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Rheological Enhancement of Pork Myofibrillar Protein– Lipid Emulsion Composite Gels via Glucose Oxidase Oxidation/Transglutaminase Cross-Linking Pathway Xu Wang, Youling L. Xiong, and Hiroaki Sato J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03007 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017
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Journal of Agricultural and Food Chemistry
Rheological Enhancement of Pork Myofibrillar Protein–Lipid Emulsion Composite Gels via Glucose Oxidase Oxidation/Transglutaminase Cross-Linking Pathway
Xu Wang,† Youling L. Xiong*,† and Hiroaki Sato§
†
Department of Animal and Food Sciences, University of Kentucky, Lexington, Kentucky 40546,
United States §
Institute of Food Sciences and Technologies, Food Products Division, Ajinomoto Company,
Inc., Kawasaki 201-8681, Japan
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ABSTRACT: Porcine myofibrillar protein (MP) was modified with glucose oxidase (GluOx)–
2
iron that produces hydroxyl radicals then subjected to microbial transglutaminase (TGase) cross-
3
linking in 0.6 M NaCl at 4 °C. The resulting aggregation and gel formation of MP were
4
examined. The GluOx-mediated oxidation promoted the formation of both soluble and insoluble
5
protein aggregates via disulfide bonds and occlusions of hydrophobic groups. The subsequent
6
TGase treatment converted protein aggregates into highly cross-linked polymers. MP–lipid
7
emulsion composite gels formed with such polymers exhibited markedly enhanced gelling
8
capacity: up to 4.4-fold increases in gel firmness and 3.5-fold increases in gel elasticity over non-
9
treated protein. Microstructural examination showed small oil droplets dispersed in a densely
10
packed gel matrix when MP was oxidatively modified, and the TGase treatment further
11
contributed to such packing. The enzymatic GluOx oxidation/TGase treatment shows promise to
12
improve the textural properties of emulsified meat products.
13 14
KEYWORDS: myofibrillar protein; glucose oxidase; transglutaminase; oxidation; gelation
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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The formation of a gel matrix, involving the interactions between protein, lipid and water, plays
18
an essential role in textural properties of processed meat.1 Myofibrillar protein (MP) is the main
19
component that dictates gel characteristics contributing to meat texture.2 Comminuted (finely
20
chopped) meat products, notably frankfurters and bolognas, consist of a three-dimensional MP
21
network filled with fat globules; therefore, a MP−lipid composite emulsion can be used to study
22
the rheological properties of such processed meats.3, 4
23
The thermal gelation process involves unfolding of native protein molecules, followed by
24
protein–protein association and further aggregation. A slow heating rate allows sufficient time
25
for protein denaturation and proper alignment to occur prior to aggregation, resulting in a stable
26
and elastic gel matrix.5 The process of gelation usually involves hydrophobic and electrostatic
27
forces, hydrogen bonds, and van der Waal's interactions, as well as covalent bonds, such as
28
disulfide linkages.6
29
Oxidation can modify amino acid residues and alter the protein structure, thus, change
30
molecular forces involved in protein–protein interactions and affect the gelation process.7 A
31
series of investigations have shown that mild protein oxidation can improve the functionality of
32
MP through altering the mode of myosin aggregation in favor of an elastic gel network, while
33
excessive oxidation tends to generate large protein aggregates that are morphologically incapable
34
of forming a fine gel network but a porous coagulum instead.8-11 Oxidants tested in these studies
35
vary from reactive oxygen species, free radicals of lipid peroxidation, and secondary lipid
36
oxidation products, to phenolic compounds. Glucose oxidase (GluOx), a commercial food
37
industry enzyme that produces H2O2 from glucose, provides a controllable enzymatic means to
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achieve deliberate oxidative modification of target proteins for functionality improvements. As
39
reported recently, GluOx-modified MP had superior rheological properties over chemically
40
(Fenton system)-modified MP because the hydroxyl radical (•OH) production in the GluOx–iron
41
system was progressive and controllable, which allows slow unfolding and subsequent
42
aggregation of proteins.12
43
The size and surface properties (hydrophobicity) of filler particles in MP composite gels
44
affect liquid retention and deformation properties of the gels.13 Transglutaminase (TGase), which
45
catalyzes an acyl transfer reaction between glutamine and lysine residues to form ε-(γ-Glu)-Lys
46
isopeptide bonds, has been extensively investigated to promote gel formation for textural
47
property enhancements in restructured muscle foods.14 Protein polymers induced by TGase
48
usually exhibit improved functionality, particularly gelation and emulsification,15 when
49
compared with proteins without TGase treatment. Because the efficacy of TGase is dependent
50
upon the accessibility of glutamine and lysine residues, Li et al.16 have introduced oxidative
51
modification with chemically generated •OH that induces protein unfolding. Indeed, aided by the
52
substrate (MP) structural change, an additional 14.8% increase (P < 0.05) in protein gel rigidity
53
attributed to TGase was reportedly achieved.
54
Because the H2O2 production from GluOx-catalyzed glucose oxidation was progressive (as
55
opposed to a sudden burst of H2O2 in the Fenton reaction), it can be hypothesized that •OH,
56
gradually formed from H2O2 reacting with Fe2+, would induce ordered MP aggregation in favor
57
of further cross-linking by TGase. The objective of this study was to test this hypothesis and,
58
specifically, investigate the rheological properties of MP gels after GluOx modification followed
59
by TGase treatment.
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Journal of Agricultural and Food Chemistry
MATERIALS AND METHODS
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Materials. Porcine Longissimus lumborum (48 h post-mortem) was obtained from the
64
University of Kentucky Meat Laboratory, a USDA-approved facility. The loin muscle samples
65
were vacuum packaged and stored in a –30 °C freezer for less than 6 months before use. MP was
66
isolated from thawed muscle at 4 °C according to Park et al.17 using a buffer of 10 mM sodium
67
phosphate, 0.1 M NaCl, 2 mM MgCl2, and 1 mM EGTA at pH 7.0. The pH of MP suspension in
68
0.1 M NaCl at the final wash was adjusted to 6.25 with 0.1 M HCl before centrifugation. Protein
69
concentration of the MP was measured by the Biuret method using bovine serum albumin as a
70
standard.18 Commercial canola oil was purchased from a local grocery. GluOx (2000 U/g),
71
TGase (1000 U/g) and organic iron (food-grade microbial iron) were donated by Ajinomoto Co.,
72
Inc. (Kawasaki, Japan).
73
GluOx Oxidation/Transglutaminase Modification of MP. Enzymatic •OH-producing
74
reagents (GluOx/glucose/iron) at final concentrations of 4, 8 and 16 µg GluOx and 50 µg glucose
75
per mg MP and 10 µM Fe were mixed into MP suspensions [20 or 40 mg/mL protein, in 15 mM
76
1,4-piperazinediethanesulfonic acid (PIPES) buffer, pH 6.25, 0.6 M NaCl]. Iron (Fe) was
77
included to produce •OH from H2O2 formed by the GluOx-catalyzed oxidation of glucose. The
78
mixtures were incubated for 12 h at 4 °C to allow oxidative modification. To investigate TGase-
79
induced protein cross-linking, control and oxidized MP samples were treated with TGase (10 µg
80
per mg MP) in the same PIPES buffer (pH 6.25) with 0.6 M NaCl at 4 °C for 2 h.
81
Measurement of Structure-Related Changes and Aggregation of MP. Samples of control
82
and GluOx-oxidized MP (40 mg/mL) with or without TGase treatment were diluted to 2 or 4
83
mg/mL protein with 15 mM PIPES containing 0.6 M NaCl (pH 6.25), then subjected to chemical
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analysis and gel electrophoresis. For hydrophobicity tests (fluorescence), MP samples were
85
diluted to 0.25 mg/mL with the same PIPES buffer solution.
86
Free Sulfhydryls. Total free sulfhydryl content (4 mg/mL MP solution) was determined as
87
described by Liu et al.19 A molar extinction coefficient of 13,600 M−1 cm−1 was used to calculate
88
the sulfhydryl concentration.
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ANS Fluorescent Spectra. Fluorescence spectra were acquired immediately after addition of
90
20 µL 8-anilinonaphthalene-1-sulfonic acid (ANS, 8.0 mM in 0.01 M phosphate buffer, pH 7.0)
91
to 4 mL 0.25 mg/mL MP solutions in 0.6 M NaCl at room temperature in the dark for 15 min.
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The emission spectra from 400 to 500 nm with an excitation wavelength of 380 nm were
93
recorded, using a FluoroMax-3 spectrofluorometer (Horiba Jobin Yvon Inc., Edison, NJ, USA).
94
Protein Solubility. MP sample solutions (2 mg/mL) were centrifuged at 5000 g and 4 °C for
95
15 min. Protein solubility was defined as the protein concentration of the supernatant divided by
96
that of the original MP suspension. The protein concentration was determined by the Biuret
97
method.
98
Electrophoresis. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)
99
was conducted on control and GluOx/TGase-modified MP samples according to the method of
100
Laemmli20 with a 4% polyacrylamide stacking gel and a 12% polyacrylamide resolving gel. For
101
all MP samples, 1 mM N-ethylmaleimide (NEM, a thiol-blocking agent) was added before
102
heating to prevent possible formation of disulfide artifacts during sample preparation. Each
103
sample well was loaded with 50 µg of protein. Formation of soluble polymers (SP) on the top of
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the stacking gel and loss of myosin heavy chain (MHC) and actin, relative to non-oxidized MP
105
without TGase, were quantified using the UN-SCAN-IT software (Silk Scientific, Orem, Utah,
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USA) to analyze the effect of GluOx oxidation on TGase cross-linking using the following
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formula:
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Relative change % =
pixel of sample – pixel of non-oxidized MP without TGase ×100. pixel of non-oxidized MP without TGase
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Evaluation of Gelling Properties of MP–Lipid Emulsion Composites. The gelling
110
properties of MP–lipid emulsion composite samples were analyzed with a small-strain dynamic
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rheological test and a large-strain extrusion measurement. Emulsions were prepared by
112
homogenizing 20 mg/mL MP suspension and canola oil (25%, w/w) with a Polytron PT 10-
113
35GT blender with PT-DA 12/2 EC-B154 aggregate (Brinkmann Instruments, Inc., Westbury,
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NY, USA) at a speed of 17,500 rpm for 2 min with the tubes submerged in an ice slurry. The
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emulsions were immediately added to the 40 mg/mL MP in 15 mM PIPES buffer containing 0.6
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M NaCl (pH 6.25) by gently stirring with a glass rod. Then the MP–lipid sols (final 30 mg/mL
117
MP and 10% lipid) were incubated with TGase (10 µg per mg MP) for 2 h at 4 °C.
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Dynamic Rheological Behavior. The viscoelastic characteristics of MP–lipid emulsion
119
composites were continuously monitored during the heat-induced gelation with a Bohlin CVO
120
100 rheometer (Malvern Instruments, Westborough, MA, USA). Sols were heated between
121
parallel plates (upper plate, 30 mm diameter) from 20 to 75 °C at a 1 °C/min rate with a fixed
122
frequency of 0.1 Hz and a controlled maximum strain of 0.02. To prevent dehydration, a thin
123
layer of silicone oil was applied to the exposed edge of samples. Changes in the storage modulus
124
(G′, an elastic force) during the sol → gel transformation were recorded.
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Gel Firmness. Aliquots of 5 g each of MP–lipid emulsion composites sols were carefully
126
transferred into glass vials (16.5 mm diameter), then heated from 20 to 75 °C at a rate of 1 °C
127
/min in a water bath. After heating, gels in the vials were immediately chilled in an ice slurry and
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stored at 4 °C overnight. The gels in vials were then equilibrated at room temperature (22 °C) for 7 ACS Paragon Plus Environment
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1 h and penetrated with a flat-faced stainless steel probe (14.8 mm diameter) at a speed of 20
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mm/s. The initial peak force required to rupture the gel was expressed as gel firmness.
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Cooking Loss. Cooking loss was calculated using the following formula, where Wgel and Wsol
132
are, respectively, the weight of the emulsion gel after heating and the weight of the original sol
133
before heating: Cooking loss (%) =
134
Wsol – Wgel Wsol
×100.
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Microstructure of MP–Lipid Emulsion Composite Gels. To observe the effect of GluOx
136
oxidation/TGase induced MP modification on the gel structure, the microstructure of MP–lipid
137
emulsion composite gels was examined.
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Light Microscopy. MP–lipid emulsion gel samples were prepared for light microscopy using
139
the paraffin section procedures as described by Wu et al.4 Approximately 5 mm3 blocks of
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samples were excised from intact gels, encased in a cassette, and fixed in 8% paraformaldehyde
141
overnight. The samples were dehydrated by immersion in a series of ethanol (50, 70, 90, and
142
100%), fixed in paraffin, and sectioned at a thickness of 8 µm using a microtome. After removal
143
of paraffin with xylene, the slides were stained with Ehrlich's hematoxylin, then imaged using a
144
MICROPHOT-FXA Nikon photomicroscope equipped with a built-in digital camera (Nikon Inc.,
145
Garden City, NY, USA).
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Scanning Electron Microscopy (SEM). Samples for SEM examination were prepared as
147
described by Feng et al.21 with slight modifications. Small cubes (approximately 5 mm3) were
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obtained from emulsion gels and fixed in 0.1 M phosphate buffer (pH 7.2) containing 4%
149
paraformaldehyde and 1% glutaraldehyde. Fixed samples were washed with 0.1 M phosphate
150
buffer (pH 7.2) 3 times and then post-fixed for 5 h in 0.1 M phosphate buffer (pH 7.2) containing
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1% osmium tetroxide. The post-fixed samples were washed with 0.1 M phosphate buffer (pH 7.2) 8 ACS Paragon Plus Environment
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3 times and dehydrated in a series of ethanol (50, 75, 90, 95, and twice 100% for 30 min, each).
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Samples were further dehydrated by submerging in acetone and dried in warm air. Dried samples
154
were sputter-coated with gold and examined under a FEI Quanta 250 (FEI Inc., Hillsboro, OR,
155
USA) with 2 kV accelerating voltage.
156
Statistical Analysis. Data obtained from two independent trials (n = 2), each employing a
157
new set of MP preparation, were submitted to the one-way analysis of variance (one-way
158
ANOVA) using the general linear model’s procedure of Statistix software 9.0 (Analytical
159
Software, Tallahassee, FL, USA). Significant (P < 0.05) differences between means were
160
identified by the least significance difference (LSD) all-pairwise multiple comparisons.
161 162
RESULTS AND DISCUSSION
163 164
Protein Structural Changes. Thiol oxidation, with disulfide bonds being the most common
165
end products, affects protein structure and protein–protein interactions.22 Reduction of the
166
sulfhydryl content and solubility of MP caused by oxidation with GluOx followed by cross-
167
linking with TGase is shown in Figure 1. The total free sulfhydryl (SH) content of non-oxidized
168
MP without TGase was 64.9 nmol/mg protein. After GluOx oxidation, the SH content decreased
169
significantly with increasing GluOx concentrations to 57.8 nmol/mg protein (P < 0.05),
170
suggesting the formation of disulfide bonds (S–S). The results were consistent with our previous
171
observation12 that high concentrations of GluOx in the presence of glucose and Fe substantially
172
modified MP, promoted S–S bond and polymer formation, and decreased the solubility (Figure
173
1B). The SH content was reduced after TGase treatment for 2 h, probably caused by reburying of
174
the cysteine residues due to TGase-catalyzed MP agglomeration.
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Surface hydrophobicity of a protein is an important determinant of its physical stability,
176
solubility, aggregation tendency, as well as adsorption behavior at the oil/water interface, which
177
affects its functionality.23 ANS, as an extrinsic fluorescent dye with an aromatic structure and a
178
negatively charged sulfonate group, can bind to the hydrophobic pockets on protein through
179
aromatic–aromatic interactions and to positively charged amino acids, e.g., histidine, lysine, and
180
arginine, via electrostatic interactions.23 Therefore, an increased ANS fluorescence intensity is
181
indicative of the exposure of hydrophobic groups of MP. The surface hydrophobicity of MP due
182
to oxidation as shown by the ANS spectra was GluOx-dose-dependent, where the fluorescence
183
intensity decreased with increasing GluOx concentrations (Figure 2A). A similar phenomenon
184
was observed previously,24 which was attributed to intramolecular S–S bonds that stabilized the
185
secondary structure of myosin.
186
With subsequent TGase treatments (Oxidation + TGase), the surface hydrophobicity
187
decreased further (Figure 2B). The steady drop in intensity of fluorescence suggests that GluOx
188
oxidation and TGase cross-linking caused MP to aggregate, resulting in a partial burial of the
189
ANS binding sites that offsets the potential effect of unfolding induced by oxidation.16 In
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addition, cross-linking between lysine and glutamine catalyzed by TGase could weaken the
191
electrostatic interactions between the negatively charged sulfonate group in ANS and the
192
positively charged amino group in lysine, leading to an additional fluorescence attenuation. A
193
slight increase of surface hydrophobicity in oxidized MP was reported by Li et al.16, where mild
194
oxidation predominantly induced protein unfolding over aggregation, which may initiate a
195
dissociated state with large clusters of hydrophobic sites exposed to allow binding with ANS,
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hence, explaining the increase in fluorescence intensity.
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Cross-Linking Patterns of MP Components. Protein polymerization after treatment with
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GluOx and TGase was analyzed by SDS–PAGE under non-reducing conditions (Figure 3); the
199
inset displays the relative content (%) of soluble polymers (SP) formed and the amount of
200
myosin heavy chain (MHC) and actin lost over non-oxidized MP without TGase. In oxidized MP,
201
the intensity of MHC and actin bands attenuated with increasing the GluOx concentration: up to
202
25.3% reductions for MHC and 16.9% for actin, and a 112% increase in SP, compared to non-
203
oxidized MP samples. To elucidate the effect of GluOx oxidation on TGase cross-linking, the
204
relative changes (∆) in band intensity (pixel) attributed to TGase were calculated. The loss of
205
actin due to TGase increased from 9.3% in non-oxidized to 14.5–21.7% in oxidized MP.
206
However, the loss of MHC due to TGase decreased from 21.7% in non-oxidized to 7.2% in
207
oxidized MP (treated with 16 µg/mg GluOx). Interestingly, the TGase treatment produced a 34.7%
208
increase of SP in non-oxidized MP, contrasting to a net loss (up to 18.3%) in the SP content in
209
GluOx-oxidized samples. This may be due to the further aggregation of SP in the latter samples
210
forming exceedingly large polymers that were unable to enter the stacking gel.
211
Under oxidative stress, protein unfolding and aggregation coexist, and the effectiveness of
212
TGase will depend on the preponderance of these specific processes. When unfolding was
213
caused in a chemically-produced •OH system, myosin cross-linking by TGase was favored.25 In
214
the present study with GluOx, protein aggregation (evidenced by the substantial loss of MHC)
215
rather than unfolding appeared to be dominant process. Enhanced intramolecular disulfide
216
bridging and occlusions of accessible glutamine and lysine residues in MP under the relatively
217
slow H2O2 production condition would render TGase less productive. When electrophoresed
218
under a reducing condition (with 5% β-mercaptoethanol), both MHC and actin were largely
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recovered in all MP samples (result not shown), indicating that disulfide was the main type of
220
covalent bond formed in oxidized samples regardless of TGase.
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Solubility provided supporting evidence for differential cross-linking in the GluOx oxidation
222
alone and the GluOx oxidation/TGase cross-linking pathway. The relative decrease of protein
223
solubility due to TGase treatment was also influenced by the oxidative status of MP. As shown
224
in Figure 1B, 88% of the protein was soluble in non-oxidized MP samples, and the solubility
225
decreased sharply to 65% (P < 0.05) following TGase treatment. While the solubility generally
226
declined with increasing GluOx concentrations, the additional changes due to TGase were much
227
less, i.e., 23.5% in non-oxidized MP and only 10% in MP oxidized with 16 µg GluOx/mg protein.
228
Rheological Behavior of MP–Lipid Emulsion Composite Gels. It is widely accepted that
229
in comminuted meat, hydrophobic regions of MP associate with fat globules, producing an
230
interfacial protein membrane that continuously interacts with the surrounding three-dimensional
231
protein network to form an interconnected cage-like gel structure during thermal processing.1
232
Such composite gel matrixes impart a viscoelastic physical barrier to hold both water and lipid in
233
a less mobile state. Wu et al.4 reported that S–S cross-linking between proteins at the oil/water
234
interface and those present in the continuous gel matrix contributed to the stabilization of
235
emulsion particles in meat systems. Since GluOx oxidation and TGase treatments can promote
236
the formation of disulfide bonds and alter the hydrophobicity of MP, the rheology of the
237
emulsion sols made from GluOx/TGase modified MP during thermal gelation was investigated.
238
During heating, all MP samples exhibited a major rheological transition in the 35–45 °C
239
temperature range (Figure 4A), which is a characteristic dynamic network-forming pattern of MP
240
attributable to the sequential unfolding of the myosin head (heavy meromyosin, HMM) followed
241
by its helical tail (light meromyosin, LMM).26,
27
The initial rise in storage modulus (G′,
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elasticity) of the MP–lipid emulsion sols was detectable at 38 °C in non-oxidized MP. This
243
temperature decreased to 36, 35, and 33 °C corresponding to 4, 8, and 16 µg GluOx per mg MP
244
treatments. The respective temperatures of the peak were also lowered from 45 to 43, 42, and
245
41 °C, demonstrating a significant GluOx-dose-dependent effect on the thermal elastic behavior
246
of emulsion gels. The facile gel formation of oxidized MP samples as indicated by the earlier
247
elastic rheological response can be explained, because pre-formed polymers in the 12-h
248
oxidation with GluOx (Figure 3) can readily aggregate into a gel network upon subsequent
249
heating.28,
250
enthalpy (determined by differential scanning calorimetry), Li et al.16 deduced an increased
251
hydrodynamic radius of myosin when exposed to oxidative stress. Such structural changes could
252
contribute to an earlier and stronger elastic response of MP during gelation, as seen in the present
253
study.
29
Based on the remarkable changes in myosin Ca2+- and K+-ATPase activity and
254
Further heating led to continuous increases of G′. At above 48 °C, the G′ of all oxidized
255
samples surpassed that of the control and reached a final value (at 75 °C) that was 1.8 to 2.9-fold
256
higher (Figure 4A; Table 1). A far more intense increase of G′ exhibited by GluOx-treated
257
samples in this temperature region (beginning at about 52 °C) when compared with at lower
258
temperatures (< 45 °C) is noteworthy. The aggregation of LMM is largely responsible for MP
259
gel network formation in this intermediate temperature zone, while that of HMM is the initiator
260
of gelation at 35–45 °C.27 Therefore, from the differential G′ development, it can be suggested
261
that •OH generated in the GluOx oxidation system likely had a stronger preference for the tail
262
portion of myosin in the later stage of intermolecular S–S linkage.30
263
When the MP emulsion composites were treated with TGase, samples modified with GluOx
264
exhibited a reduced gel onset temperature from the non-oxidized control (Figure 4B). Structural
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changes induced by •OH as well as accumulations of soluble polymers (which appeared to be
266
excellent target of TGase, Figure 3) were obviously the main causative factors. Promotion of the
267
elastic protein network by TGase was most pronounced for samples treated with 4 µg GluOx/mg
268
MP. This can be seen because the G′ gain in the transition temperature range (28.5−46 °C) from
269
the TGase treatment for the 4 µg GluOx sample was much greater than for the 8 µg GluOx-
270
treated sample when compared with the counterpart samples without TGase treatment (Figure
271
4B). The result suggests that moderately unfolded MP in the mild GluOx oxidation system
272
facilitated TGase-catalyzed cross-linking and aggregation. The decreased transition temperature
273
from 46 °C (non-oxidized MP) to 41 °C (GluOx/TGase-treated MP) further supported the
274
hypothesis that mild oxidation facilitated the TGase efficacy. However, with TGase treatment,
275
the sample oxidized by 16 µg GluOx per mg MP showed the lowest final G′ (75 °C), indicating
276
that extensive and macroscopic aggregations and insolubilization impeded gel formation. A
277
similar finding was reported earlier19 that excessive cross-linkages of MP before heating
278
produced large protein aggregates unsuitable for a fine gel network, hence, a low final G′.
279
Firmness of MP-Lipid Emulsion Composite Gels. The “set gels” (cooked and then chilled)
280
were subjected to extrusion, and the initial rupture force was recorded as gel firmness (Table 1).
281
GluOx oxidation increased gel firmness by as much as 2-fold, while with the combined
282
GluOx/TGase treatment, gel firmness rose to a 4.4-fold level when compared with the non-
283
oxidized sample without TGase. The effect of GluOx oxidative modification was more obvious
284
in samples without TGase (2-fold) than with TGase (up to 1.4-fold), agreeing with the
285
corresponding findings on gel elasticity (G′ at 75 °C) (Figure 4). Gel firmness enhancements due
286
to TGase (∆Due to TGase, noted in Table 1) in samples with GluOx oxidation were higher (up to
287
0.48 N) than that in non-oxidized samples (0.37 N).
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The emulsion gels with MP oxidized by GluOx had no significant difference in cooking loss
289
compared with the non-oxidized sample (Table 1). However, the GluOx/TGase treatment
290
aggravated cooking loss from merely 0.61% in non-oxidized control to 5.29% for MP samples
291
oxidized with 16 µg GluOx per mg MP (P < 0.05). The increased cooking loss may be attributed
292
to extensive aggregation of MP induced by the combined treatment of oxidation and subsequent
293
TGase cross-linking, which was consistent with the decreased final G′ as well as the rigidity of
294
the final gel. Further TGase cross-linking of oxidatively modified protein may result in
295
microstructural disruption and generate porous water channels during thermal gelation. A similar
296
phenomenon was observed by Jia et al.31 for a two-step TGase treatment where further TGase
297
cross-linking of pre-formed polymers was found to increase the cooking loss. The remarkable
298
reduction in surface hydrophobicity due to GluOx/TGase treatment (Figure 2) would lead to
299
weaker hydrophobic interactions which were critical to MP gel network,1,6 hence, less potential
300
to physically restrain bulk water.
301
Microstructure of Gels. Morphological examination of the MP–lipid emulsion gels showed
302
remarkable differences due to GluOx oxidation regardless of TGase treatment. Light microscopy
303
revealed a proteinaceous matrix structure in which lipid droplets were imbedded. The control gel
304
contained intact and round lipid particles stabilized with a visible (dark) coating and were
305
connected to the protein network (Figure 5). However, for gels made of oxidatively stressed MP,
306
some parts of the interfacial membrane appeared to be disrupted, and irregularly shaped oil
307
particles and some void spaces (holes) were visibly present. This was most noticeable in gels
308
with MP that was modified with 4 µg GluOx. Similar but more substantial structural
309
irregularities occurred with increasing the dosage of GluOx (8 and 16 µg GluOx per mg MP).
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The treatment with TGase did not change the distribution pattern of oil particles in the gels and
311
GluOx remained to be the dominant factor for the morphological changes.
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312
SEM images of emulsion gels were also obtained to visualize structural details of the protein
313
network and oil droplet distribution within the gel. For control gel, oil droplets resembling a
314
‘water balloon’ with diameters mostly within the 8–40 µm range were confined within the
315
protein matrix (Figure 5). Compared with the smooth surface of lipid droplets seen in control
316
samples, more rugged and uneven surfaces were noticed in the samples with GluOx oxidation.
317
Layers of protein strands separated from the bulk gel matrix and remaining on the lipid surface
318
as interfacial protein films would account for the surface irregularity. As myosin has a molecular
319
length of ~180 nm;32 micrometer-scale protein strands can result from GluOx oxidation/TGase
320
cross-linking, thereby providing physical barriers for the lipid globules. Some small oil droplets
321
(< 10 µm) were adhered to bigger ones through proteinaceous strands instead of lipid fusion in
322
samples with GluOx oxidation, which may explain the irregular lipid pocket formation seen
323
under light microscopy, as a composite of different sizes of lipid droplets bundled with MP
324
strands.
325
The structure of the composite gel formed by non-oxidized MP was influenced by TGase: oil
326
droplets with heterogeneous sizes were densely packed, differing from the control gel without
327
TGase treatment (Figure 5). On the other hand, smaller oil droplets anchoring into a ‘cement’
328
was the dominant structural feature of the gels made from MP oxidized with 4 µg GluOx and
329
treated with TGase. At higher GluOx concentrations, a certain degree of lipid fusion was
330
observed on the gel samples (arrow-pointed, Figure 5). This may be attributed to the formation
331
of intensely clumped aggregates within the proteinaceous matrix instead of moderate protein
332
strands in the interfacial protein films for lipid immobility. The spherical shape of the oil droplets
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333
also became less distinctive. Overall, smaller lipid droplets immobilized with a densely packed
334
matrix is a general pattern of the gels produced from the combined GluOx–TGase treatment,
335
distinguishing from the control. Evidently, the aggregation induced by GluOx oxidation was
336
primarily responsible for the protein strands formed on the surface of oil droplets, and their
337
presence as an interfacial constituent provided an additional physical barrier to maintain the
338
emulsion stability.
339
As reported previously, in meat emulsion, fat globules act as fillers to enforce the protein gel
340
(batter).1 Such physical effects are strongly reinforced when the membrane proteins interact
341
actively with the surrounding proteins (continuous phase) via disulfide linkages.4 A strong filler-
342
size-dependent Young’s modulus of particle-filled MP gels was described by Gravelle et al.13
343
Hence, compared to non-oxidized samples, the oxidation-induced thicker interfacial protein layer
344
and the shielding of larger fat particles (10–40 µm) by the smaller ones (< 10 µm) were the
345
reasons why oxidized MP composite gels were significantly firmer than the control gel.
346
In conclusion, the GluOx oxidation/TGase cross-linking coupling allowed a controllable
347
structural modification and aggregation of MP, enabling an elastic gel matrix and a firm gel
348
texture. Disulfide linkages formed from oxidation and isopeptide bonds produced by the TGase
349
reaction not only reinforced the structure of the proteinaceous interfacial membrane but also
350
promoted its interaction with the gel matrix, thereby producing a firm emulsion composite gel. A
351
plausible mechanism of GluOx oxidation followed by TGase cross-linking to promote the
352
gelation of MP (predominantly myosin) is therefore proposed (Figure 6) to summarize the main
353
findings. Overall, as an enzymatic method to structurally modify muscle proteins, the GluOx
354
oxidation/TGase pathway offers great potential for the enhancement of gelling properties of
355
muscle proteins and modification of the texture of communicated meat products.
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356 357
AUTHOR INFORMATION
358
Corresponding Author
359
*(Y.L.X.) Phone: (859) 257-3822. Fax: (859) 257-5318. Email:
360
[email protected].
361
ORCID
362
Youling L. Xiong: 0000-0002-2164-4783
363
Funding
364
This research was supported by the USDA National Institute of Food and Agriculture (Hatch
365
Project 1005724), Ajinomoto Co., Inc., Japan, and an Oversea Study Fellowship from the China
366
Scholarship Council (to X.W.). Approved for publication as journal article no. 17-07-061 by the
367
Director of the Kentucky Agricultural Experiment Station.
368
Notes
369
The authors declare no competing financial interest.
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REFERENCES (1) Gravelle, A. J.; Marangoni, A. G.; Barbut, S. Insight into the mechanism of myofibrillar protein gel stability: Influencing texture and microstructure using a model hydrophilic filler. Food Hydrocoll. 2016, 60, 415–424. (2) Lan, Y. H.; Novakofski, J.; McCusker, R. H.; Brewer, M. S.; Carr, T. R.; McKeith, F. K. Thermal gelation properties of protein fractions from pork and chicken breast muscles. J. Food Sci. 1995, 60, 742–747. (3) Dickinson, E. Emulsion gels: The structuring of soft solids with protein-stabilized oil droplets. Food Hydrocoll. 2012, 28, 224−241. (4) Wu, M.; Xiong, Y. L.; Chen, J. Role of disulphide linkages between protein-coated lipid droplets and the protein matrix in the rheological properties of porcine myofibrillar proteinpeanut oil emulsion composite gels. Meat Sci. 2011, 88, 384–390. (5) Liu, W.; Lanier, T. C. Rapid (microwave) heating rate effects on texture, fat/water holding, and microstructure of cooked comminuted meat batters. Food Res. Int. 2016, 81, 108–113. (6) Sun, X. D.; Arntfield, S. D. Molecular forces involved in heat-induced pea protein gelation: Effects of various reagents on the rheological properties of salt-extracted pea protein gels. Food Hydrocoll. 2012, 28, 325–332. (7) Liu, Z.; Xiong, Y. L.; Chen, J. Identification of restricting factors that inhibit swelling of oxidized myofibrils during brine irrigation. J. Agric. Food Chem. 2009, 57, 10999–1007. (8) Xiong, Y. L.; Blanchard, S. P.; Ooizumi, T.; Ma, Y. Hydroxyl radical and ferryl-generating systems promote gel network formation of myofibrillar protein. J. Food Sci. 2010, 75, C215−21.
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(9) Li, Y.; Li, X.; Wang, J.-z.; Zhang, C.-h.; Sun, H.-m.; Wang, C.-q.; Xie, X.-l. Effects of oxidation on water distribution and physicochemical properties of porcine myofibrillar protein gel. Food Biophys. 2014, 9, 169–178. (10) Zhou, F.; Zhao, M.; Su, G.; Cui, C.; Sun, W. Gelation of salted myofibrillar protein under malondialdehyde-induced oxidative stress. Food Hydrocoll. 2014, 40, 153‒162. (11) Chanarat, S.; Benjakul, S.; Xiong, Y. L. Physicochemical changes of myosin and gelling properties of washed tilapia mince as influenced by oxidative stress and microbial transglutaminase. J. Food Sci. Tech. 2015, 52, 3824–3836. (12) Wang, X.; Xiong, Y. L.; Sato, H.; Kumazawa, Y. Controlled cross-Linking with glucose oxidase for the enhancement of gelling potential of pork myofibrillar protein. J. Agric. Food Chem. 2016, 64, 9523–9531. (13) Gravelle, A. J.; Barbut, S.; Marangoni, A. G. Influence of particle size and interfacial interactions on the physical and mechanical properties of particle-filled myofibrillar protein gels. RSC. Adv. 2015, 5, 60723–60735. (14) Zhu, Y.; Rinzema, A.; Tramper, J.; Bol, J. Microbial transglutaminase-a review of its production and application in food processing. Appl. Microbiol. Biotechnol. 1995, 44, 277–282. (15) Gaspar, A. L.; de Goes-Favoni, S. P. Action of microbial transglutaminase (MTGase) in the modification of food proteins: a review. Food Chem. 2015, 171, 315–22. (16) Li, C.; Xiong, Y. L.; Chen, J. Oxidation-induced unfolding facilitates myosin cross-linking in myofibrillar protein by microbial transglutaminase. J. Agric. Food Chem. 2012, 60, 8020−8027.
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(17) Park, D.; Xiong, Y. L.; Alderton, A. L. Concentration effects of hydroxyl radical oxidizing systems on biochemical properties of porcine muscle myofibrillar protein. Food Chem. 2007, 101, 1239–1246. (18) Gornall, A. G.; Bardawill, C. J.; David, M. M. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 1949, 177, 751−766. (19) Liu, G.; Xiong, Y.; Butterfield, D. A. Chemical physical and gel forming properties of oxidized myofibrils and whey- and soy-protein isolates. J. Food Sci. 2000, 65, 811−818. (20) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. (21) Feng, J.; Xiong, Y. L.; Mikel, W. B. Textural properties of pork frankfurters containing thermally/enzymatically modified soy proteins. J. Food Sci. 2003, 68, 1220–1224. (22) Soladoye, O. P.; Juárez, M. L.; Aalhus, J. L.; Shand, P.; Estévez, M. Protein oxidation in processed meat: Mechanisms and potential implications on human health. Compr. Rev. Food Sci. Food Saf. 2015, 14, 106–122. (23) Hawe, A.; Sutter, M.; Jiskoot, W. Extrinsic fluorescent dyes as tools for protein characterization. Pharm. Res. 2008, 25, 1487–99. (24) Liu, C.; Xiong, Y. L. Oxidation-initiated myosin subfragment cross-linking and structural instability differences between white and red muscle fiber types. J. Food Sci. 2015, 80, C288– 297. (25) Li, C.; Xiong, Y. L.; Chen, J. Protein oxidation at different salt concentrations affects the cross-linking and gelation of pork myofibrillar protein catalyzed by microbial transglutaminase. J. Food Sci. 2013, 78, C823−31.
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(26) Samejima, K.; Ishioroshi, M.; Yasui, T. Relative roles of the head and tail portions of the molecule in heat‐induced gelation of myosin. J. Food Sci. 1981, 46, 1412‒1418. (27) Egelandsdal, B.; Fretheim, K.; Samejima, K. Dynamic rheological measurements on heatindunced myosin gels: Effect of ionic strength, protein concentration and addition of adenosine triphosphoate or pyrophosphate. J. Sci. Food Agric. 1986, 37, 915–926. (28) Ziegler, G. R.; Foegeding, E. A. The gelation of proteins. Adv. Food Nutr. Res. 1990, 34, 203‒298. (29) Xiong, Y. L., Structure-function relationships of muscle proteins. In Food Proteins and Their Applications, Damodaran, S., Ed. CRC Press: New York, 1997; pp 341–392. (30) Ooizumi, T.; Xiong, Y. L. Identification of cross-linking site(s) of myosin heavy chains in oxidatively stressed chicken myofibrils. J. Food Sci. 2006, 71, 196–199. (31) Jia, D.; Huang, Q.; Xiong, S. Chemical interactions and gel properties of black carp actomyosin affected by MTGase and their relationships. Food Chem. 2016, 196, 1180–1187. (32) Scholz, T.; Altmann, S. M.; Antognozzi, M.; Tischer, C.; Horber, J. K.; Brenner, B. Mechanical properties of single myosin molecules probed with the photonic force microscope. Biophys. J. 2005, 88, 360–71.
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Table of content
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Table 1. Gel firmness, rigidity and cooking loss of myofibrillar protein (MP)–lipid emulsion composite gels. Prior to gelation, MP was modified by oxidation (12 h) with GluOx followed by cross-linking with TGase (2 h) at 4 °C. Oxidation was carried out with 0– 16 µg GluOx and 50 µg glucose per mg MP in the presence of 10 µM Fe. GluOx (µg/mg MP) 0 4 8 16
Gel rigidity (G′ at 75 °C, Pa)
Gel firmness (N) Oxid only
Oxid + TGase E
0.17 ± 0.01 0.26 ± 0.04D 0.27 ± 0.05D 0.34 ± 0.12C
B
0.54 ± 0.05 0.68 ± 0.05A 0.74 ± 0.06A 0.75 ± 0.11A
∆Due to TGase 0.37 0.43 0.48 0.41
Oxid only Oxid + TGase ∆Due to TGase 396 709 929 1173
1163 1326 1392 1017
A–E
767 617 400 −150
Cooking loss (%) Oxid only
Oxid + TGase C
0.54 ± 0.20 0.55 ± 0.32C 0.55 ± 0.13C 0.75 ± 0.01C
0.61 ± 0.48C 2.79 ± 0.81B 3.74 ± 0.79AB 5.29 ± 3.62A
Means without a common letter differ significantly (P < 0.05). Relative changes of gel firmness and gel rigidity due to TGase treatment: ∆Due to TGase = (sample with oxidation and TGase) – (sample with oxidation only).
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Free sulfhydryl (nmol/mg protein)
70
(A)
Oxidation only Oxidation + TGase
A AB
65
AB
60
BC
AB
55
BC
BC
50
C
0
4
8
16
Glucose oxidase (µ µg/mg protein) 100
Solubility (%)
90
(B)
Oxidation only Oxidation + TGase
A
BC
80 BC
70
DE
60
CD CDE
DE E
50 0
4
8
16
Glucose oxidase (µ µg/mg protein)
Figure 1. Reduction of the sulfhydryl content (A) and solubility (B) of myofibrillar protein (MP) caused by oxidation (12 h) with GluOx followed by cross-linking with TGase (2 h) at 4 °C. Oxidation was carried out with 0–16 µg GluOx and 50 µg glucose per mg MP in the presence of 10 µM Fe. Means without a common letter differ significantly (P < 0.05).
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8
6 RFI (c.p.s., ×10 )
0 µg GluOx/mg MP
6
4 µg GluOx/mg MP 0 µg GluOx/mg MP
8 µg GluOx/mg MP 16 µg GluOx/mg MP
4/8 µg GluOx/mg MP
4 16 µg GluOx/mg MP
2 (A) Oxidation only
(B) Oxidation + TGase
0 400
420
440
460
480
500
400
420
Wavelength (nm)
440
460
480
500
Wavelength (nm)
Figure 2. Changes in the relative intensity (RFI) of ANS fluorescent emission spectra of myofibrillar protein (MP) caused by oxidation (12 h) with GluOx followed by cross-linking with TGase (2 h) at 4 °C. Oxidation was carried out with 0–16 µg GluOx and 50 µg glucose per mg MP in the presence of 10 µM Fe. A: Oxidation only; B: Oxidation + TGase; c.p.s., counts per second.
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Soluble polymers (SP) formed and myosin heavy chain (MHC) and actin lost relative (%) to non-oxidized samples without TGase Glucose oxidase (µg/mg MP) 0 4 8 16 D
MHC
BC
Oxid only
0.0
11.8
Oxid + TGase ∆ Due to TGase
21.7 21.7
Oxid only
0.0
CD
D
Actin Oxid + TGase
CD
6.2
ABC
∆ Due to TGase
9.3 9.3
Oxid only
0.0
Oxid + TGase ∆ Due to TGase
34.7 34.7
AB
EF
66.7 DE
BC
32.5 16.6 BC
11.7
21.2 15.1
F
SP
BC
26.5 14.7
AB
15.9
BC
55.8 −10.9
A
26.2 14.5
A
25.3 AB
32.4 7.1 AB
16.9 A
38.6 21.7
DE
78.8
CD
112.2
B
A
61.3 −17.5
93.8 −18.4
Figure 3. SDS–PAGE patterns of myofibrillar protein (MP) modified by oxidation (12 h) with GluOx followed by cross-linking with TGase (2 h) at 4 °C. Oxidation was carried out with 0–16 µg GluOx and 50 µg glucose per mg MP in the presence of 10 µM Fe. Relative changes of pixel intensity by TGase treatment: ∆ Due to TGase = (sample with oxidation and TGase) – (sample with oxidation only).
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1200
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16 µg GluOx/mg MP
(A) Oxidation only
1000
8 µg GluOx/mg MP
G' (Pa)
800 4 µg GluOx/mg MP
600 400
0 µg GluOx/mg MP
200
∆GluOx = 776.9 Pa
0 20
30
40
50
60
70
80
Temperature (°C)
G' (Pa)
1600
(B) Oxidation + TGase
1400
8 µg GluOx/mg MP 4 µg GluOx/mg MP
1200
0 µg GluOx/mg MP
1000
16 µg GluOx/mg MP
800
46 °C 41 °C
28.5 °C
600 400 200
∆GluOx=270.0 Pa
0 20
30
40
50
60
70
80
Temperature (°C)
Figure 4. Representative storage modulus (G′) development of myofibrillar protein (MP)–lipid emulsion composite sols during thermal gelation. Prior to gelation, MP was modified by oxidation (12 h) with GluOx followed by cross-linking with TGase (2 h) at 4 °C. Oxidation was carried out with 0–16 µg GluOx and 50 µg glucose per mg MP in the presence of 10 µM Fe. A: Oxidation only; B: Oxidation + TGase. Calculated differences of G′ at final 75 °C (∆GluOx): between highest G′ and the control for each treatment.
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Figure 5. Microstructure of myofibrillar protein (MP)–lipid emulsion composite gels. Prior to gelation, MP was modified by oxidation (12 h) with GluOx followed by cross-linking with TGase (2 h) at 4 °C. Oxidation was carried out with 0–16 µg GluOx and 50 µg glucose per mg MP in the presence of 10 µM Fe. LM: light microscopy with 20 µm scale bar; SEM: scanning electron microscope with 100 µm scale bar.
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Glucose
Myosin Fe3+
Fe2+
GluOx
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0.6 M NaCl
• OH
H2O2
Unfolded
S
S
S
TGase
More cross-linking
Control gel (Loose matrix)
S
S
S
S
: S−S : Gln-Lys
Enhanced cross-linking
GluOx gel (Dense matrix)
GluOx + TGase gel (Packed matrix)
Figure 6. Proposed mechanism of GluOx oxidation coupled with TGase cross-linking to promote the gelation of MP (predominantly myosin) in a protein–lipid emulsion composite system where the microstructure of the gels was suggested. Disulfide bonds (S–S) and Gln-Lys isopeptide bonds are marked.
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