Subscriber access provided by - Access paid by the | UCSF Library
Food and Beverage Chemistry/Biochemistry
Emulsifying Properties of Polysaccharide Conjugates Prepared from Chin-brick Tea Xiaoqiang Chen, Yuntian Zhang, Yu Han, Qian Li, Li Wu, Jia Zhang, Xiaoling Zhong, Jianchun Xie, Shengrong Shao, Yinjun Zhang, and Zhengqi Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03161 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 40
Journal of Agricultural and Food Chemistry
1
Emulsifying Properties of Polysaccharide Conjugates Prepared from
2
Chin-brick Tea
3
Xiaoqiang Chen*1,2,Yuntian Zhang§1, Yu Han§1, Qian Li1, Li Wu3, Jia Zhang1,
4
Xiaoling Zhong1, Jianchun Xie2, Shengrong Shao1, Yinjun Zhang4, Zhengqi Wu1
5 6 7 8 9 10 11 12 13 14
1National
"111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei University of Technology, Wuhan 430068, China 2Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU),Beijing 100048,China 3Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan 430060, China 4College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China
15 16 17 18 19 20 21
§Equal
contribution * Corresponding author Tel: (+86) 27-87950483 Fax: (+86) 27-87950483 E-mail address:
[email protected] 22 23 24 25 26 27 28 29 30
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
31
ABSTRACT: Chin-brick tea polysaccharide conjugates (TPC-C) were prepared to
32
study their emulsion capabilities. The interfacial tension and the effects of some
33
factors, such as storage time, metal ion concentrations (Na+, Ca2+), pH (2.0-8.0), and
34
heat treatment (70-100 °C) on the emulsions stabilized by TPC-C were studied. The
35
interfacial tension of TPC-C (10.88 mN/m) was lower than that of gum arabic (15.18
36
mN/m) at the concentration of 0.08%. As TPC-C concentration increased from 0.1
37
wt% to 3.0 wt%, the mean particle diameter (MPD) (d32) of emulsions stabilized by
38
TPC-C decreased from 1.88 μm to 0.16 μm. Furthermore, at a concentration of 0.5
39
wt% or higher, the MPD (d32) of emulsions stabilized by TPC-C at 25 °C and 60 °C
40
for 10 days were between 0.20 μm and 0.50 μm. In the tested pH conditions from 2.0
41
to 8.0, the MPD (d32) of emulsions stabilized by 2.0 wt% TPC-C was less than 0.20
42
μm. At Na+ concentration conditions between 0.10 mol/L to 0.50 mol/L, the MPD (d32)
43
of emulsions were between 0.19 μm to 0.20 μm, and the zeta potential values varied
44
from -34.10 mV to -32.60 mV. However, with increasing Ca2+ concentrations from
45
0.01 mol/L to 0.05 mol/L, the MPD (d32) of emulsions were between 0.20 μm to
46
21.65 μm, and the zeta potential raised sharply from -34.10 mV to -28.46 mV. The
47
emulsions stabilized by TPC-C have decent storage stability after high temperature
48
heat treatment. Overall, tea polysaccharide conjugates (TPC) strongly stabilized the
49
emulsions, which support their new application as natural emulsifiers.
50 51
KEYWORDS: tea polysaccharide conjugates; Chin-brick tea ; emulsifier; stability
52
ACS Paragon Plus Environment
Page 2 of 40
Page 3 of 40
Journal of Agricultural and Food Chemistry
53
Introduction
54
Oil-in-water emulsions are widely utilized in many food products, including milk,
55
yogurt, dressings, desserts, ice cream, sauces, and beverages. Because they are
56
thermodynamically unstable systems, they require the addition of emulsifiers acting
57
on the interface to provide kinetic stability by reducing the interfacial tension and
58
generating repulsive interactions (steric or electrostatic).1 At present, emulsifiers used
59
in food, cosmetics, and pesticide products are primarily synthetic, including fatty acid
60
monoglycerides, sucrose esters, Tween-80, Span, and polysorbates.2,3 With the
61
growing desire to consume healthy foods, the development of natural emulsifiers has
62
received much attention.4 To date, protein-based emulsifiers have
63
utilized in the food industry due to their abundance and good emulsifying ability.
64
However, the stability of emulsion stabilized by protein-based emulsifiers is easily
65
influenced by high ionic strength, elevated temperature, and pH values close to their
66
isoelectric point.5,6
been widely
67
Polysaccharide-based emulsifiers are also commonly used in the food industry as
68
natural emulsifiers. Tea produced from the buds and leaves of Camellia sinensis L.
69
has been widely utilized in dietary and medicinal applications for thousands of years.7
70
There are six major categories of teas: green, white, yellow, Oolong, black, and dark
71
teas.8 Chin-brick tea, a dark tea similar to Pu'er tea, is primarily produced in Hubei,
72
China. Chin-brick tea is prepared from sundried green tea by commonly used methods,
73
such as pile-fermentation, sieving, shaping, and drying. The pile-fermentation process
74
lasts for several weeks and has greatly improved the quality and functional
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
75
components in tea.9 Tea polysaccharide conjugates (TPCs), an important bioactive
76
compounds in six categories of tea, have received increased attention because of their
77
various health-promoting activities, including hypoglycemic, immunomodulatory, and
78
anti-oxidation.10,11,12 TPCs are macromolecules containing a small amount of
79
covalently bound protein. It is thought that the binding protein in TPC is coated by the
80
carbohydrate chain and forms a hydrophobic "core",13-16and this polysaccharide and
81
protein conjugate could induce an emulsification function to TPC. To date, it is
82
difficult to find
reports on the emulsification ability of TPC.
83
Therefore, the objectives of this investigation were to quantify the emulsifying
84
properties and emulsion stability of polysaccharide conjugates from Chin-brick tea
85
(TPC-C). To achieve this objective, emulsions stabilized by different levels of TPC-C
86
were generated by high speed blender and microfluidizer. Droplet sizes, zeta potential,
87
microstructure, and interfacial properties of the oil-in-water emulsions were measured.
88
This work could provide valuable information about the potential utilization of TPC
89
as a natural emulsifier.
90
MATERIALS AND METHODS
91
Materials. Chin-brick tea was purchased from Yichang, Hubei province, China.
92
The monosaccharide standards (D-glucuronic acid, D-galacturonic acid, L-fucose,
93
D-arabinose, D-mannose, D-xylose, D-fructose, L-rhamnose, D-galactose, D-ribose,
94
and D-glucose), gum arabic (GA), and nile red were purchased from Sigma-Aldrich
95
(St. Louis, MO, USA). All other reagents used were of analytical grade and were
96
obtained from either Sigma-Aldrich (St. Louis, MO, USA) or Aladdin (Shanghai,
ACS Paragon Plus Environment
Page 4 of 40
Page 5 of 40
Journal of Agricultural and Food Chemistry
97
China). Double distilled water was used to prepare all solutions and emulsions.
98
Polysacchride conjugates prepared from Chin-brick tea. Chin-brick tea (1.0 kg)
99
was ground using a blender. The processed material was extracted by hot water (1:16
100
m/v) under 95 °C for 3.3 h with continuous stirring. After centrifugation at 5000 rpm
101
for 15 min, the supernatant was collected and concentrated via a rotary evaporator at
102
50 °C under reduced pressure. Three volumes of ethanol were added to the
103
concentrated solution to precipitate the polysaccharide overnight at 4 °C. The
104
obtained polysaccharide was re-dissolved in double distilled water, was concentrated
105
again to remove the residual ethanol, and then lyophilized to give TPC-C.10
106
Chemical properties of TPC-C. The monosaccharide composition, amino acid
107
composition, and relative molecular weight of tea polysaccharide conjugates were
108
determined according to the reported methods. The sample was derivatized with
109
acetic anhydride, and the monosaccharide fraction was determined by gas
110
chromatography. Gel Permeation Chromatography-Multi angle light scattering (GPC-
111
MALS) was used to analyze the molecular weight distribution of the TPC-C. After the
112
sample was hydrolyzed by HCl, the amino acid composition was determined by the
113
external standard method of Hitachi L-8900 Amino Acid Auto Analyzer.17 The pH of
114
0.1-3.0 wt% TPC aqueous solution was determined by pH meter (Mettler
115
Toledo,Switzerland).
116
Interfacial tension measurements. The interfacial tension of TPC-C solutions
117
with different concentrations was measured at MCT oil-water interfaces using a Drop
118
Tensiometer (Teclis, France).15 The interfacial tension was measured by performing
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
119
an online automatic analysis of the oil drop profile using the Laplace equation:
p
120
2
r
121
Where γ is the specific surface free energy, p is the additional pressure and r is the
122
radius of curvature.
123
Ten microliters of oil droplet was delivered using a micro syringe (Exmire, Japan)
124
through a U-shaped stainless steel needle (internal diameter 0.56 mm) into an optical
125
glass cuvette containing 5 mL TPC-C solutions maintained at 25 °C. The
126
measurement was continued at 25 °C for 12000 s. The concentrations of TPC-C
127
ranged from 0.02 wt% to 0.10 wt% at an interval of 0.02, because the polysaccharide
128
conjugate solution was not transparent enough at concentrations above 0.10 wt%, and
129
the measurement results were not reliable. The GA solution with a concentration of
130
0.08 wt% was used as a control. During the detection process, the entire system
131
should be balanced to avoid external vibration interference measurements.
132
Emulsion stabilized by different concentration of TPC-C.
133
i) Emulsion preparation. Emulsions were produced by homogenizing 8.0 wt%
134
MCT with 92.0 wt% phosphate buffer solution (PBS, pH 7.0, 0.01M) containing 0.1 -
135
3.0 wt% TPC-C using a high-speed shearer (25,000 rpm) for 3 min (T18, IKA,
136
Germany). Next, the solution was passed through a high-pressure homogenizer
137
(Microfluidics M-110L, USA) at 75 MPa for three cycles. Sodium azide (0.02 % w/v)
138
was added to final emulsions to prevent microorganism growth. The concentration
139
gradient of TPC-C was set to 0.10 wt%, 0.25 wt%, 0.50 wt%, 1.00 wt%, 2.00 wt%, and
140
3.00 wt%.
ACS Paragon Plus Environment
Page 6 of 40
Page 7 of 40
Journal of Agricultural and Food Chemistry
141
ii) Particle size (d32) and zeta potential measurements. The particle size
142
distribution of emulsions was determined using static light scattering (Mastersizer
143
2000, Malvern Instruments Ltd., UK). Emulsions were dispersed in phosphate buffer
144
solutions at the same pH until an obscuration rate of 10-20% was obtained. The mean
145
particle size was reported as the surface-weighted mean diameter (d32). The zeta
146
potential of emulsions was measured using Zetasizer Nano-ZS (Malvern Instruments,
147
UK). Prior to analysis, samples were diluted with 10 mM phosphate buffer at the
148
same pH to avoid multiple scattering effects. All tests were carried out at 25 °C and
149
repeated in triplicate. It is worth noting that the sample was vortexed for 5 s before
150
each measurement.
151
Influencing factors of emulsion stability
152
i) Effect of storage time on TPC-C emulsion stability. The emulsions stabilized
153
by 2.0 wt% TPC-C (pH 7.0) were prepared and distributed into different beakers as
154
test samples. These samples were stored at 25 °C and 60 °C, while their zeta potential
155
and MPD (d32) were determined daily during the storage of 10 days as the methods
156
described above. Their emulsion appearance was observed daily until the 21st day.
157
ii)
Effect of metal ions on the stability of TPC-C emulsions. The emulsions
158
stabilized by 2.0 wt% TPC-C containing 0.10, 0.20, 0.30, 0.40, and 0.50 mol/L NaCl
159
and 0.01, 0.02, 0.03, 0.04, and 0.05 mol/L CaCl2 (pH 7.0) were prepared. The
160
emulsion samples were placed in glass test tubes and then incubated at 25 °C for 10
161
days. The stability of the emulsions was characterized by observing their appearance,
162
measuring the particle size distribution, mean particle diameter (d32), and zeta
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
163
Page 8 of 40
potential. The measurement methods were performed as described above.
164
The microstructure of TPC-C emulsions treated by metal ions was determined by
165
laser scanning confocal microscope (LSCM) with a SIM scanner for simultaneous
166
laser stimulation and observed with a 100 × oil immersion objective (Olympus Corp.,
167
Tokyo, Japan). The oil phase of emulsions was dyed by nile red solution (1 mg/mL
168
ethanol). The excitation and emission wavelength for nile red were 488 nm and 520
169
nm, respectively. All the microstructure images were acquired and analyzed using
170
Olympus viewer (Olympus Fluoview Ver.3.1 Viewer).
171
iii) Effect of pH on the stability of TPC-C emulsion. The emulsions stabilized by
172
2.0 wt% TPC-C (pH 7.0) were distributed into different beakers, and then each
173
sample was adjusted to a different pH value ranging from 2.0 to 8.0 at an interval of
174
1.0 using HCl or NaOH solutions. The emulsion samples were placed in glass test
175
tubes and then incubated at 25 °C for 10 days. The stability of the emulsions was
176
characterized by observing their appearance, measuring their particle size distribution,
177
mean particle diameter (d32), zeta potential, and the microstructure. These
178
measurement methods were performed as described above.
179
iv) Effect of thermal treatment on TPC-C emulsion stability.
The emulsions
180
stabilized by 2.0 wt% TPC-C (pH 7.0) were prepared and distributed into different
181
beakers, and then each sample was bathed at 70 °C, 80 °C, 90 °C, and 100 °C for 30
182
min. After heat treatment, these TPC-C emulsions were cooled to room temperature
183
and placed at 25 °C for 24 h. The particle size distribution and zeta potential were
184
measured. Their mean particle diameter (d32) and appearance were investigated during
ACS Paragon Plus Environment
Page 9 of 40
Journal of Agricultural and Food Chemistry
185
the storage of 10 days at 25 °C. The measurement methods were as described above.
186
Statistical analysis. The statistical analysis was carried out using the statistical
187
software Statistical Package for Social Sciences 21.0 (SPSS Inc., 2012). The
188
comparisons between groups were analyzed by Dunnett’s two-tailed t-test after
189
one-way ANOVA.
190
RESULTS
191
Chemical properties of TPC-C. GPC-MALLS results indicated that the average
192
molecular weight of TPC-C was 31580 Da. The monosaccharide and uronic acid
193
compositions of polysaccharides from Chin-brick tea were analyzed by complete acid
194
hydrolysis. The results indicated that the monosaccharide compositions were found to
195
be arabinose, rhamnose, galactose, glucose, xylose, mannose, and galacturonic acid in
196
the molar ratio of 1.00, 1.30, 2.65, 0.85, 0.39, 1.40, and 2.15, respectively. The free
197
amino acids (Cf.a.a) were not detected in TPC-C. The amino acid composition of
198
TPC-C protein moiety were Asp, Glu, Ser, Gly, Thr, Arg, Ile, Phe, Val, Tyr, Pro, Ala,
199
Leu, and Lys with the mass content (mg/g) of 7.12, 9.00, 5.72, 5.12, 4.34, 3.77, 2.19,
200
2.88, 3.72, 2.72, 2.96, 4.17, 3.36, and 1.29, respectively. The total amount of these
201
amino acids (Ca.a.) were 5.84%. The content of the protein moiety of TPC-C was
202
calculated using the formula: (Ca.a. − Cf.a.a) × 110/128 with 5.02% of TPC-C. 13
203
Interfacial tension of TPC-C. An effective emulsifier must be surface-active and
204
have the capacity to reduce the oil-water interfacial tension. Thus, the interfacial
205
tension of emulsifiers is closely related to the formation and stabilization of
206
emulsions.18,19 Therefore, in order to investigate the potential emulsifying activity of
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
207
TPC-C, we measured the dynamic interfacial tension of TPC-C at different
208
concentrations (0.02 - 0.10 wt%) and 0.08 wt% GA in solution with an MCT oil
209
droplet (Figure 1). As shown in Figure 1A, the interfacial tension decreased rapidly at
210
the beginning of adsorption, indicating that TPC-C can quickly adsorb onto the oil-
211
water interfaces. In addition, the interfacial tension decreased as the TPC-C
212
concentrations increased, with the highest concentration of TPC-C having the lowest
213
interfacial tension. As shown in Figure 1B, at the concentration of 0.08%, the
214
interfacial tension of TPC-C (10.88 mN/m) was lower than that of GA (15.18 mN/m),
215
which indicated that TPC-C can more quickly adsorb onto the oil-water interface and
216
more effectively decrease the interfacial tension. Reports have suggested that the
217
formation of emulsion is dependent on the ability of emulsifiers to absorb onto the
218
oil-water interface and to undergo a conformational rearrangement to form a
219
viscoelastic film surrounding the oil droplets.20 Therefore, TPC-C has the potential to
220
form emulsions as a natural emulsifier.
221
Effect of TPC-C concentration on the emulsion formation. A well- established
222
polysaccharide-based emulsifier, GA (5.0 wt%), was used as a control to compare to
223
the emulsifying capability of TPC-C. An oil-in-water emulsion was generated by
224
passing oil (8.0 wt%) and aqueous phase (92.0 wt%) through a high-pressure
225
homogenizer at 75MPa. The mean particle diameter (MPD) (d32) and particle size
226
distribution of emulsions stabilized by different concentrations of TPC-C and GA
227
were measured.
228
Particle size of emulsions plays an important role in their application.21 As shown
ACS Paragon Plus Environment
Page 10 of 40
Page 11 of 40
Journal of Agricultural and Food Chemistry
229
in Figure 2A, the emulsion stabilized by 5.0 wt% GA had the widest particle size
230
distribution, followed by 0.1 wt% TPC-C; both stabilized emulsions had bimodal
231
distributions. The particle size distribution of the emulsions stabilized by 0.25-3.0
232
wt% TPC-C exhibited unimodal distribution. The higher the concentration of TPC-C,
233
the smaller the particle size. If there was insufficient emulsifier molecules to cover the
234
oil droplet surface, the emulsions were more susceptible to bridging flocculation.22,23
235
Large droplet surface areas can be covered by increasing concentrations of TPC-C
236
within the homogenizer, leading to the quick formation of a smaller droplet size and
237
the reduction of droplet re-coalescence, indicating that TPC-C possesses excellent
238
emulsifying properties.1
239
As shown in Figure 2B, the MPD (d32) of emulsions stabilized by TPC-C
240
decreased with the increasing concentrations. Specifically, the MPD (d32) value of
241
emulsions decreased from 1.88 to 0.16 µm as the TPC-C concentration increased from
242
0.1 to 3.0 wt%. This phenomenon may occur, because more emulsifiers can cover a
243
larger droplet surface area, leading to smaller droplets sizes within the homogenizer.
244
Additionally, oil droplet surfaces can be covered more quickly, thereby decreasing
245
droplet re-coalescence during homogenization.24,25 The MPD (d32) value of emulsion
246
stabilized by GA was 8.68 µm, which was significantly higher than that of the
247
emulsions stabilized by TPC-C. This suggested that the emulsifying capacity of
248
TPC-C was more effective than that of the commercial polysaccharide-based
249
emulsifiers and that TPC-C can be used as a novel emulsifier.
250
Zeta potential is an indicator of the emulsion stability induced by the electrostatic
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
251
repulsive interactions.1 As shown in Figure 2B, the surface charges of droplets in
252
TPC-C-stabilized emulsions were above -35.0 mV. In addition, the negative surface
253
charge of emulsion droplets decreased with increasing concentrations of TPC-C.
254
Because TPC is weakly acidic, the pH of the solution decreases slightly as the
255
concentration of TPC increases (Figure 2C), resulting in a slight decrease in the zeta
256
potential. Previous studies have also reported that the zeta potential of emulsion
257
droplets decreases with a reduction of emulsion pH.26,27
258
Emulsion storage stability. Emulsion storage stability is imperative for the shelf
259
life of commercial food and beverage products. In order to further investigate the
260
potential of TPC-C as a natural emulsifier, we measured the storage stability of
261
emulsions stabilized by TPC-C and GA at 25 °C and 60 °C for 10 days.
262
As shown in Figure 3, there was no significant change in the mean particle
263
diameter of a droplet in emulsion stabilized with TPC-C (concentrations of 0.5 to 2.0
264
wt%) at 25 °C for up to 10 days of storage. Particle size distribution analysis showed
265
a unimodal distribution at higher TPC-C concentrations during storage, indicating that
266
TPC-C possess excellent emulsion-stabilizing properties (Shown in Figure 4). In
267
addition, visual observation results showed that TPC-C-stabilized emulsions were still
268
stable, and no visible cream layers were observed on the top of the emulsions when
269
the TPC-C concentration was above 0.5 wt% after 10 days of storage. GA-stabilized
270
emulsions formed visible white-creaming layers on the top after 24 h of storage. The
271
relatively high stability of emulsions stabilized by TPC-C at certain concentrations
272
maybe due to their small droplet size; the creaming rate is proportional to the square
ACS Paragon Plus Environment
Page 12 of 40
Page 13 of 40
Journal of Agricultural and Food Chemistry
273
of the droplet diameter, and the rate of gravitational separation decreases with a
274
reduction in droplet size.1
275
No significant difference was observed in the d32 values of emulsions stabilized by
276
TPC-C at 0.5, 1.0 and 2.0 wt% at 60 °C after 10 days of storage (Shown in Figure 3).
277
As shown in Figure 5, there were no significant changes in the microstructures of
278
droplets in emulsions at higher TPC-C concentration during 10 days of storage.
279
Interestingly, the particle size distribution of emulsions stabilized by TPC-C
280
(concentrations from 0.5 to 2.0 wt%) remained unimodal throughout storage.
281
Furthermore, negligible or no creaming was observed in emulsions at higher TPC-C
282
concentrations due to the small droplet size in these systems. Nevertheless,
283
GA-stabilized emulsions significantly separated into two phases, with a white cream
284
layer on top and a relative clear phase at the bottom. The above results indicated that
285
the emulsion stabilized by TPC-C had excellent long-term stability; thus, TPC-C
286
could be used as a natural emulsifier in commercial food and beverage applications.
287
Effects of metal ions on the stability of TPC-C emulsion. Na+ has a small impact
288
on the particle size distribution, zeta potential, and MPD (d32) of 2.0 wt% TPC-C
289
stabilized emulsions. As the concentration of Na+ increased from 0 to 0.5 mol/L, the
290
particle size distribution of the emulsions showed single peaks that almost overlapped
291
each other. All MPD (d32) values were lower than 0.30 μm (Shown in Figure 6A). The
292
absolute values of the zeta potential of the emulsions slightly decreased from 34.10 to
293
32.60 mV (Shown in Figure 6B). These emulsions maintained stability during storage
294
for 10 days at 25 °C (As captured in Figure 7). Calcium ion concentrations above 0.03
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
295
mol/L have a destructive effect on TPC-C stabilized emulsions. Calcium ions cause
296
droplet aggregation and demulsification during the storage for 10 days at 25 °C
297
(Shown in Figure 7). As the concentration of calcium ions increased from 0 to 0.05
298
mol/L, the MPD (d32) of the emulsions also increased from 0.2 to 21.65 μm (Shown
299
in Figure 8A,and Figure 9), and the absolute values of the zeta potentials of the
300
emulsions decreased linearly from 34.10 to 28.46 (Shown in Figure 8B). The linear
301
equation was Y=11.98X-32.26, R2=0.99, where Y is the Zeta potential, and X is the
302
concentration of calcium ions. The increased calcium ion concentration neutralizes the
303
negative charges on the surface of TPC-C and reduces the absolute value of the zeta
304
potential in a concentration- dependent manner.
305
Effects of pH on the stability of TPC-C emulsion. The effect of pH on the stability
306
of TPC-C emulsions is shown in Figure 10 and Figure 11. The size of the emulsion
307
droplets did not change significantly under different acidity and alkalinity (pH2-8),
308
and the emulsion had a single peak distribution with particle sizes of less than 0.20
309
μm (shown in Figure 10A, 11B). The zeta potential values ranged from -2.95 to -35.4
310
when the pH value increased from 2 to 8 (shown in Figure 10B), which suggests that
311
the emulsion will be more stable in a higher pH solution environment. At a pH greater
312
than 4.0, the absolute value of the zeta potential of the TPC-C emulsions was higher
313
than 25 mV. As shown in Figure 11B, the size of the emulsion droplets did not change
314
significantly under different acidity and alkalinity, and was less than 0.20 μm.
315
Thermal stability of TPC-C emulsion. In order to better promote the commercial
316
application of emulsions, the thermal stability of emulsions during production,
ACS Paragon Plus Environment
Page 14 of 40
Page 15 of 40
Journal of Agricultural and Food Chemistry
317
processing, transportation, and storage should be studied. The effect of heat treatment
318
at 70-100 °C on the stability of TPC-C emulsions was investigated. The emulsion
319
stabilized by 2.0 wt% TPC-C was stored at 60 °C for 10 days to maintain good
320
stability as a control. All the particle size distributions of TPC-C emulsions
321
heat-treated at different temperatures overlap with that of the control, have a single
322
peak distribution, and maintained a small particle size (Figure 12A). The zeta
323
potential values of the heat-treated emulsions were measured on the first, seventh, and
324
tenth days of storage at 25 °C (as shown in Figure 12B). The zeta potential values of
325
the heat-treated emulsions at different temperatures were close and maintained at
326
approximately -32.5 mV. The magnitude of the change in the absolute values of the
327
zeta potential was 0.30-1.96 during storage for 10 days, indicating that the high
328
temperature heat treatment did not destroy the electrostatic repulsion and steric
329
hindrance between the droplets in the emulsions. The average particle size d32 of the
330
TPC-C emulsions treated at different temperatures was not significantly changed with
331
the increase of storage days, and was maintained near 0.2 μm (Figure 12C). The
332
TPC-C emulsions did not appear to be layered at 25 °C by the 10th day after heat
333
treatment of 70-100 °C (Figure 12D), and they were apparently indistinguishable from
334
the unheated control. The TPC-C emulsions maintained storage stability after high
335
temperature heat treatment.
336 337
DISCUSSION
338
The emulsification properties of TPC have been discovered in this work, which will
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
339
greatly enhance its development and application value. At present, emulsifiers used in
340
food industry, such as fatty acid monoglycerides and sucrose esters, are primarily
341
synthetic.3, 4 However, with the increasing concern about healthy foods, the
342
development of natural emulsifiers has received much attention.2 Many current
343
studies on natural emulsifiers focus on protein or its complex with exogenous
344
polysaccharides.28 A complex in which a polysaccharide is covalently or
345
non-covalently bound to a protein is typically used as an emulsion stabilizer to
346
synergistically exert the emulsification activity of the protein and the stability of the
347
polysaccharide over a wider range of pH and ionic strength.30 TPC is composed of a
348
polysaccharide and a covalently bound protein, which allows it to have a decent
349
emulsification properties without the need to be attached to external proteins. The
350
polysaccharide moiety provided pH stability to the TPC-C stabilized emulsions .
351
Calcium ions, unlike sodium ions, destroyed the emulsion stability. The Ca2+-induced
352
protein aggregation developed because of three effects: 1) electrostatic shielding, 2)
353
crosslinking of adjacent anionic molecules by forming protein-Ca2+-protein bridges,
354
and 3) ion-specific hydrophobic interaction.29,30 In this study, the absolute value of the
355
zeta potential of the emulsion decreased in a calcium ion concentration-dependent
356
manner. The positively charged calcium ions neutralize the negative charge on the
357
surface of the TPC-C, which resulted in cross-linking between the molecules and
358
emulsion aggregation .
359 360
Funding This research was financially supported by the National Natural Science Foundation
ACS Paragon Plus Environment
Page 16 of 40
Page 17 of 40
Journal of Agricultural and Food Chemistry
361
of China (grant number 31871813) and the Beijing Advanced Innovation Center for
362
Food Nutrition and Human Health (grant number 20161012).
363
Notes The authors declare no competing financial interest.
364
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
366
REFERENCE (1) McClements, D. J.; Gumus, C. E. Natural emulsifiers-Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance. Adv Colloid Interface Sci. 2016, 234, 3-26. (2) Krog, N.; Sparsø, F.; Friberg, S.; Larsson, K.; Sjöblom, J. Food emulsifiers: their chemical and physical properties. Food Emulsions. 2004. (3) Wang, F.; Marangoni, A. Advances in the application of food emulsifier α-gel phases: saturated monoglycerides, polyglycerol fatty acid esters, and their derivatives. J Colloid Interface Sci, 2016, 483, 394-403. (4) Mcclements, D. J.; Bai, L.; Chung, C. Recent advances in the utilization of natural emulsifiers to form and stabilize emulsions. Annual Review of Food Science and Technology. 2017, 8(1), 205-236. (5) Dickinson, E. Interfacial structure and stability of food emulsions as affected by protein–polysaccharide interactions. Soft Matter. 2008, 4(5), 932. (6) McClements, D. J.; Gumus, C.E. Natural emulsifiers - Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance. Adv Colloid Interface Sci. 2016, 234, 3-26. (7) Nie, S.P.; Xie, M.-Y. A review on the isolation and structure of tea polysaccharides and their bioactivities. Food Hydrocolloids. 2011, 25(2), 144-149. (8) Zhu, Y. F.; Chen, J. J.; Ji, X. M.; Hu, X.; Ling, T. J.; Zhang, Z. Z.; Bao, G. H.; Wan, X. C. Changes of major tea polyphenols and production of four new B-ring fission metabolites of catechins from post-fermented Jing-Wei Fu brick tea. Food Chem. 2015, 170, 110-117. (9) Lu, S. F.; Zheng, P. C.; Liu, P. P.; Wang, S. P.; Teng, J.;Feng, L.; Gong, Z.M. Research Progress on Qingzhuan Tea. Acta Tea Sinica. 2018, 59 (3),162 -167. (10) Chen, X. Q.; Fang, Y. P.; Nishinari, K.; We, H.; Sun, C. C.; Li, J. R.; Jiang, Y. W. Physicochemical characteristics of polysaccharide conjugatesprepared from fresh tea leaves and their improving impaired glucose tolerance. Carbohydrate Polymers. 2014, 112, 77-84. (11) Chen, X. Q.; Zhang, Z. F.; Gao, Z. M.; Huang, Y.; Wu, Z. Q. Physicochemical properties and cell-based bioactivity of Pu’erh teapolysaccharide conjugates. International Journal of Biological Macromolecules. 2017, 104, 1294– 1301. (12) Chen, X. Q.; Ye, Y.; Cheng, H.; Jiang, Y. W.; Wu, Y. L. Thermal Effects on the Stability and Antioxidant Activity of an Acid Polysaccharide Conjugate Derived from Green Tea. J. Agric. Food Chem. 2009, 57, 5795–5798. (13) Chen, X. Q.; Du, Y.; Wu, L.; Xie, J. C.; Chen, X. L.; Hu, B. B.; Wu, Z. Q.; Yao, Q. F.; Li, Q. Effects of Tea Polysaccharide Conjugates and Metal Ions on Precipitate Formation by Epigallocatechin Gallate and Caffeine, the Key Components of Green Tea Infusion. J. Agric. Food Chem 2019, 67(13), 3744-3751. (14) Chen, X. Q.; Xie, J. C.; Huang, W.; Shao, S. R.; Wu, Z. Q.; Wu, L.; Li, Q. Comparative analysis of physicochemical characteristics of green tea polysaccharide conjugates and its decolored fraction and their effect on HepG2 cell proliferation. Industrial Crops and
ACS Paragon Plus Environment
Page 18 of 40
Page 19 of 40
Journal of Agricultural and Food Chemistry
Products. 2019, 131, 243-249. (15) Chen, X. Q.; Shao, S. R.; Xie, J. C.; Yuan, H.; Li, Q.; Wu, L; Wu, Z. Q.; Yuan, H. B.; Jiang, Y. W. Analysis of Protein Moiety of Polysaccharide Conjugates Water-extracted from Low Grade Green Tea. Chem. Res. Chin. Univ. 2018, 3(44), 691-696. (16) Chen, X. Q.; Song, W.; Zhao, J.; Zhang, Z. F.; Zhang, Y. T. Some physical properties of protein moiety of alkali-extracted tea polysaccharide conjugates were shielded by its polysaccharide. Molecules 2017, 22(6), 914 (17) Chen, X. Q.; Lin, Z.; Ye, Y.; Zhang, R.; Yin, J. F.; Jiang, Y. W.; Wan, H. T.; Suppression of diabetes in non-obese diabetic (NOD) mice by oral administration of water-soluble and alkali-soluble polysaccharide conjugates prepared from green tea. Carbohydrate Polymers, 2010, 82, 28–33. (18) Dickinsion, E. Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids. 2003, 17, 25-39 (19) Jiang, J.; Jin, Y.; Liang, X.; Piatko, M.; Campbell, S.; Lo, S. K.; Liu, Y. Synergetic interfacial adsorption of protein and low-molecular-weight emulsifiers in aerated emulsions. Food Hydrocolloids. 2018, 81, 15-22. (20) Uruakpa, F. O.; Arntfield, S.D. Emulsifying characteristics of commercial canola protein– hydrocolloid systems. Food Research International. 2005, 38(6), 659-672. (21) Xu, X.; Zhong, J.; Chen, J.; Liu, C.; Luo, L.; Luo, S.; Wu, L.; McClements, D. J. Effectiveness of partially hydrolyzed rice glutelin as a food emulsifier: Comparison to whey protein. Food Chem. 2016, 213, 700-707. (22) Dickinson, E. Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocolloids. 2009,.23(6), 1473-1482. (23) Mc, C.; David, J. Enhanced delivery of lipophilic bioactives using emulsions: a review of major factors affecting vitamin, nutraceutical, and lipid bioaccessibility. Food & function, 2018, 9, 22-41. (24) Dickinson, E. Strategies to control and inhibit the flocculation of protein-stabilized oil-in-water emulsions. Food Hydrocolloids. 2019, 96, .209-223 (25)Tisserand, C.; Brambilla G.; Meunier G.; Parker A. Predicting the long-term stability of depletion-flocculated emulsions by static multiple light scattering (SMLS). Journal of Dispersion Science and Technology. 2019, 1-8. (26) Ma, F.; Zhang, Y.; Yao, Y.; Wen, Y.; Hu, W.; Zhang, J.; Liu, X.; Bell, A. E.; Tikkanen-Kaukanen, C. Chemical components and emulsification properties of mucilage from Dioscorea opposita Thunb. Food Chem. 2017, 228, 315-322. (27) Nakauma, M.; Funami, T.; Noda, S.; Ishihara, S.; Al-Assaf, S.; Nishinari, K.; Phillips, G. O. Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying properties. Food Hydrocolloids. 2008, 22(7), 1254-1267. (28) Krstonosic, V.; Dokic, L.; Nikolic, I.; Milanovic, M. Influence of xanthan gum on oil-in-water emulsion characteristics stabilized by OSA starch. Food Hydrocolloids. 2015, 45, 9-17. (29) Ju, Z. Y.; Kilara,A. Aggregation induced by calcium chloride and subsequent thermal gelation of whey protein isolate, J Dairy Sci ,1998, 81, 925-931.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
(30) Wang, C. H.; S. Damodaran. Thermal gelation of globular proteins: influence of protein conformation on gel strength. J. Agric. Food Chem. 1991, 39, 434-442.
ACS Paragon Plus Environment
Page 20 of 40
Page 21 of 40
Journal of Agricultural and Food Chemistry
Figure Captions Figure 1. Interfacial tension at different concentrations of TPC-C (A) and 0.08 wt% GA (B) at 25 °C . Figure 2. Particle size distribution (A), the mean particle diameter (d32) and zeta potential of emulsions stabilized by different concentrations of TPC-C at 25 °C (B); pH of different concentrations of TPC-C aqueous solution at 25 °C (C). Figure 3. The mean particle diameter (d32) of emulsions stabilized by different concentrations of TPC-C during 10 days of storage at 25 °C (A) and 60° C (B). Figure 4. Particle size distribution of emulsions stabilized by different concentrations of TPC-C during 10 days storage at 25 °C (A) and 60 °C (B). Figure 5. Appearance changes were observed on day 0 and day 10 (A) and day 21 and day 31 (B) for different concentrations of TPC-C emulsions and 5.00% GA emulsion. (The numbers 1, 2, 3, 4, 5, 6, and 7, in pictures represent 0.10% TPC-C emulsion, 0.25% TPC-C emulsion, 0.5% TPC-C emulsion, 2.0% TPC-C emulsion, 3.0% TPC-C emulsion, and 5.00% GA emulsion, respectively. Figure 6. Particle size distribution (A) and zeta potential (B) of emulsions stabilized by 2.0 wt% TPC-C with different concentrations of NaCl. Figure 7. Appearance change of emulsions stabilized by 2.0 wt% TPC-C with different metal ion concentrations over 10 days at 25 °C. The numbers 1-5 in pictures represent the concentration of NaCl from 0.10 to 0.50 mol/L, respectively. The numbers 6-10 in pictures represent the concentration of CaCl2 from 0.01mol/L to 0.05 mol/L, respectively. Figure 8. Particle size distribution (A) and zeta potential (B) of emulsions stabilized by 2.0 wt% TPC-C with different concentrations of CaCl2. Figure 9. Microscopic appearance of 2.0 wt% TPC-C emulsions stained with nile red dye and observed by using LSCM under different metal ion concentrations on the 10th day of storage at 25 °C. The ionic strength of the solution was prepared by NaCl (0.10-0.50 mol/L) and CaCl2 (0.01-0.05 mol/L). Figure 10. Particle size distribution (A) and zeta potential (B) of the emulsions stabilized by 2.0 wt% TPC-C under different pH conditions on the 10th day at 25 °C. Figure 11. Appearance (A) and 10th-day microscopic appearance (B) of emulsions stabilized by 2.0 wt% TPC-C under the conditions of pH 2-8 and 25 °C, stained with nile red dye and observed using LSCM. Figure 12. Particle size distribution (A) and zeta potential (B) of 2.0 wt% TPC-C stabilized emulsion under the thermal treatment at 70-100 °C during 24 h storage at 25 °C, MPD (d32) (C) and appearance (D) during the 10 day storage at 25 °C .The emulsion stabilized by 2.0 wt% TPC-C was stored at 60 °C as an untreated control.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 22 of 40
Figure 1.
A 0.10% TPC-C 0.08% TPC-C 0.06% TPC-C 0.04% TPC-C 0.02% TPC-C
Interfacial tension (mN/m)
40
30
20
10
0
2000
4000
6000
Time (s)
ACS Paragon Plus Environment
8000
10000
12000
Page 23 of 40
Journal of Agricultural and Food Chemistry
B
32
0.08% GA 0.08% TPC-C
30
Interfacial tension (mN/m)
28 26 24 22 20 18 16 14 12 10 0
2000
4000
6000
8000
10000
12000
Time(s)
Figure 2. 5.00% GA 0.10% TPC-C 0.25% TPC-C 0.50% TPC-C 1.00% TPC-C 2.00% TPC-C 3.00% TPC-C
A 70 60
Volume (%)
50 40 30 20 10 0 0.01
0.1
1
10
100
Particle size m)
ACS Paragon Plus Environment
1000
10000
Journal of Agricultural and Food Chemistry
B
Page 24 of 40
Mean particle size D[3,2] 2.0
-44
Zeta potential
D[3,2] (m)
1.5
-40 -38
1.0
-36 -34
0.5
-32 0.0
-30
0.10%
0.25%
0.50%
1.00%
2.00%
3.00%
Concentration (%)
C
7.1 7.0 6.9
pH
6.8 6.7 6.6 6.5 6.4 6.3
0.10%
0.25%
0.50%
1.00%
2.00%
Concentration (%)
ACS Paragon Plus Environment
3.00%
Zeta Potential (mV)
-42
Page 25 of 40
Journal of Agricultural and Food Chemistry
Figure 3. 0.10% TPC-C 0.25% TPC-C 0.50% TPC-C 1.00% TPC-C 2.00% TPC-C
A 3.5
D[3,2]-25C
3.0
D[3,2] (m)
2.5 2.0 1.5 1.0 0.5 0.0 0
2
4
6
8
10
Time (Day)
B 7
0.10% TPC-C 0.25% TPC-C 0.50% TPC-C 1.00% TPC-C 2.00% TPC-C
D[3,2]-60C
6
D[3,2] (m)
5 4 3 2 1 0 0
2
4
6
8
Time (Day)
ACS Paragon Plus Environment
10
Journal of Agricultural and Food Chemistry
Page 26 of 40
Figure 4. 40
Day0 Day1 Day3 Day5 Day7 Day10
0.10%-25C
30
30
25
25
20 15
20 15
10
10
5
5
0
0
0.01
0.1
1
10
100
1000
Day0 Day1 Day3 Day5 Day7 Day10
0.25%-25C
35
Volume (%)
Volume (%)
35
40
0.01
10000
0.1
1
Day0 Day1 Day3 Day5 Day7 Day10
0.50%-25C
40
30
30
25
25
20 15
5
5
0
0 10
100
1000
10000
0.01
0.1
Day0 Day1 Day3 Day5 Day7 Day10
2.00%-25C
35
Volume (%)
30 25 20 15 10 5 0 0.01
0.1
1
10
1
10
Partical Size (m)
Partical Size (m) 40
Day0 Day1 Day3 Day5 Day7 Day10
1.00%-25C
15 10
1
10000
20
10
0.1
1000
35
Volume (%)
Volume (%)
35
0.01
100
Partical Size (m)
Partical Size (m) 40
10
100
1000
10000
Partical Size (m)
A
ACS Paragon Plus Environment
100
1000
10000
Page 27 of 40
Journal of Agricultural and Food Chemistry
40
Day0 Day1 Day3 Day5 Day7 Day10
0.10%-60C
30
30
25
25
20 15
15 10
5
5 0
0.01
0.1
1
10
100
1000
Partical Size (m) 40
0.01
10000
0.50%-60C
0.1
1
10
100
1000
Partical Size (m)
Day0 Day1 Day3 Day5 Day7 Day10
35
40
10000 Day0 Day1 Day3 Day5 Day7 Day10
1.00%-60C
35 30
30 25
Volume (%)
Volume (%)
0.25%-60C
20
10
0
20 15
25 20 15 10
10
5
5
0
0 0.01
0.1
1
10
100
1000
0.01
10000
40
Day0 Day1 Day3 Day5 Day7 Day10
2.00%-60C
35 30 25 20 15 10 5 0 0.01
0.1
1
10
0.1
1
10
Partical Size (m)
Partical Size (m)
Volume (%)
Day0 Day1 Day3 Day5 Day7 Day10
35
Volume (%)
Volume (%)
35
40
100
1000
10000
Partical Size (m)
B
ACS Paragon Plus Environment
100
1000
10000
Journal of Agricultural and Food Chemistry
Figure 5.
A
B
ACS Paragon Plus Environment
Page 28 of 40
Page 29 of 40
Journal of Agricultural and Food Chemistry
Figure 6.
A Fresh emulsion 0.10 mol/L NaCl 0.20 mol/L NaCl 0.30 mol/L NaCl 0.40 mol/L NaCl 0.50 mol/L NaCl
10
Volume (%)
8
6
4
2
0 0.01
0.1
1
10
100
1000
Particle Size (m)
B
-40
Zeta Potential (mV)
-35
-30
-25
-20
-15 0.00
0.10
0.20
0.30
0.40
Concentration (mol/L)
ACS Paragon Plus Environment
0.50
10000
Journal of Agricultural and Food Chemistry
Figure 7.
ACS Paragon Plus Environment
Page 30 of 40
Page 31 of 40
Journal of Agricultural and Food Chemistry
Figure 8.
A
Fresh emulsion 0.01 mol/L CaCl2 0.02 mol/L CaCl2 0.03 mol/L CaCl2 0.04 mol/L CaCl2 0.05 mol/L CaCl2
35 30
Volume (%)
25 20 15 10 5 0 0.01
0.1
1
10
Partical Size (m)
ACS Paragon Plus Environment
100
1000
10000
Journal of Agricultural and Food Chemistry
B
Page 32 of 40
-40
Zeta Potential (mV)
-35
-30
-25
-20
-15 0.00
0.01
0.02
0.03
0.04
Concentration (mol/L)
ACS Paragon Plus Environment
0.05
Page 33 of 40
Journal of Agricultural and Food Chemistry
Figure 9.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 34 of 40
Figure 10. pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 pH 8
A 10
Volume (%)
8
6
4
2
0 0.01
0.1
1
10
100
Partical Size (m)
ACS Paragon Plus Environment
1000
10000
Page 35 of 40
Journal of Agricultural and Food Chemistry
B
-40 -35
Zeta Potential (mv)
-30 -25 -20 -15 -10 -5 0 2
3
4
5
6
pH
ACS Paragon Plus Environment
7
8
Journal of Agricultural and Food Chemistry
Figure 11.
ACS Paragon Plus Environment
Page 36 of 40
Page 37 of 40
Journal of Agricultural and Food Chemistry
Figure 12.
A
12
CK 70C 80C 90C 100C
24h
10
Volume (%)
8
6
4
2
0 0.01
0.1
1
10
100
Partical Size (m)
ACS Paragon Plus Environment
1000
10000
Journal of Agricultural and Food Chemistry
Page 38 of 40
CK 70℃ 80℃ 90℃ 100℃
B -40
Zeta Potention (mv)
-35
-30
-25
-20
-15
-10 0
2
4
6
8
10
Time (Day)
C
CK 70℃ 80℃ 90℃ 100℃
0.40 0.35
D[3,2] (m)
0.30 0.25 0.20 0.15 0.10 0.05 0.00 0
2
4
6
Time (Day)
ACS Paragon Plus Environment
8
10
Page 39 of 40
Journal of Agricultural and Food Chemistry
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Table of Contents (TOC) Graphic
ACS Paragon Plus Environment
Page 40 of 40