI conjugates were
142
taken out and stored at 4C for further analysis.
143 144
2.3. Determination of the browning intensity
145
Browning intensity(BI) of FSG-WPI conjugates collected at selected intervals were
146
measured to determine the progress of the reaction(Zhao and Zhou et al., 2016). Briefly,
147
FSG-WPI conjugates dispersions (5 mg/mL) were prepared using Milli-Q water, and the
148
absorbance was measured at 420 nm on a SpectraMax M2 (Molecular Devices, Silicon
149
Valley, USA). The experiments were performed in triplicate.
150 151
2.4. Determination of free amino group content
152
To determine free amino group (FAG) content in the FSG-WPI conjugates, 1mL of 5
153
mg/mL FSG-WPI conjugate solution was mixed with 1 mL of 4% NaHCO 3 (pH 8.5)
154
solution and 1 mL of 0.1% (w/w) 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) solution,
155
and then stored at 40C for 2 h. Subsequently, 1 mL of 10% sodium dodecyl sulfate
156
solution and 0.5 mL of 1 M HCl solution were added to terminate the reaction. The
157
absorbance of the resultant solution was measured at 340 nm. The FAG concentration
158
was calculated through the following equation:
159
FAG(%) = 𝐴 𝑡 × 100%
160
where A0 is the absorbance of the FSG-WPI mixture, At are the absorbances of the
161
conjugate solutions with different incubation times. The FAG concentration was
162
measured in triplicate.
𝐴
(1)
0
163 164
2.5. Determination of sulfhydryl content on the protein surface
7
165
Total surface sulfhydryl content on the protein surface was analyzed according to a
166
modified method(Qi and Xiao et al., 2017). The samples were dissolved in the buffer
167
solution (PBS containing 1.0 mM EDTA, pH 8.0) to a concentration of 20 mg/mL. The
168
sample solution (500 µL) and buffer solution (2.25 mL) were mixed with 50 µL of 1.0
169
mM 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman reagent) solution. Afterward,
170
the reaction solution was kept in the dark for 20 min at 25C. The absorbance was
171
measured at 412 nm against a blank, which is 500 µL of reaction buffer solution. All
172
experiments were performed in triplicate. The sulfhydryl group content, [SH], was
173
calculated through the following formula:
174
[SH] (µmol/g) =
175
where A412 is the absorbance at 412 nm, 13,600 is the extinction coefficient, C is the
176
protein concentration (mg/mL), and D is the dilution factor of the sample solution.
106 ×A412 ×D
(2)
13,600×C
177 178
2.6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
179
SDS-PAGE using 15% (w/v) acrylamide separating gel was performed according to a
180
modified
181
Radioimmunoprecipitation (RIPA) lysis buffer (strong) and with 10% (w/v) SDS were
182
prepared. The conjugate solution (20 μL) was mixed with 5 μL of 5× loading buffer, and
183
the mixture was denatured at 100C for 5 min. Electrophoresis was carried out at 90 V
184
for 20 min and then at a voltage of 150 V for 70 min. Subsequently, the gel was stained
185
using Coomassie brilliant blue R-250. The electropherograms were obtained using a
186
Bio-Rad GS-800 gel scanner (California, USA).
method
described
by
Laemmli(Laemmli,
1970).
Samples
in
187 188
2.7. Fourier transform infrared (FT-IR) spectroscopy
189
For FT-IR spectroscopy, samples were freeze-dried for 24 h and then stored in a
190
decicator. The dry powder samples were mixed with potassium bromide (KBr) at a ratio 8
191
of 1:100 and then fully ground and compressed under 20 MPa for 2-3 min to form
192
uniform slices. All samples were scanned from 4000-400 cm−1 at a resolution of 4 cm-1
193
using a Perkin Elmer FT-IR 1600 instrument (Norwalk, USA).
194 195
2.8. Ultraviolet-visible (UV-VIS) spectroscopy
196
Sample dispersions at a concentration of 0.5 mg/mL in PBS (pH = 7.2-7.4) were
197
prepared. Absorbance spectra were obtained on a SpectraMax M2 (Molecular Devices,
198
Silicon Valley, USA), and the wavelengths were recorded from 220 to 400 nm.
199 200
2.9. Antioxidant activity
201
The scavenging efficiency of FSG-WPI conjugates for DPPH• radical was
202
evaluated through the method(Cheng and Yu et al., 2019), with slight modifications. In
203
brief, 0.1 mM of DPPH• in ethanol solution and FSG-WPI conjugate solutions of
204
different concentrations subjected to reactions for different durations were prepared.
205
The butylated hydroxytoluene (BHT) in ethanol solution (0.5 mg/mL) was used as
206
control. Afterward, 1 mL of FSG-WPI conjugate solution was mixed with 1 mL of
207
DPPH• of solution and shaken well. The reaction system was placed away from light for
208
30 min at room temperature. At was measured at 517 nm on a SpectraMax M2. Milli-Q
209
water (1 mL) was used instead of sample solution in the same operation, and the reading
210
was recorded as Ac. In addition, the absorbance of a 1 mL sample solution was mixed
211
with 1 mL of absolute alcohol, and the reading was recorded as Ab. Experiments were
212
conducted in triplicate. The scavenging effect of FSG-WPI conjugates on DPPH•
213
radical was calculated as follows:
214
Scavenging Efficiency (%) =
215
Where At is the absorbance of sample solution with DPPH• solution, Ac is the
216
absorbance of Milli-Q water with DPPH• solution, and Ab is the absorbance of sample
217
solution with absolute alcohol.
𝐴𝑐 −(𝐴𝑡 −𝐴𝑏 ) 𝐴𝑐
× 100
9
(3)
218 219
2.10. Thermal properties
220
The thermal properties of conjugates were characterized by differential scanning
221
calorimetry (DSC) using a TA Instruments Q2000 (New Castle, USA). About 6 mg of
222
conjugate powder was loaded in a sample pan in a nitrogen atmosphere, and an empty
223
sample pan was used as reference. The temperature was raised from 30 oC to 250 oC at a
224
rate of 5 oC/min. The variations of heat flow with temperature were recorded.
225 226
2.11 Emulsifying activity index (EAI) and emulsion stability index (ESI)
227
EAI and ESI were determined by the turbidimetric method of Pearce and Kinsella
228
(Pearce and Kinsella, 1978) with minor modifications. For emulsion formation, 30 mL of
229
0.5 % (w/v) protein solutions in 100 mM PBS (pH 7.0) and 10 mL of soybean oil were
230
homogenized in Ultra-Turrax T25 homogenizer (IKA Werke GmbH & Co. KG, Staufen,
231
Germany) at 10,000 rpm for 1 min. One hundred microliters of the resultant emulsion
232
was withdrawn at 0 min and 10 min, diluted (1:100, v/v) with 0.1 % (w/v) SDS solution.
233
After shaking in a vortex mixer for 5 s, the absorbance of dilute emulsions was
234
determined at 500 nm using a UV2300 spectrophotometer (Techcomp, Shanghai, China)
235
immediately against a blank (0.1 % (w/v) SDS solution instead of the emulsion). The
236
EAI was determined from the absorbance measured immediately after the emulsion had
237
formed (0 min). The EAI and ESI were calculated using the following equations:
2 2.303 A0 N m2 ) g c L 10000
238
EAI (
239
ESI (min)
240
where, N was the dilution factor, c was the protein concentration (g/m3), Φ is the
241
oil volume fraction (v/v) in the emulsion, L is the optical path (1 cm), and A0 and A10
242
are the absorbance of diluted emulsions at 0 and 10 min, respectively; t is 10 min.
A0 t A0 A10
10
243
Measurements were performed in triplicate.
244 245
2.12. Emulsion preparation and analysis
246
The samples were dissolved in Milli-Q water to a concentration of 0.5% (w/w), and
247
then MCT was added slowly to the solutions to a concentration of 5%. The emulsions
248
were homogenized using an IKA T25 digital Ultra-Turrax (Staufen, Germany) at 15,000
249
rpm for 3 min in an ice bath. The particle size of the emulsions was measured on a
250
Mastersizer 2000 (Malvern, UK) and the zeta potential was determined on a Malvern
251
Zetasizer Nano-ZS90 (Malvern, UK). The refractive indexes of the oil and aqueous
252
phases were set at 1.54 and 1.33, respectively.
253
2.13. Evaluation of emulsion stability
254
The stability of the emulsion samples was evaluated as described previously (Bi and Yang
255
et al., 2017), with slight modification. The emulsions were stored at 4 oC for 13 days.
256
Then observation of macroscopic features and measurement of particle size distribution
257
were conducted (section 2.12). All experiments were performed in triplicate.
258 259
2.14 Statistical analysis
260
All the samples were measured in triplicate and results were expressed as mean ±
261
standard deviation (SD).
262
263
3. Results and discussion
264
3.1. Formation of the FSG-WPI conjugates
265
3.1.1. BI and FAG content
266
Maillard reaction involves a series of complex non-enzymatic browning reactions. The
267
extent of the Maillard reaction can be indicated by the decrease in the protein or 11
268
carbohydrates involved in the reaction and the degree of browning. Therefore, the BI
269
and FAG content of the protein and polysaccharide conjugates have been commonly
270
used to characterize the progress of the Maillard reaction(Chen and Xue et al., 2014;
271
Setiowati and Vermeir et al., 2016; Han and Yi et al., 2017) . As shown in Fig. 1 (a), elongated
272
heating led to increased BI and decreased FAG content, indicating the Maillard reaction
273
was occurred during dry heating process(Guan and Qiu et al., 2006; Jafar and Fadia, 2010).
274
The FAG content decreased rapidly from time zero to 22 h, and then became nearly
275
constant at 65%. These observations were supported by Bi et al.(Bi and Yang et al., 2017),
276
who studied the conjugation of β-lactoglobulin (β-LG) with gum from Acacia Seyal.
277 278
3.1.2. Sulfhydryl content of the protein surface
279
As shown in Fig. 1 (b), a reduction of sulfhydryl content of the WPI was observed after
280
48 h of heating in comparison with the native WPI, which was mainly due to protein
281
denaturation and aggregation. However, the surface sulfhydryl content increased in the
282
FSG-WPI system during dry heating process, indicating the intramolecular sulfhydryl
283
groups could be exposed to the surface due to the changes of protein structure during
284
Maillard reaction. The broken disulfide bond induced by heating also contributes to the
285
increase of sulfhydryl groups(Creamer and Bienvenue et al., 2004). In addition, the
286
sulfhydryl activity on the molecule surface was enhanced during the reaction(Schmidt
287
and Illingworth et al., 1979) due to thermal denaturation, which causes its exposure and
288
participation in sulfhydryl/disulfide linkages interchange reaction, involving both β-LG
289
and α-LG(Wang and Ismail, 2012). It is therefore not surprising that the result led to an
290
increasing trend of sulfhydryl content on the protein surface, albeit it was less than that
291
on the native WPI.
292 293
3.1.3. SDS-PAGE
294
SDS-PAGE was used to confirm the covalent cross-linking between FSG and WPI due 12
295
to the formation of high-molecular-weight conjugates(Schmidt and Pietsch et al., 2016). As
296
shown in Fig. 2, the FSG-WPI mixture without dry-heating treatment (lane 1) produced
297
prominent bands for α-lactalbumin, β-LG, β-LG dimer, and bovine serum albumin
298
consistent with literature(Schmidt and Pietsch et al., 2016; Setiowati and Vermeir et al., 2016) ..
299
The molecular weights of all protein-containing substances were below 130 kDa.
300
Low-molecular-weight bands in lanes 2-4 seemed to be gradually fading.
301
Simultaneously, high-molecular-weight bands appeared at the boundary between the
302
separating gel and stacking gel. These phenomena became more obvious with prolonged
303
incubation time. Therefore, we could conclude that there was covalent conjugation of
304
FSG and WPI mixtures by dry heating in agreement with previous studies (Liu and Ma et
305
al., 2016; Mengibar and Miralles et al., 2017). Similar SDS-PAGE patterns for egg
306
white-pectin conjugates were reported by Al-Hakkak(Jafar and Fadia, 2010). The
307
SDS-PAGE profiles are consistent with the reduction in FAG content of the FSG-WPI
308
conjugates (Fig. 1(a)). Based on these results, it was clear that conjugation of FSG and
309
WPI had occurred where the carbonyl group of FSG was bonded to the amino groups of
310
WPI through the Maillard reaction.
311 312
3.1.4. FT-IR spectroscopy
313
FT-IR spectroscopy can be useful to investigate the molecular structure and interactions
314
of protein polysaccharide systems(Liu and Shen et al., 2016). The characteristic absorption
315
peaks of FSG located at wavenumbers 3438, 2928, and 1639 cm−1 (Fig. 3) were
316
attributed to O–H, C–H, and C=O stretching vibrations respectively(Zhou and Hu et al.,
317
2016). An absorption peak for WPI at 2928 m−1 is due to anti-symmetric stretching of –
318
CH2. The most distinctive absorption features for WPI were observed at 1647 cm−1
319
(C=O stretching) and 1541 cm−1 (N–H bending)(Gu and Jin et al., 2010). In the spectra of
320
the FSG-WPI mixture, the intensity of the band at 2928 cm−1 was markedly reduced.
321
This may be due to the intermolecular interactions resulting in the reduction of
322
methylene vibrations. After incubation of the FSG-WPI mixture for 48 h, the spectra
323
showed apparent differences in intermolecular interaction between FSG and WPI. It can 13
324
be observed that the characteristic band of O–H at 3456 cm−1 became flatter, indicating
325
the formation of hydrogen bonds. Additionally, the characteristic band of FSG at 1639
326
cm−1 (C=O stretching) disappeared, and the intensity of the band for WPI at 1541 cm−1
327
(N–H bending) decreased remarkably while a new band appeared at 1637 cm−1 (amide
328
bond). These changes were ascribed to the Maillard reaction between FSG and
329
WPI(Golkar and Nasirpour et al., 2016).
330 331
3.1.5. UV-VIS spectroscopy
332
To further explore the interaction between FSG and WPI, the UV-VIS absorption
333
spectra of FSG after 48h of heating (FSG-48h), WPI after 48h heating (WPI-48h),
334
FSG-WPI mixture, and conjugates were studied (Fig. 4). The main absorption peaks of
335
FSG-48h and WPI-48h were observed at 260 and 280 nm, respectively. The absorbance
336
intensity of the FSG-WPI conjugates increased with the reaction time, and the UV-VIS
337
absorption maximum showed a characteristic red shift with the incubation period. This
338
red shift may be explained by a difference in the Schiff base environments, which led to
339
formation of Schiff base products. Meanwhile, it was also noticed that the sample
340
solutions gradually turned into yellow color with prolong incubation time from 22 to 72
341
hours. These changes further indicate that FSG and WPI had interacted to produce
342
Maillard products. Similar results were published by Zhu et al.(Dan and Srinivasan et al.,
343
2008), in which they reported a red shift of the UV absorption maximum of WPI-dextran
344
conjugates.
345 346
3.2. DPPH• scavenging activity
347
Results show that FSG-WPI conjugates had the ability to scavenge DPPH• (Fig. 5). The
348
scavenging capacity of the conjugates increased with the incubation time and
349
concentration of conjugates. A dramatic increase in the antioxidant activity of the
350
conjugates was observed during incubation for 22 to 48 h, especially for higher 14
351
concentration of conjugates. After 48 hours of incubation, the scavenging capacity of
352
the 6 mg/mL conjugate reached 91.17%. To give a better comparison, we had conducted
353
DPPH assay for the commercial antioxidant BHT and observed its scavenging capacity
354
as 87.12% at 0.5 mg/mL. Current results also show that the antioxidant activity of
355
conjugates at a concentration of 2 mg/mL was limited, and the DPPH• scavenging
356
ability did not apparently improve with longer incubation period. In a previous study,
357
ovalbumin-glucose conjugates and ovalbumin-maltose conjugates formed by Maillard
358
reaction had shown to exhibit a drastic increase in DPPH• scavenging activity under
359
heat/moisture treatment at 120 C for 20 min(Huang and Tu et al., 2012).
360 361
3.3. Thermal properties
362
It has been reported that Maillard reaction between proteins and polysaccharides can
363
improve the thermal stability of proteins and their mixtures(Liu and Ma et al., 2016). DSC
364
can detect alterations of the sample in the heat flow during temperature changes, and it
365
can provide very useful information related to thermal stability of the sample.
366
From the DSC characteristics of the FSG-WPI mixture and the conjugates at different
367
incubation times (Fig. 6), we observed that the denaturation temperatures of the
368
conjugates were markedly higher than that of the mixture. The denaturation temperature
369
of the conjugate after 22 h of incubation increased from 129 ºC to 165 ºC. Higher
370
denaturation temperature means better thermal stability. The result indicates that the
371
thermal stability of the FSG-WPI mixture was improved markedly through the Maillard
372
reaction. This phenomenon may be explained by the increase in the steric repulsion
373
forces between WPI molecules due to glycation(Liu and Ma et al., 2016). The onset
374
temperature of FSG and WPI was 108 ºC while the FSG-WPI conjugates increased with
375
incubation time, from 140 ºC to 150 ºC. Previous studies had reported that the glycation
376
could enhance the heat stability of protein, thus our results were in agreement with the
377
others(Huang and Tu et al., 2012).
378 15
379
3.4. Emulsifying properties
380
3.4.1 Emulsifying activity index (EAI) and emulsion stability index (ESI)
381
The EAI and ESI of WPI and FSG-WPI mixture were investigated. As can be observed
382
from Fig.7(a), with the increase of reaction time, the EAI of FSG-WPI mixture
383
increased gradually, and then descended slightly. The same result has also been reported
384
in a previous literature(Li and Wang et al., 2015; Chen and Lv et al., 2019; Xu and Huang et al.,
385
2019). The slight decline of EAI might be attributed to the developing of polymerization
386
products, which could decrease the molecules mobility and make the conjugates
387
absorbed in the oil/water interface much slower. The ESI of FSG-WPI mixture dropped
388
sharply, and then had a slight increase. Significant increase in EAI of FSG-WPI
389
conjugates could be mainly attributed to the conformation ability of protein to expose
390
the hydrophobic groups and lysyl residues buried in the interior which react easily with
391
the reducing-end carbonyl group in polysaccharides. A new balance value of
392
hydrophobic and hydrophilic groups consents to be achieved and favor of emulsion
393
formation. Additionally, it can also be attributed to conjugating WPI with highly soluble
394
and charged saccharides, proteins in the emulsions were able to provide a better
395
potentiality for the adsorption at the oil-water interface, resulting in enhancement of
396
emulsion properties. After Millard reaction, there was a slight increase trend of ESI of
397
FSG-WPI mixture as a function of reaction time. Millard reaction facilitated the
398
emulsion stability of FSG-WPI mixture.
399
3.4.2 Emulsion stabilized by FSG-WPI mixture
400
The partical size of FSG-WPI-0h mixture and FSG-WPI conjugates storaged at 4 oC for
401
2, 6, and 13 days are shown in Fig. 7(b). The droplet size distributions of the fresh
402
emulsions in Fig. 7(c) show substantial differences in particle size distribution of the
403
emulsions stabilized by the FSG-WPI mixture and the conjugates incubated for different
404
durations. The emulsions stabilized by the FSG-WPI conjugates after Maillard reaction
405
(especially those conjugated for 48 and 72 h) had fewer large droplets as compared with
406
that stabilized by the FSG-WPI mixture. The results clearly indicate that the 16
407
emulsifying activity of the FSG-WPI conjugates improved with the progress of Maillard
408
reaction. The droplet size distribution of the emulsions stored for 13 days (Fig. 7(d))
409
produced with FSG-WPI-48h and FSG-WPI-72h conjugates did not change noticeably
410
when compared with that of the fresh sample (Fig. 7(c)). However, the particle size
411
distribution of the FSG-WPI mixture had a notable change because of droplets
412
aggregation. These findings indicate that the stability of the emulsions is improved by
413
the FSG-WPI conjugates produced via Maillard reaction, especially with a reaction time
414
of 72 h. Visual observation of macroscopic appearance show that the emulsions
415
stabilized by the conjugates (after 48 and 72 h of incubation) exhibited uniform
416
behavior after storage at 4°C and for 13 days. However, creaming was observed in the
417
emulsions stabilized by the native WPI and FSG-WPI mixture and the conjugates
418
produced by 22 h of incubation, indicating lesser emulsion stability, which conformed to
419
the results of ESI.
420
The changes in zeta potential of the emulsions stabilized by WPI, the FSG-WPI
421
mixture, and the FSG-WPI conjugates is as shown in Table 1. The zeta potential values
422
of the FSG-WPI conjugates after 48 and 72 h of incubation were −40.1 mV and −41.9
423
mV, respectively. These values were higher than those of WPI and the FSG-WPI
424
mixture, indicating better emulsion stability of the FSG-WPI conjugates than the
425
formers. These phenomena, once again, illustrating that Maillard reaction has improved
426
the emulsifying properties of the FSG-WPI conjugates and the incubation time has an
427
obvious impact on this properties.
428
Our results are in agreement with the work reported by Kato et al. (Kato and Minaki et
429
al., 1993). Their study showed that the conjugate of egg white protein and galactomannan
430
formed by controlled dry-heating reaction had dramatically enhanced the emulsifying
431
activity and stability as compared with their mixture. In addition, they also observed
432
that the conjugate had better emulsifying properties than some commercial emulsifiers
433
including sucrose-fatty acid ester and decaglyceryl monostearate. The emulsifying
434
properties of conjugates obtained via reaction between β-LG and gum from Acacia
435
Seyal were found to improve with the extent of Maillard reaction(Bi and Yang et al., 2017), 17
436
consistent
with
our
findings.
The
improvement
of
emulsion
stability
of
437
protein-polysaccharide conjugates by Maillard reaction may be mainly due to the
438
presence of hydrophilic polysaccharide, which provides sufficient steric repulsion and
439
electrostatic repulsion around the oil droplets, this protecting the oil droplets and
440
preventing their aggregation(Zhou and Wang et al., 2016). Similar results were also
441
reported by Xu et al.(Xu and Qi et al., 2017), who studied the effect of FSG on WPI
442
stabilized β-carotene emulsions, and they proved that FSG at a concentration of 0.1 wt%
443
exhibited a remarkable increase in physical stability.
444
4. Conclusions
445
Current study was conducted to investigate the functional properties of FSG-WPI
446
conjugates produced via Maillard reaction in an attempt to explore the potential
447
application of FSG in food industry. Our results show that BI and surface sulfhydryl
448
content increased with increasing incubation time, while the FAG content first
449
decreased rapidlybefore approaching a constant value. Both SDS-PAGE and FT-IR
450
spectroscopy confirmed the formation of large-molecular-weight fragments and the
451
binding of carbonyl groups and amino groups and thus the occurrence of Maillard
452
reaction. The conjugates showed substantially improved antioxidant activity and
453
thermal stability, especially that obtained after 72 h of incubation time. Furthermore,
454
this conjugate also showed the best emulsifying activity after 22 h and stability as
455
reflected by emulsion particle size distribution and zeta potential measurements. The
456
conjugate, thus, appears to have promising applications for nutrient delivery and for
457
effective emulsifiers because of their improved functional performance.
458 459
Acknowledgements
460
This work was supported by the National Natural Science Foundation of China
461
(Grant No 31701643 and 31171661), National High Technology Research and
462
Development Program of China (863 Program: 2017YFD0400200). 18
463 464
Conflict of interest
465
We declares that he has no conflict of interest.
466
19
467 468
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583 584 585
23
586
Table 1
587
Zeta potentials of freshly prepared emulsions with whey protein isolate (WPI) (0 h), FSG–WPI
588
mixture (0 h), and FSG–WPI Maillard reaction products (22, 48, and 72 h) at 25ºC.
Aqueous phase
Z-potential (mV)
WPI-0h
−36.6 ± 0.56
FSG-WPI-0h
−36.7 ± 0.21
FSG-WPI-22h
−35.6 ± 1.35
FSG-WPI-48h
−40.1 ± 1.81
FSG-WPI-72h
−41.9 ± 2.02
589
24
590
Figures captions
591
Fig. 1 Effect of incubation time on the formation of the FSG-WPI conjugates. a: Change in free
592
amino group (FAG) content and browning intensity (BI) of FSG-WPI conjugates during dry heating.
593
Samples include a mixture at 0 h incubation time, as well as the conjugates after 22, 48, and 72 h of
594
incubation time; b: Soluble sulfhydryl content ([SH]) of WPI, WPI at 48 h, FSG-WPI mixture, and
595
conjugates as determined using Ellman reagent.
596
Fig. 2 SDS-PAGE pattern of FSG-WPI conjugates at different incubation times. Lane 1 contains a
597
mixture of FSG and WPI. Lanes 2, 3, and 4 contain the conjugates of FSG-WPI subjected to dry
598
heating for 22, 48, and 72 h, respectively. MW contains the protein markers that indicate molecular
599
weights (kDa).
600
Fig. 3. FT-IR spectra of FSG, WPI, FSG-WPI-0h mixture, and FSG-WPI-48h conjugate.
601
Fig. 4. UV-VIS spectra of FSG-48h, WPI-48h, FSG-WPI mixture, and conjugates. The concentration
602
of all samples was 0.5 mg/mL.
603
Fig. 5. Changes in DPPH• scavenging efficiency of the FSG-WPI conjugates at concentrations of 2,
604
4, and 6 mg/mL and at different incubation times.
605
Fig. 6. DSC analysis of FSG-WPI-0h mixture and FSG-WPI conjugates incubated for 22, 48, and 72
606
h.
607
Fig. 7. Emulsifying properties of FSG-WPI mixture and FSG-WPI conjugates. The EAI and ESI of
608
FSG-WPI mixture and FSG-WPI conjugates (a); Partical size of FSG-WPI-0h mixture and
609
FSG-WPI conjugates after storage at 4 oC for 2, 6, and 13 days (b); Particle size distribution of the
610
emulsions stabilized by the FSG-WPI mixture and the conjugates incubated for different durations (c)
611
after 13 days (d).
612
Table 1 Zeta potentials of freshly prepared emulsions with whey protein isolate (WPI) (0 h),
613
FSG-WPI mixture (0 h), and FSG-WPI Maillard reaction products (22, 48, and 72 h) at 25ºC.
614 25
615 616
26
617 618
27
619 620
28
621 622
29
623 624
30
625 626
31
627
32