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Fabrication of caseins nanoparticles to improve the stability of cyanidin 3-O-glucoside
Journal Pre-proofs Fabrication of caseins nanoparticles to improve the stability of cyanidin 3-Oglucoside Yongzhong Ouyang, Lei Chen, Liu Qian, Xiujun Lin, Xiaoyun Fan, Hui Teng, Hui Cao PII: DOI: Reference:
S0308-8146(20)30278-8 https://doi.org/10.1016/j.foodchem.2020.126418 FOCH 126418
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Food Chemistry
Received Date: Revised Date: Accepted Date:
17 November 2019 10 February 2020 14 February 2020
Please cite this article as: Ouyang, Y., Chen, L., Qian, L., Lin, X., Fan, X., Teng, H., Cao, H., Fabrication of caseins nanoparticles to improve the stability of cyanidin 3-O-glucoside, Food Chemistry (2020), doi: https://doi.org/ 10.1016/j.foodchem.2020.126418
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1
Fabrication of caseins nanoparticles to improve the stability of cyanidin
2
3-O-glucoside
3
Yongzhong Ouyang1, Lei Chen2,#, Liu Qian2,#, Xiujun Lin2, Xiaoyun Fan2, Hui
4
Teng2,*, Hui Cao3,4*
5
1School
6
528000, China.
7
2College
8
China
9
3Guangdong-Macau
of Environmental and Chemical Engineering, Foshan University, Foshan
of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002,
Traditional Chinese Medicine Technology Industrial Park
10
Development Co., Ltd, Hengqin New Area, Zhuhai 519031, China
11
4Institute
12
Chinese Medicine, University of Macau, Macau
13
*Corresponding author: Hui Teng, [email protected]; Hui Cao,
14
[email protected]
15
#authors contributed equally to this study
of Chinese Medical Sciences, State Key Laboratory of Quality Research in
16 17
Abstract
18
The influence of encapsulation with caseins on the stability of cyanidin 3-O-glucoside
19
(C3G) was investigated. The modified casein nanoparticles (MCs) prepared at pH 5.5
20
after heated at 80 °C for 30 min was applied to encapsulate C3G. The diameter of
21
nanoparticle (MCs-C3G) was 110±0.31 nm and zeta-potential was -8.83±0.52 mV.
22
The molecular weight of α-casein (32 kDa) and β-casein (25 kDa) increased along 1
23
with the encapsulation of C3G. The interactions of MCs with C3G were examined at
24
pH 6.3 by fluorescence spectroscopy and IR spectroscopy. MCs encapsulated C3G
25
mainly via the hydrophobic interaction. The secondary structures of caseins were
26
changed along with the combination of C3G, with a decreasing in α-helix, turn
27
random, and coil structure, as well as increased β-sheet. In addition, the MCs-C3G
28
interaction appeared to have a positive effect on the thermal, oxidation and photo
29
stability of C3G.
30
Key words: cyanidin 3-O-glucoside; caseins; stability; nanoparticles; encapsulation
31 32
1. Introduction
33
Caseins (Cs) consist of phosphorylated α-, κ-, β-format, are the major proteins in
34
bovine milk (approximately 80% of the total milk proteins) (Dalgleish, 2011).
35
α-Casein contains two tryptophan (Trp) residues, while β-casein has one and with
36
stronger hydrophobic bond than α-casein and κ -casein (Dalgleish & Corredig, 2012).
37
As water soluble pigments, anthocyanins have been reported to present numerous
38
benefits for human health (Tang, Li, Bi, & Gao, 2016). There are six common
39
glycosidic and acylglycosidic derivatives of anthocyanidins, namely pelargonidin,
40
cyanidin, malvidin, delphinidin, peonidin, and petunidin, which are classified
41
according to the number and position of hydroxyl group on the flavan nucleus
42
(Sinopoli, Calogero & Bartolotta, 2019). Anthocyanins can exist in a stable form of
43
astragalus salt cations under acidic conditions, while with the increase of pH
44
gradually, the anthocyanins form become unstable chalcone. 2
45
Stability of polyphenols is crucial for the nutrition of the food and is directly
46
associated with the chemical structures of polyphenols (Xiao & Högger, 2015). The
47
physicochemical conditions such as pH, temperature, light, oxygen availability, metal
48
ions, chemical modification, enzyme, proteins, nitrite salt, sulfur dioxide as well as
49
ascorbic acid must be taken into account for the polyphenols’ stability (Xiao, 2018;
50
Cao et al., 2020). Our group's previous experiments optimized the extraction and
51
purification of anthocyanins in raspberry, and found that cyanidin 3-O-glucoside
52
(C3G) showed anti-diabetic and anti-obesity activities. However, C3G is susceptible
53
to the surrounding environment, such as temperature, pH, light intensity, and metal
54
ions, which greatly reduce its bioactivity and nutrition. Therefore, improving the
55
stability of anthocyanins is a current technical problem that needs to be solved. Based
56
on the important influence of the low stability of C3G on daily use, this study intends
57
to use C3G as the research object and α-casein as the conjugate, spectroscopic
58
techniques are used to analyze the combination mode, the binding distance and the
59
structural changes; the stability changes in different environments after their binding
60
were studied to determine the degradation mode and kinetic parameters. We aimed to
61
explain the influence mechanism of C3G and α-casein on the stability of anthocyanin
62
after binding, and provide a scientific theoretical basis for the rational and effective
63
development of the anthocyanins resources and its application in the food industry.
64 65
2. Materials and Methods
66
2.1.
Materials and chemicals 3
67
Casein with a protein content of 86% (41% α-casein, 35% β-casein) was purchased
68
from Kerry Group (Beloit, WI, USA). α-Casein with purity of 70% and β-casein with
69
purity of 98%，and C3G purity of 97% were purchased from Sigma-Aldrich Chemical
70
Co. (St. Louis, MO, USA). All other chemicals were of analytical grade and were
71
purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
72 73
2.2. Modified casein preparation
74
Modified casein (MCs) preparation was acted as following: first, casein (Cs) was
75
dissolved in 10 mM PBS buffer (pH 7.4) to obtain the solutions with the
76
concentrations of 1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL. Then, the solution was heated in
77
40-120 °C for 30 min, 60 min, 90 min and 120 min. After that, the mixtures were
78
placed in a water bath at 50 °C and stirred continuously for 60 min, and then acidified
79
with HCl 1 M to obtain MCs. The mean zeta potential value of Cs and MCs (0.05%
80
w/w) used in this study were measured by the Zetasizer Nano series from Malvern
81
Instruments Ltd (Worcs, UK). The data was analyzed using the Zetasizer Nano v3.30
82
software. The average value was taken from three readings and all measurements
83
were performed three times.
84 85
2.2. MCs-C3G preparation
86
MCs-C3G was prepared following previous study with minor modification (He et al.,
87
2016). To ensure complete solubilization of powders, all dispersions were first stirred
88
at 50 °C for 1 h using a magnetic stirrer and then kept at 4 °C for 24 h. The MCs-C3G 4
89
mixture was prepared with 1 mg/mL MCs and 100 μM C3G in PBS (pH 7.0). Final
90
sample powder was obtained by freeze-drying.
91 92
2.3. Transmission electron microscopy (TEM) analysis
93
The nanosphere aggregates was observed by using a HT 7700 transmission electron
94
microscopy (Tokyo, Japan) according to the method of McMahon, Du, McManus,
95
and Larsen (2009) with modifications. Dried MCs-C3G samples were diluted 10-fold
96
with ultra-pure water. The diluted sample and ammonium molybdate solution (2
97
g/100 mL) (1:1) were mixed and left for 3 min at room temperature. A drop of this
98
solution was placed on a copper mesh for 5 min before the excess liquid was drawn
99
off using filter papers. The mesh was examined using a TEM at an operating voltage
100
of 200 KV.
101 102
2.4. FTIR spectroscopic measurement
103
FTIR spectra were recorded on a Bruker Vertex 70 FTIR spectrometer (Beijing,
104
China). Sample powders were blended with KBr at a mass ratio of 1:100 and pressed
105
into a tablet for FTIR analysis. Interferograms were obtained at the spectral range of
106
4000-400 cm-1 with a resolution of 2 cm-1 and 32 scans. Additionally, PeakFit v4.12
107
software was used to separate peaks in the FTIR spectrogram to obtain peak area of
108
each absorption peak.
109 110
2.5. Fluorescence spectroscopy 5
111
Fluorescence spectroscopy was recorded on a Hitachi F-4600 Spectrometer (Tokyo,
112
Japan). The final concentration of MCs in each mixture was 1 mg/mL, and the
113
concentrations of C3G in the mixtures were 0, 10, 20, 30, 40, 50 and 60 μM. The
114
mixture solutions were determined by scanning emission wavelengths from 300 to
115
400 nm, with excitation wavelength of 280 nm at 293, 303 and 313 K to obtain the
116
intrinsic fluorescence spectroscopy of MCs. The excitation and emission slit widths
117
were 5.0 nm.
118 119
2.6 Data processing
120
In order to further understand the binding mechanism for MCs-C3G, the fluorescence
121
data were performed using the Stern-Volmer equation:
122
F0 1 K SV cq 1 K q 0C q F
(1)
123
where F0 and F are the fluorescence intensities in the absence and presence of
124
quencher, respectively. Kq is the bimolecular quenching constant, KSV is the
125
Stern-Volmer dynamic quenching constant, cq is the concentration of the quencher
126
and τ0 is the average lifetime of the molecule without any quencher [τ0 =10−8 s]. Ksv
127
and Kq are determined from the slope of the regression curves of F0/F against cq. The
128
quenching mechanism is divided into static quenching and dynamic quenching. When
129
Kq calculated according to Eq. (1) was much higher than the limiting diffusion rate
130
constant of the biomolecules (2×1010 M-1 s-1), indicated that static, and not dynamic
131
quenching was the main quenching mechanism between MCs and C3G.
132
For the static quenching mechanism, the binding constant (KS) of the complexes 6
133
and the binding sites numbers (n) were calculated by using the double logarithmic
134
Stern-Volmer equation (Eq. (2)).
135
log
136
The non-covalent forces of protein-ligand binding were calculated by the
137
thermodynamic parameters calculated from the following equations (Eqs. (3)-(5)).
138
G 1 H d d T T
139
G RT ln K s
140
G H TS
141
Where H is the enthalpy change, S is the entropy change and G is the free energy
142
change, R is the gas constant (8.314 J/mol K), T is the absolute temperature (K), and
143
KS is the binding constant at the corresponding temperature.
F0 F log K s n log cq F
(2)
(3) (4) (5)
144 145
2.7. Thermal, oxidation, photo and storage stability
146
The MCs-C3G mixtures and their corresponding MCs samples were subjected to
147
thermal, oxidation, photo and storage stability by following previous study (He et al.,
148
2016). The thermal stability test was performed by heating the samples in 10 mL
149
tubes wrapped in a water bath for 30 °C, 60 °C, 90 °C with 30 min, and then rapidly
150
cooled down for further analysis. The oxidation stability test was carried out by
151
addition of H2O2 into the samples at a final concentration of 0.5 %, 1%, 1.5% and
152
oxidizing for 1 h at room temperature in the dark. The photo stability test was
153
measured by illuminating the samples in 10 mL transparent tubes for 6 h, 18 h, 30 h at 7
154
room temperature in YZ18RR fluorescent lamps (Osram Co., Foshang, China). The
155
storage stability test was determined by placing in brown tubes for 15 days at -4 °C in
156
the dark. During the storage period, the content was analyzed (Lee, Durst, &
157
Wrolstad, 2005). The method based on the structural change of the C3G chromophore
158
between pH 1.0 and 4.5, calculated as follows:
159
A A 530 nm A 700 nm pH 1.0 A 530 nm A 700 nm pH 4.5
160
TAC (mg/L)
161
where TAC is the total anthocyanins contents (mg/L); MW (the molecular
162
weight)=449.2 g/mol; DF is the dilution factor; ε (molar extinction coefficient) =
163
26900; b is the path length in cm.
A MW DF 103 b
164 165
2.8. Statistical analysis
166
All experiments were repeated at least three times. Data were expressed as the mean
167
± the standard deviation (SD). Significant differences (p < 0.05) were identified by
168
the least significant difference procedure between the means.
169 170
3. Results and discussion
171
3.1 MCs structure
172
The intermolecular aggregation was generated through the hydrophobic interaction to
173
form unstable nanoparticles (Vemula, Li & John, 2006). As shown in Fig. 1, when the
174
concentration of casein is more than 2 mg/mL, casein will start to form polymers with 8
175
the increased particle size. The optimum formulation of MCs was 2 mg/mL casein at
176
80 °C for 30 min (Fig. 1) with the particle size of 110±0.31 nm and zeta potential of
177
-8.83±0.52 mV. When the pH of casein solution approached to the isoelectric point,
178
the protonation of amino acids on the protein led to enhance hydrogen and ionic bond
179
in the process of casein micelle formation (Ding, Huang, Cai & Wang, 2019). The
180
special interaction of π electrons provided by amino acid residues such as
181
phenylalanine, tryptophan, and tyrosine with cations provided by protonation of side
182
chains of amino acid residues could also result in a smaller particle size (Gallivan &
183
Dougherty, 2000). Meanwhile, the reduction of electrostatic repulsion interaction led
184
to the reduction of intermolecular distance, and the micellar structure became more
185
compact, which was manifested as the reduction of particle size.
186 187
3.2 Analysis of transmission electron microscopic (TEM) in the presence of C3G
188
As shown in Fig. 2, the TEM results clearly indicated the acidification and thermal
189
treatment cause the change in the structural features and associated function of caseins
190
(Fig. 2A, B). When C3G added to MCs solution, a thin coated layer covered upon the
191
interface (Fig. 2C), indicating the core-shell structure consequently formed. Caseins
192
underwent a series of physical and chemical alterations by the guidance of acid-heat
193
treatment, and made the C3G and MCs bind more closely by the ultrasonic dispersion
194
and shearing effect. It was supposed that the lighter spot (Fig. 2D) in the center of
195
MCs-C3G nanocomplexe was C3G core, which was in agreement with published
196
results (Chitkara and Kumar, 2013; Fang et al., 2014). It was further confirmed that 9
197
the encapsulation of C3G with MCs and the formation of core–shell nano-complexes.
198
The changes of nanoparticles size are in agreement with the findings from casein
199
modification studies, which casein tends to shrink in acidic solution and swell in
200
alkaline solution (Liu & Guo, 2008).
201 202
3.3 FTIR spectroscopy of MCs-C3G
203
FTIR spectroscopy is an effective method to determine the secondary structure of
204
caseins. Both intensity variations and spectral shifting for the protein amide I band at
205
1600–1700 cm-1 (C=O stretch) and the amide II band, which occurs in the region
206
<1548 cm-1 (C–N stretching coupled with N–H bending), have been widely used upon
207
polyphenol interaction and are related to the secondary structure of the proteins (Peng
208
et al., 2015; Zhang, Wright, & Zhong, 2013). As shown in Fig. 3A, the peak position
209
of amide I moved from 1656.63 to 1653.47 cm-1 and that of amide II moved from
210
1538.17 to 1543.05 cm-1 in the MCs IR spectrum after interaction with C3G,
211
respectively. Changes of amide I and II bands were due to C3G binding to the caseins
212
C=O, C–N and N–H groups and modified casein via hydrogen bonding and
213
hydrophobic attraction. In addition, the intensities of the amide I of casein decreased
214
after binding with C3G, indicating that the α-helical content in the casein structure
215
was reduced. A peak position of amide A moved from 3411.2 to 3408.1 cm-1 in the
216
MCs and MCs-C3G, the shifting of the amide A band at 3300-3400 cm-1 (N–H
217
stretching mode) could be the additional evidence on caseins structure changes (Qi,
218
Ren, Xiao, & Tomasula, 2015). 10
219
A quantitative analysis of the caseins secondary structure for the MCs and MCs-C3G
220
has been carried out and the results are shown in Fig. 3C and 3D. The MCs has 18%
221
α-helix, 28.3% β-sheet, 36.5% turn structure and random 17.2% coil. The MCs-C3G
222
has 15.9% α-helix, 24.0% β-sheet, 44.9% turn structure and random 15.2% coil.
223
These results are consistent with spectroscopic study of caseins previously reported
224
(Menikh & Fragata, 1994). Upon the interaction, the α-helix, β-sheet and random
225
decreased, whilst turn structure increased in the MCs-C3G complexes, which are
226
indicative of larger perturbations of caseins secondary structure by C3G. The changes
227
of the caseins secondary structure will be discussed with fluorescence data further on.
228 229
3.4 Fluorescence spectroscopy of MCs-C3G
230
Figure 3B showed the fluorescence emission spectra of MCs in the presence of C3G
231
(0–60 μM) with an excitation wavelength of 280 nm. The fluorescence intensities of
232
caseins decreased with the increasing concentration of C3G, which is ascribed to the
233
quenching effect of C3G. The quenching efficiency of 60 μM C3G on MCs
234
fluorescence was 66.7%. The λmax of MCs appeared a blue shift with higher C3G
235
concentrations. This indicated that the polarity of microenvironment around the Trp
236
and Tyr residues in modified caseins had increased and the hydrophobicity had
237
decreased after the addition of C3G. In addition, the results revealed that modified
238
caseins conformations changes were ascribed to binding with C3G (Chen, Xie, Jiang,
239
& Yao, 2008).
240 11
241
3.5 The mechanism of encapsulation
242
The quenching parameters of caseins with different concentrations of C3G at 293, 303
243
and 313 K are shown in Figure 4 and Table 1. The values of KSV were Kq values were
244
all much higher than the dynamic quenching constant 2×1010 M-1 s-1, which indicated
245
that C3G could quench the fluorescence of MCs via the static quenching process
246
predominantly caused by the formation of complex. The Ks of MCs bound with C3G
247
fluorescence quenched at 293 K, 303 K, 313 K were all in the order of 105, with the
248
values of 2.54, 3.03, 1.04, respectively, which confirmed the strong binding affinity
249
between C3G and MCs. Additionally, all of the values of n were approximately 1,
250
suggesting there was around one binding site in the MCs for C3G and the static
251
complex was formed with a molar ratio of about 1:1. As shown in the Table 1, G
252
values were all negative, which revealed that the binding process of MCs with C3G
253
was spontaneous. H < 0 and S < 0 indicated that the Van der Waals forces or
254
hydrogen binding were the dominant binding force (Xiao et al., 2007). However, the
255
result is different from the findings of He, Xu, Zeng, Qin, & Chen, (2016) that
256
showed the quenching parameters of caseins were decreased with increasing
257
temperature, possibly due to the structural difference of the phenolic ligands.
258 259
3.6 Stability of C3G as influenced by complexing with MCs
260
Color and anthocyanin content are the two important quality traits for natural
261
anthocyanins pigment products. The stability of natural anthocyanin pigments can be
262
evaluated by measuring the changes of color and anthocyanin content in the 12
263
accelerated tests. The effects of different concentrations of casein on the thermal,
264
oxidation, and photo stability of the C3G solution at pH 6.3 are shown in Table 2.
265
Caseins could increase the thermal stability of C3G, oxidation and illumination
266
treatment dramatically. For instance, C3G was significantly affected by heating
267
treatments at 60 °C for 30 min; contrastingly, desirable protective effect of MCs on
268
C3G was found. Nevertheless, when the heating temperature was set at 90°C, the
269
stabilities of C3G and MCs-C3G declined sharply, while the MCs played an
270
important role in protecting C3G from heat destruction. In addition, it can be found
271
that more than half C3G degraded with illuminating for 30 min. After adding 0.1
272
mg/mL MCs, C3G degradation in the PBS solution at pH 6.3 induced by thermal
273
treatment (30-90 °C /2 h), oxidation (0.5-1.5% H2O2/1 h) and photo illumination
274
(6-30 h). The C3G in the presence of MCs had significantly (p < 0.05) higher contents
275
than the C3G samples without MCs after the stability testing, which indicated that the
276
degradation of C3G alone was greater than that of the samples with MCs. C3G
277
degradation probably because the pyrylium ring of anthocyanin opened and produced
278
a chalcone structure via hydrolysis of the carbon atom at position C2 position
279
(Norman, Bartczak, Zdarta, Ehrlich, & Jesionowski, 2016). The caseins can be used
280
as a natural nano-delivery vehicle and interacted with C3G via hydrogen bonding and
281
hydrophobic reaction to form complexes, thereby effectively protecting C3G from
282
degradation and improving their thermal, oxidation photo and storage stability.
283 284
4. Conclusions 13
285
In summary, the optimal modification for caseins were performed at the pH value of
286
5.5 after heated at 80 °C for 30 min. The spectroscopic analyses showed that MCs
287
bound with C3G and formed complexes via hydrophobic interactions. MCs indicated
288
stronger binding affinity toward C3G. Binding of C3G caused different alterations of
289
the secondary structures of the MCs, with a decrease in α-helix, turn random, coil
290
structure and an increase in β-sheet. The addition of casein significantly (p < 0.05)
291
prevented the thermal (90 °C/30 min), oxidation (15% H2O2 / 1 h), photo (30 h)
292
degradation and storage stability of C3G. These results may be helpful in expanding
293
the protection mechanism of C3G and caseins as natural embedding medium used
294
with small molecular substances.
295 296
Acknowledgments
297
This work is supported by the National Natural Science Foundation of China
298
(NSFC, Grant No. 31801459 and 31701520), China Postdoctoral Science Foundation
299
Funded Project (No. 2018M642551), the Funds for Distinguished Young Scientists
300
(Grant No. kxjq17012) at Fujian agriculture and forestry university of China.
301 302 303
Conflict of interest The authors declare no conflict of interest
304 305 306
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389
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390
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391 392 393 394 18
395 396
Figure legends
397
Figure 1. Particle size and zeta-potential of casein at the different concentration (A)
398
(pH=6.5). Effects of heating temperature (B) on the particle size and
399
zeta-potential of casein for heating 30min, and different heating time (C) at
400
80 °C ( 2 mg/mL casein concentration at pH 5.5).
401
Figure 2. TEM images of native Cs (A) and MCs (B) showing the caseins structure
402
changes induced by acidification and thermal treatment. The nanoparticle
403
changes of C3G were shown the significant changes with MCs (C, D).
404
Figure 3. FTIR spectra of native Cs and MCs powder in the absence and presence of
405
C3G for heating 80 °C with 30 min at pH 5.5 (A); Fluorescence emission
406
spectra of modified caseins at excitation wavelength 280 nm in presence of
407
C3G (0, 10, 20, 30, 40, 50, 60 μmol/L) (1–7) (B); second derivative resolution
408
enhancement and curve-fitted amide I region (1700–1600 cm -1) for MCs (C)
409
and MCs-C3G (D).
410
Figure 4. The Stern-Volmer curves of C3G (A) at 297K, 317K and 337 K. The double
411
logarithm regression curve of log [(F0-F)/F] versus log [cq] of
412
Cyanidin-3-o-glucoside (B) at 297 K, 317 K and 337K.; the effect of storage
413
stability on the C3G and MCs-C3G that kept away from light at -4°C after
414
15days (C).
415 416 19
417 418 419 420
Table 1. The quenching constants (KSV), bimolecular quenching constant (Kq), binding constants (KS), binding sites numbers (n) and thermodynamic parameters for C3G binding to caseins at 293, 303 and 313 K. 293
T(K) 303
313
KSV(×104 M-1)
2.95±0.01
2.69±0.04
2.47±0.02
Kq (×1012 M-1·S-1)
2.95±0.01
2.69±0.04
2.47±0.02
KS (×105 M-1)
2.54±0.13
3.03±0.10
1.04±0.12
n △H (kJ·mol-1)
1.21±0.00
1.23±0.00 -33.51
1.14±0.01
△G (kJ·mol-1)
-30.32
-31.80
-30.06
△S (J·mol-1·K-1)
-10.89
-5.65
-11.01
Parameters
421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 20
Table 2. Effect of casein on stability of C3G at pH 6.3 after heat, oxidation and illumination treatment.
MCs
C3G (μM)
Temperature
Photo illuminate
H2O2
Untreated 30°C
60°C
90°C
6h
18h
30h
0.5%
1.0%
1.5%
100
11.66±0.06a
10.87±0.22a
5.23±0.1b
0.24±0.02c
9.76±0.3b
6.27±0.29c
3.34±0.12d
0.55±0.02b
0.33±0.07bc
0.17±0.03c
200
22.1±0.89a
21.59±0.15a
12.08±0.08b
0.27±0.18c
19.01±0.08a
12.77±0.28b
8.13±0.11c
2.66±0.13b
1.68±0.4c
0.65±0.04d
300
32.28±0.89a
31.01±0.37a
17.18±0.42b
0.34±0.15c
27.76±0.58a
18.99±0.3b
11.66±0.66c
4.54±0.1b
2.8±0.05c
1.51±0.14d
400
42.84±0.26a
41.19±1.27a
19.36±0.33b
0.37±0.24c
36.23±0.59b
25.04±0.24c
17.29±0.16d
6.48±0.04b
4.37±0.82b
2.62±0.22c
100
9.26±0.25a
8.45±0.39a
7.66±0.44a
2.67±0.1b
7.93±0.48a
7.33±0.63a
5.39±0.52b
0.32±0.05b
0.24±0.02b
0.15±0.02b
200
20.48±0.78a
18.86±1.42ab
17.8±1.33ab
3.37±0.46b
17.66±0.66a
16.77±0.46a
12.25±0.89b
2.62±0.07b
1.11±0.13bc
0.73±0.08c
300
31.94±0.78a
28.82±1.51a
26.62±1.6a
9.43±2.3b
27.4±1.14a
26.32±2.13a
19.83±0.57a
7.76±0.66b
3.01±0.16c
1.76±0.11d
400
44.72±0.39a
38.96±1.36a
36.83±1.45a
9.61±1.39b
37.46±2.6a
35.39±1.5ab
27.48±1.02b
11.97±0.25b
5.62±1.05bc
3.89±0.05c
Non
1 mg/mL
Values are expressed as the mean ± standard deviation. Different letters in the same column for each treatment indicate significant differences (p < 0.05).
21
Fig. 1.
0
116
-2
150
-2
125
145
-4
114
-4
112
-6
110
-8
108
-10
106 104
-12 40
60
80
100
120
Temperature（℃） particle size zeta-potential
particle size（d.nm）
155
zeta-potential（mV）
0
particle size（d.nm）
118
130
120
-6
135
-8
130
-10
100
-12
95
125 1
2
3
4
5
Concentration（μM） particle size zata-potential
22
-2 -4
115
140
0
-6
110
-8
105
zeta-potential（mV）
C
B zeta-potential（mV）
particle size（d.nm）
A
-10 -12 0
30
60
Time（min） particle size
90
120
zeta-potential
Fig. 2. A
B
C
D
23
Fig. 3. 100
A
2000
% Transmittance
100
80
MCs MCs-C3G 1538.17
1656.63
60
3408.10
1543.05
80
1538.17
2000
3000
7 1000
500
0
4000
300
320
Wavenumbers（cm )
1656.63
3408.10
340
360
380
Wavelength（nm）
3411.20
1653.47
0.15
0.15 α-Helix β-Sheet Turn 4000 Random coil
C 1000
1500
-1
60 1543.05
3411.20
1653.47
1000
B 1
Fluorescence intensity
% Transmittance
MCs MCs-C3G
2000
3000
MCs R2 =0.9994 -1) Wavenumbers（cm 0.10
18.0% 28.3% 36.5% 17.2%
0.10
0.05
0.00 1600
α-Helix β-Sheet Turn Random coil
D MCs-C3G R2 =0.9994
15.9% 24.0% 44.9% 15.2%
0.05
1620
1640
1660
1680
0.00 1600
1700
Wavelength（cm-1）
1620
1640
1660
Wavelength（cm-1）
24
1680
1700
Fig. 4. Highlights
A
3.50
-
MCs
bound with
F0 /F
3.00
C3G and formed
2.50
complexes
2.00
293K
via
303K
1.50
313K
hydrophobic
1.00 0
80
interactions -
Casein
significantly
B
-0.8
Log[(F0 -F)/F]
20 40 60 C3G concentration (10 -6 M)
prevented the
-0.6
thermal,
-0.4
oxidation,
-0.2
photo degradation
293K
0
303K
and storage
313K
0.2
stability of
0.4 -4
-4.2
-4.4 -4.6 -4.8 Log[C3G](M)
-5
-5.2
C3G. -
C
Binding
of C3G caused alterations of the secondary structures of the MCs.
25
CRediT author statement Yongzhong Ouyang: Conceptualization, Methodology, Software Lei Chen: Writing- Original draft preparation Liu Qian: Investigation Xiujun Lin: Investigation, Validation Xiaoyun Fan: Investigation Hui Teng: Data curation, Supervision Hui Cao: Reviewing and Editing,
26
S0308-8146(20)30278-8 https://doi.org/10.1016/j.foodchem.2020.126418 FOCH 126418
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
17 November 2019 10 February 2020 14 February 2020
Please cite this article as: Ouyang, Y., Chen, L., Qian, L., Lin, X., Fan, X., Teng, H., Cao, H., Fabrication of caseins nanoparticles to improve the stability of cyanidin 3-O-glucoside, Food Chemistry (2020), doi: https://doi.org/ 10.1016/j.foodchem.2020.126418
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© 2020 Published by Elsevier Ltd.
1
Fabrication of caseins nanoparticles to improve the stability of cyanidin
2
3-O-glucoside
3
Yongzhong Ouyang1, Lei Chen2,#, Liu Qian2,#, Xiujun Lin2, Xiaoyun Fan2, Hui
4
Teng2,*, Hui Cao3,4*
5
1School
6
528000, China.
7
2College
8
China
9
3Guangdong-Macau
of Environmental and Chemical Engineering, Foshan University, Foshan
of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002,
Traditional Chinese Medicine Technology Industrial Park
10
Development Co., Ltd, Hengqin New Area, Zhuhai 519031, China
11
4Institute
12
Chinese Medicine, University of Macau, Macau
13
*Corresponding author: Hui Teng, [email protected]; Hui Cao,
14
[email protected]
15
#authors contributed equally to this study
of Chinese Medical Sciences, State Key Laboratory of Quality Research in
16 17
Abstract
18
The influence of encapsulation with caseins on the stability of cyanidin 3-O-glucoside
19
(C3G) was investigated. The modified casein nanoparticles (MCs) prepared at pH 5.5
20
after heated at 80 °C for 30 min was applied to encapsulate C3G. The diameter of
21
nanoparticle (MCs-C3G) was 110±0.31 nm and zeta-potential was -8.83±0.52 mV.
22
The molecular weight of α-casein (32 kDa) and β-casein (25 kDa) increased along 1
23
with the encapsulation of C3G. The interactions of MCs with C3G were examined at
24
pH 6.3 by fluorescence spectroscopy and IR spectroscopy. MCs encapsulated C3G
25
mainly via the hydrophobic interaction. The secondary structures of caseins were
26
changed along with the combination of C3G, with a decreasing in α-helix, turn
27
random, and coil structure, as well as increased β-sheet. In addition, the MCs-C3G
28
interaction appeared to have a positive effect on the thermal, oxidation and photo
29
stability of C3G.
30
Key words: cyanidin 3-O-glucoside; caseins; stability; nanoparticles; encapsulation
31 32
1. Introduction
33
Caseins (Cs) consist of phosphorylated α-, κ-, β-format, are the major proteins in
34
bovine milk (approximately 80% of the total milk proteins) (Dalgleish, 2011).
35
α-Casein contains two tryptophan (Trp) residues, while β-casein has one and with
36
stronger hydrophobic bond than α-casein and κ -casein (Dalgleish & Corredig, 2012).
37
As water soluble pigments, anthocyanins have been reported to present numerous
38
benefits for human health (Tang, Li, Bi, & Gao, 2016). There are six common
39
glycosidic and acylglycosidic derivatives of anthocyanidins, namely pelargonidin,
40
cyanidin, malvidin, delphinidin, peonidin, and petunidin, which are classified
41
according to the number and position of hydroxyl group on the flavan nucleus
42
(Sinopoli, Calogero & Bartolotta, 2019). Anthocyanins can exist in a stable form of
43
astragalus salt cations under acidic conditions, while with the increase of pH
44
gradually, the anthocyanins form become unstable chalcone. 2
45
Stability of polyphenols is crucial for the nutrition of the food and is directly
46
associated with the chemical structures of polyphenols (Xiao & Högger, 2015). The
47
physicochemical conditions such as pH, temperature, light, oxygen availability, metal
48
ions, chemical modification, enzyme, proteins, nitrite salt, sulfur dioxide as well as
49
ascorbic acid must be taken into account for the polyphenols’ stability (Xiao, 2018;
50
Cao et al., 2020). Our group's previous experiments optimized the extraction and
51
purification of anthocyanins in raspberry, and found that cyanidin 3-O-glucoside
52
(C3G) showed anti-diabetic and anti-obesity activities. However, C3G is susceptible
53
to the surrounding environment, such as temperature, pH, light intensity, and metal
54
ions, which greatly reduce its bioactivity and nutrition. Therefore, improving the
55
stability of anthocyanins is a current technical problem that needs to be solved. Based
56
on the important influence of the low stability of C3G on daily use, this study intends
57
to use C3G as the research object and α-casein as the conjugate, spectroscopic
58
techniques are used to analyze the combination mode, the binding distance and the
59
structural changes; the stability changes in different environments after their binding
60
were studied to determine the degradation mode and kinetic parameters. We aimed to
61
explain the influence mechanism of C3G and α-casein on the stability of anthocyanin
62
after binding, and provide a scientific theoretical basis for the rational and effective
63
development of the anthocyanins resources and its application in the food industry.
64 65
2. Materials and Methods
66
2.1.
Materials and chemicals 3
67
Casein with a protein content of 86% (41% α-casein, 35% β-casein) was purchased
68
from Kerry Group (Beloit, WI, USA). α-Casein with purity of 70% and β-casein with
69
purity of 98%，and C3G purity of 97% were purchased from Sigma-Aldrich Chemical
70
Co. (St. Louis, MO, USA). All other chemicals were of analytical grade and were
71
purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
72 73
2.2. Modified casein preparation
74
Modified casein (MCs) preparation was acted as following: first, casein (Cs) was
75
dissolved in 10 mM PBS buffer (pH 7.4) to obtain the solutions with the
76
concentrations of 1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL. Then, the solution was heated in
77
40-120 °C for 30 min, 60 min, 90 min and 120 min. After that, the mixtures were
78
placed in a water bath at 50 °C and stirred continuously for 60 min, and then acidified
79
with HCl 1 M to obtain MCs. The mean zeta potential value of Cs and MCs (0.05%
80
w/w) used in this study were measured by the Zetasizer Nano series from Malvern
81
Instruments Ltd (Worcs, UK). The data was analyzed using the Zetasizer Nano v3.30
82
software. The average value was taken from three readings and all measurements
83
were performed three times.
84 85
2.2. MCs-C3G preparation
86
MCs-C3G was prepared following previous study with minor modification (He et al.,
87
2016). To ensure complete solubilization of powders, all dispersions were first stirred
88
at 50 °C for 1 h using a magnetic stirrer and then kept at 4 °C for 24 h. The MCs-C3G 4
89
mixture was prepared with 1 mg/mL MCs and 100 μM C3G in PBS (pH 7.0). Final
90
sample powder was obtained by freeze-drying.
91 92
2.3. Transmission electron microscopy (TEM) analysis
93
The nanosphere aggregates was observed by using a HT 7700 transmission electron
94
microscopy (Tokyo, Japan) according to the method of McMahon, Du, McManus,
95
and Larsen (2009) with modifications. Dried MCs-C3G samples were diluted 10-fold
96
with ultra-pure water. The diluted sample and ammonium molybdate solution (2
97
g/100 mL) (1:1) were mixed and left for 3 min at room temperature. A drop of this
98
solution was placed on a copper mesh for 5 min before the excess liquid was drawn
99
off using filter papers. The mesh was examined using a TEM at an operating voltage
100
of 200 KV.
101 102
2.4. FTIR spectroscopic measurement
103
FTIR spectra were recorded on a Bruker Vertex 70 FTIR spectrometer (Beijing,
104
China). Sample powders were blended with KBr at a mass ratio of 1:100 and pressed
105
into a tablet for FTIR analysis. Interferograms were obtained at the spectral range of
106
4000-400 cm-1 with a resolution of 2 cm-1 and 32 scans. Additionally, PeakFit v4.12
107
software was used to separate peaks in the FTIR spectrogram to obtain peak area of
108
each absorption peak.
109 110
2.5. Fluorescence spectroscopy 5
111
Fluorescence spectroscopy was recorded on a Hitachi F-4600 Spectrometer (Tokyo,
112
Japan). The final concentration of MCs in each mixture was 1 mg/mL, and the
113
concentrations of C3G in the mixtures were 0, 10, 20, 30, 40, 50 and 60 μM. The
114
mixture solutions were determined by scanning emission wavelengths from 300 to
115
400 nm, with excitation wavelength of 280 nm at 293, 303 and 313 K to obtain the
116
intrinsic fluorescence spectroscopy of MCs. The excitation and emission slit widths
117
were 5.0 nm.
118 119
2.6 Data processing
120
In order to further understand the binding mechanism for MCs-C3G, the fluorescence
121
data were performed using the Stern-Volmer equation:
122
F0 1 K SV cq 1 K q 0C q F
(1)
123
where F0 and F are the fluorescence intensities in the absence and presence of
124
quencher, respectively. Kq is the bimolecular quenching constant, KSV is the
125
Stern-Volmer dynamic quenching constant, cq is the concentration of the quencher
126
and τ0 is the average lifetime of the molecule without any quencher [τ0 =10−8 s]. Ksv
127
and Kq are determined from the slope of the regression curves of F0/F against cq. The
128
quenching mechanism is divided into static quenching and dynamic quenching. When
129
Kq calculated according to Eq. (1) was much higher than the limiting diffusion rate
130
constant of the biomolecules (2×1010 M-1 s-1), indicated that static, and not dynamic
131
quenching was the main quenching mechanism between MCs and C3G.
132
For the static quenching mechanism, the binding constant (KS) of the complexes 6
133
and the binding sites numbers (n) were calculated by using the double logarithmic
134
Stern-Volmer equation (Eq. (2)).
135
log
136
The non-covalent forces of protein-ligand binding were calculated by the
137
thermodynamic parameters calculated from the following equations (Eqs. (3)-(5)).
138
G 1 H d d T T
139
G RT ln K s
140
G H TS
141
Where H is the enthalpy change, S is the entropy change and G is the free energy
142
change, R is the gas constant (8.314 J/mol K), T is the absolute temperature (K), and
143
KS is the binding constant at the corresponding temperature.
F0 F log K s n log cq F
(2)
(3) (4) (5)
144 145
2.7. Thermal, oxidation, photo and storage stability
146
The MCs-C3G mixtures and their corresponding MCs samples were subjected to
147
thermal, oxidation, photo and storage stability by following previous study (He et al.,
148
2016). The thermal stability test was performed by heating the samples in 10 mL
149
tubes wrapped in a water bath for 30 °C, 60 °C, 90 °C with 30 min, and then rapidly
150
cooled down for further analysis. The oxidation stability test was carried out by
151
addition of H2O2 into the samples at a final concentration of 0.5 %, 1%, 1.5% and
152
oxidizing for 1 h at room temperature in the dark. The photo stability test was
153
measured by illuminating the samples in 10 mL transparent tubes for 6 h, 18 h, 30 h at 7
154
room temperature in YZ18RR fluorescent lamps (Osram Co., Foshang, China). The
155
storage stability test was determined by placing in brown tubes for 15 days at -4 °C in
156
the dark. During the storage period, the content was analyzed (Lee, Durst, &
157
Wrolstad, 2005). The method based on the structural change of the C3G chromophore
158
between pH 1.0 and 4.5, calculated as follows:
159
A A 530 nm A 700 nm pH 1.0 A 530 nm A 700 nm pH 4.5
160
TAC (mg/L)
161
where TAC is the total anthocyanins contents (mg/L); MW (the molecular
162
weight)=449.2 g/mol; DF is the dilution factor; ε (molar extinction coefficient) =
163
26900; b is the path length in cm.
A MW DF 103 b
164 165
2.8. Statistical analysis
166
All experiments were repeated at least three times. Data were expressed as the mean
167
± the standard deviation (SD). Significant differences (p < 0.05) were identified by
168
the least significant difference procedure between the means.
169 170
3. Results and discussion
171
3.1 MCs structure
172
The intermolecular aggregation was generated through the hydrophobic interaction to
173
form unstable nanoparticles (Vemula, Li & John, 2006). As shown in Fig. 1, when the
174
concentration of casein is more than 2 mg/mL, casein will start to form polymers with 8
175
the increased particle size. The optimum formulation of MCs was 2 mg/mL casein at
176
80 °C for 30 min (Fig. 1) with the particle size of 110±0.31 nm and zeta potential of
177
-8.83±0.52 mV. When the pH of casein solution approached to the isoelectric point,
178
the protonation of amino acids on the protein led to enhance hydrogen and ionic bond
179
in the process of casein micelle formation (Ding, Huang, Cai & Wang, 2019). The
180
special interaction of π electrons provided by amino acid residues such as
181
phenylalanine, tryptophan, and tyrosine with cations provided by protonation of side
182
chains of amino acid residues could also result in a smaller particle size (Gallivan &
183
Dougherty, 2000). Meanwhile, the reduction of electrostatic repulsion interaction led
184
to the reduction of intermolecular distance, and the micellar structure became more
185
compact, which was manifested as the reduction of particle size.
186 187
3.2 Analysis of transmission electron microscopic (TEM) in the presence of C3G
188
As shown in Fig. 2, the TEM results clearly indicated the acidification and thermal
189
treatment cause the change in the structural features and associated function of caseins
190
(Fig. 2A, B). When C3G added to MCs solution, a thin coated layer covered upon the
191
interface (Fig. 2C), indicating the core-shell structure consequently formed. Caseins
192
underwent a series of physical and chemical alterations by the guidance of acid-heat
193
treatment, and made the C3G and MCs bind more closely by the ultrasonic dispersion
194
and shearing effect. It was supposed that the lighter spot (Fig. 2D) in the center of
195
MCs-C3G nanocomplexe was C3G core, which was in agreement with published
196
results (Chitkara and Kumar, 2013; Fang et al., 2014). It was further confirmed that 9
197
the encapsulation of C3G with MCs and the formation of core–shell nano-complexes.
198
The changes of nanoparticles size are in agreement with the findings from casein
199
modification studies, which casein tends to shrink in acidic solution and swell in
200
alkaline solution (Liu & Guo, 2008).
201 202
3.3 FTIR spectroscopy of MCs-C3G
203
FTIR spectroscopy is an effective method to determine the secondary structure of
204
caseins. Both intensity variations and spectral shifting for the protein amide I band at
205
1600–1700 cm-1 (C=O stretch) and the amide II band, which occurs in the region
206
<1548 cm-1 (C–N stretching coupled with N–H bending), have been widely used upon
207
polyphenol interaction and are related to the secondary structure of the proteins (Peng
208
et al., 2015; Zhang, Wright, & Zhong, 2013). As shown in Fig. 3A, the peak position
209
of amide I moved from 1656.63 to 1653.47 cm-1 and that of amide II moved from
210
1538.17 to 1543.05 cm-1 in the MCs IR spectrum after interaction with C3G,
211
respectively. Changes of amide I and II bands were due to C3G binding to the caseins
212
C=O, C–N and N–H groups and modified casein via hydrogen bonding and
213
hydrophobic attraction. In addition, the intensities of the amide I of casein decreased
214
after binding with C3G, indicating that the α-helical content in the casein structure
215
was reduced. A peak position of amide A moved from 3411.2 to 3408.1 cm-1 in the
216
MCs and MCs-C3G, the shifting of the amide A band at 3300-3400 cm-1 (N–H
217
stretching mode) could be the additional evidence on caseins structure changes (Qi,
218
Ren, Xiao, & Tomasula, 2015). 10
219
A quantitative analysis of the caseins secondary structure for the MCs and MCs-C3G
220
has been carried out and the results are shown in Fig. 3C and 3D. The MCs has 18%
221
α-helix, 28.3% β-sheet, 36.5% turn structure and random 17.2% coil. The MCs-C3G
222
has 15.9% α-helix, 24.0% β-sheet, 44.9% turn structure and random 15.2% coil.
223
These results are consistent with spectroscopic study of caseins previously reported
224
(Menikh & Fragata, 1994). Upon the interaction, the α-helix, β-sheet and random
225
decreased, whilst turn structure increased in the MCs-C3G complexes, which are
226
indicative of larger perturbations of caseins secondary structure by C3G. The changes
227
of the caseins secondary structure will be discussed with fluorescence data further on.
228 229
3.4 Fluorescence spectroscopy of MCs-C3G
230
Figure 3B showed the fluorescence emission spectra of MCs in the presence of C3G
231
(0–60 μM) with an excitation wavelength of 280 nm. The fluorescence intensities of
232
caseins decreased with the increasing concentration of C3G, which is ascribed to the
233
quenching effect of C3G. The quenching efficiency of 60 μM C3G on MCs
234
fluorescence was 66.7%. The λmax of MCs appeared a blue shift with higher C3G
235
concentrations. This indicated that the polarity of microenvironment around the Trp
236
and Tyr residues in modified caseins had increased and the hydrophobicity had
237
decreased after the addition of C3G. In addition, the results revealed that modified
238
caseins conformations changes were ascribed to binding with C3G (Chen, Xie, Jiang,
239
& Yao, 2008).
240 11
241
3.5 The mechanism of encapsulation
242
The quenching parameters of caseins with different concentrations of C3G at 293, 303
243
and 313 K are shown in Figure 4 and Table 1. The values of KSV were Kq values were
244
all much higher than the dynamic quenching constant 2×1010 M-1 s-1, which indicated
245
that C3G could quench the fluorescence of MCs via the static quenching process
246
predominantly caused by the formation of complex. The Ks of MCs bound with C3G
247
fluorescence quenched at 293 K, 303 K, 313 K were all in the order of 105, with the
248
values of 2.54, 3.03, 1.04, respectively, which confirmed the strong binding affinity
249
between C3G and MCs. Additionally, all of the values of n were approximately 1,
250
suggesting there was around one binding site in the MCs for C3G and the static
251
complex was formed with a molar ratio of about 1:1. As shown in the Table 1, G
252
values were all negative, which revealed that the binding process of MCs with C3G
253
was spontaneous. H < 0 and S < 0 indicated that the Van der Waals forces or
254
hydrogen binding were the dominant binding force (Xiao et al., 2007). However, the
255
result is different from the findings of He, Xu, Zeng, Qin, & Chen, (2016) that
256
showed the quenching parameters of caseins were decreased with increasing
257
temperature, possibly due to the structural difference of the phenolic ligands.
258 259
3.6 Stability of C3G as influenced by complexing with MCs
260
Color and anthocyanin content are the two important quality traits for natural
261
anthocyanins pigment products. The stability of natural anthocyanin pigments can be
262
evaluated by measuring the changes of color and anthocyanin content in the 12
263
accelerated tests. The effects of different concentrations of casein on the thermal,
264
oxidation, and photo stability of the C3G solution at pH 6.3 are shown in Table 2.
265
Caseins could increase the thermal stability of C3G, oxidation and illumination
266
treatment dramatically. For instance, C3G was significantly affected by heating
267
treatments at 60 °C for 30 min; contrastingly, desirable protective effect of MCs on
268
C3G was found. Nevertheless, when the heating temperature was set at 90°C, the
269
stabilities of C3G and MCs-C3G declined sharply, while the MCs played an
270
important role in protecting C3G from heat destruction. In addition, it can be found
271
that more than half C3G degraded with illuminating for 30 min. After adding 0.1
272
mg/mL MCs, C3G degradation in the PBS solution at pH 6.3 induced by thermal
273
treatment (30-90 °C /2 h), oxidation (0.5-1.5% H2O2/1 h) and photo illumination
274
(6-30 h). The C3G in the presence of MCs had significantly (p < 0.05) higher contents
275
than the C3G samples without MCs after the stability testing, which indicated that the
276
degradation of C3G alone was greater than that of the samples with MCs. C3G
277
degradation probably because the pyrylium ring of anthocyanin opened and produced
278
a chalcone structure via hydrolysis of the carbon atom at position C2 position
279
(Norman, Bartczak, Zdarta, Ehrlich, & Jesionowski, 2016). The caseins can be used
280
as a natural nano-delivery vehicle and interacted with C3G via hydrogen bonding and
281
hydrophobic reaction to form complexes, thereby effectively protecting C3G from
282
degradation and improving their thermal, oxidation photo and storage stability.
283 284
4. Conclusions 13
285
In summary, the optimal modification for caseins were performed at the pH value of
286
5.5 after heated at 80 °C for 30 min. The spectroscopic analyses showed that MCs
287
bound with C3G and formed complexes via hydrophobic interactions. MCs indicated
288
stronger binding affinity toward C3G. Binding of C3G caused different alterations of
289
the secondary structures of the MCs, with a decrease in α-helix, turn random, coil
290
structure and an increase in β-sheet. The addition of casein significantly (p < 0.05)
291
prevented the thermal (90 °C/30 min), oxidation (15% H2O2 / 1 h), photo (30 h)
292
degradation and storage stability of C3G. These results may be helpful in expanding
293
the protection mechanism of C3G and caseins as natural embedding medium used
294
with small molecular substances.
295 296
Acknowledgments
297
This work is supported by the National Natural Science Foundation of China
298
(NSFC, Grant No. 31801459 and 31701520), China Postdoctoral Science Foundation
299
Funded Project (No. 2018M642551), the Funds for Distinguished Young Scientists
300
(Grant No. kxjq17012) at Fujian agriculture and forestry university of China.
301 302 303
Conflict of interest The authors declare no conflict of interest
304 305 306
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391 392 393 394 18
395 396
Figure legends
397
Figure 1. Particle size and zeta-potential of casein at the different concentration (A)
398
(pH=6.5). Effects of heating temperature (B) on the particle size and
399
zeta-potential of casein for heating 30min, and different heating time (C) at
400
80 °C ( 2 mg/mL casein concentration at pH 5.5).
401
Figure 2. TEM images of native Cs (A) and MCs (B) showing the caseins structure
402
changes induced by acidification and thermal treatment. The nanoparticle
403
changes of C3G were shown the significant changes with MCs (C, D).
404
Figure 3. FTIR spectra of native Cs and MCs powder in the absence and presence of
405
C3G for heating 80 °C with 30 min at pH 5.5 (A); Fluorescence emission
406
spectra of modified caseins at excitation wavelength 280 nm in presence of
407
C3G (0, 10, 20, 30, 40, 50, 60 μmol/L) (1–7) (B); second derivative resolution
408
enhancement and curve-fitted amide I region (1700–1600 cm -1) for MCs (C)
409
and MCs-C3G (D).
410
Figure 4. The Stern-Volmer curves of C3G (A) at 297K, 317K and 337 K. The double
411
logarithm regression curve of log [(F0-F)/F] versus log [cq] of
412
Cyanidin-3-o-glucoside (B) at 297 K, 317 K and 337K.; the effect of storage
413
stability on the C3G and MCs-C3G that kept away from light at -4°C after
414
15days (C).
415 416 19
417 418 419 420
Table 1. The quenching constants (KSV), bimolecular quenching constant (Kq), binding constants (KS), binding sites numbers (n) and thermodynamic parameters for C3G binding to caseins at 293, 303 and 313 K. 293
T(K) 303
313
KSV(×104 M-1)
2.95±0.01
2.69±0.04
2.47±0.02
Kq (×1012 M-1·S-1)
2.95±0.01
2.69±0.04
2.47±0.02
KS (×105 M-1)
2.54±0.13
3.03±0.10
1.04±0.12
n △H (kJ·mol-1)
1.21±0.00
1.23±0.00 -33.51
1.14±0.01
△G (kJ·mol-1)
-30.32
-31.80
-30.06
△S (J·mol-1·K-1)
-10.89
-5.65
-11.01
Parameters
421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 20
Table 2. Effect of casein on stability of C3G at pH 6.3 after heat, oxidation and illumination treatment.
MCs
C3G (μM)
Temperature
Photo illuminate
H2O2
Untreated 30°C
60°C
90°C
6h
18h
30h
0.5%
1.0%
1.5%
100
11.66±0.06a
10.87±0.22a
5.23±0.1b
0.24±0.02c
9.76±0.3b
6.27±0.29c
3.34±0.12d
0.55±0.02b
0.33±0.07bc
0.17±0.03c
200
22.1±0.89a
21.59±0.15a
12.08±0.08b
0.27±0.18c
19.01±0.08a
12.77±0.28b
8.13±0.11c
2.66±0.13b
1.68±0.4c
0.65±0.04d
300
32.28±0.89a
31.01±0.37a
17.18±0.42b
0.34±0.15c
27.76±0.58a
18.99±0.3b
11.66±0.66c
4.54±0.1b
2.8±0.05c
1.51±0.14d
400
42.84±0.26a
41.19±1.27a
19.36±0.33b
0.37±0.24c
36.23±0.59b
25.04±0.24c
17.29±0.16d
6.48±0.04b
4.37±0.82b
2.62±0.22c
100
9.26±0.25a
8.45±0.39a
7.66±0.44a
2.67±0.1b
7.93±0.48a
7.33±0.63a
5.39±0.52b
0.32±0.05b
0.24±0.02b
0.15±0.02b
200
20.48±0.78a
18.86±1.42ab
17.8±1.33ab
3.37±0.46b
17.66±0.66a
16.77±0.46a
12.25±0.89b
2.62±0.07b
1.11±0.13bc
0.73±0.08c
300
31.94±0.78a
28.82±1.51a
26.62±1.6a
9.43±2.3b
27.4±1.14a
26.32±2.13a
19.83±0.57a
7.76±0.66b
3.01±0.16c
1.76±0.11d
400
44.72±0.39a
38.96±1.36a
36.83±1.45a
9.61±1.39b
37.46±2.6a
35.39±1.5ab
27.48±1.02b
11.97±0.25b
5.62±1.05bc
3.89±0.05c
Non
1 mg/mL
Values are expressed as the mean ± standard deviation. Different letters in the same column for each treatment indicate significant differences (p < 0.05).
21
Fig. 1.
0
116
-2
150
-2
125
145
-4
114
-4
112
-6
110
-8
108
-10
106 104
-12 40
60
80
100
120
Temperature（℃） particle size zeta-potential
particle size（d.nm）
155
zeta-potential（mV）
0
particle size（d.nm）
118
130
120
-6
135
-8
130
-10
100
-12
95
125 1
2
3
4
5
Concentration（μM） particle size zata-potential
22
-2 -4
115
140
0
-6
110
-8
105
zeta-potential（mV）
C
B zeta-potential（mV）
particle size（d.nm）
A
-10 -12 0
30
60
Time（min） particle size
90
120
zeta-potential
Fig. 2. A
B
C
D
23
Fig. 3. 100
A
2000
% Transmittance
100
80
MCs MCs-C3G 1538.17
1656.63
60
3408.10
1543.05
80
1538.17
2000
3000
7 1000
500
0
4000
300
320
Wavenumbers（cm )
1656.63
3408.10
340
360
380
Wavelength（nm）
3411.20
1653.47
0.15
0.15 α-Helix β-Sheet Turn 4000 Random coil
C 1000
1500
-1
60 1543.05
3411.20
1653.47
1000
B 1
Fluorescence intensity
% Transmittance
MCs MCs-C3G
2000
3000
MCs R2 =0.9994 -1) Wavenumbers（cm 0.10
18.0% 28.3% 36.5% 17.2%
0.10
0.05
0.00 1600
α-Helix β-Sheet Turn Random coil
D MCs-C3G R2 =0.9994
15.9% 24.0% 44.9% 15.2%
0.05
1620
1640
1660
1680
0.00 1600
1700
Wavelength（cm-1）
1620
1640
1660
Wavelength（cm-1）
24
1680
1700
Fig. 4. Highlights
A
3.50
-
MCs
bound with
F0 /F
3.00
C3G and formed
2.50
complexes
2.00
293K
via
303K
1.50
313K
hydrophobic
1.00 0
80
interactions -
Casein
significantly
B
-0.8
Log[(F0 -F)/F]
20 40 60 C3G concentration (10 -6 M)
prevented the
-0.6
thermal,
-0.4
oxidation,
-0.2
photo degradation
293K
0
303K
and storage
313K
0.2
stability of
0.4 -4
-4.2
-4.4 -4.6 -4.8 Log[C3G](M)
-5
-5.2
C3G. -
C
Binding
of C3G caused alterations of the secondary structures of the MCs.
25
CRediT author statement Yongzhong Ouyang: Conceptualization, Methodology, Software Lei Chen: Writing- Original draft preparation Liu Qian: Investigation Xiujun Lin: Investigation, Validation Xiaoyun Fan: Investigation Hui Teng: Data curation, Supervision Hui Cao: Reviewing and Editing,
26