Rheological Enhancement of Pork Myofibrillar ProteinâLipid Emulsion

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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

1 ACS Paragon Plus Environment


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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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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

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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

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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



38

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

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The size and surface properties (hydrophobicity) of filler particles in MP composite gels

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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

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property enhancements in restructured muscle foods.14 Protein polymers induced by TGase

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usually exhibit improved functionality, particularly gelation and emulsification,15 when

49

compared with proteins without TGase treatment. Because the efficacy of TGase is dependent

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upon the accessibility of glutamine and lysine residues, Li et al.16 have introduced oxidative

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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

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attributed to TGase was reportedly achieved.

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Because the H2O2 production from GluOx-catalyzed glucose oxidation was progressive (as

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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

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of further cross-linking by TGase. The objective of this study was to test this hypothesis and,

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specifically, investigate the rheological properties of MP gels after GluOx modification followed

59

by TGase treatment.

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MATERIALS AND METHODS

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Materials. Porcine Longissimus lumborum (48 h post-mortem) was obtained from the

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University of Kentucky Meat Laboratory, a USDA-approved facility. The loin muscle samples

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were vacuum packaged and stored in a –30 °C freezer for less than 6 months before use. MP was

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isolated from thawed muscle at 4 °C according to Park et al.17 using a buffer of 10 mM sodium

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phosphate, 0.1 M NaCl, 2 mM MgCl2, and 1 mM EGTA at pH 7.0. The pH of MP suspension in

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0.1 M NaCl at the final wash was adjusted to 6.25 with 0.1 M HCl before centrifugation. Protein

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concentration of the MP was measured by the Biuret method using bovine serum albumin as a

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standard.18 Commercial canola oil was purchased from a local grocery. GluOx (2000 U/g),

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TGase (1000 U/g) and organic iron (food-grade microbial iron) were donated by Ajinomoto Co.,

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Inc. (Kawasaki, Japan).

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GluOx Oxidation/Transglutaminase Modification of MP. Enzymatic •OH-producing

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reagents (GluOx/glucose/iron) at final concentrations of 4, 8 and 16 µg GluOx and 50 µg glucose

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per mg MP and 10 µM Fe were mixed into MP suspensions [20 or 40 mg/mL protein, in 15 mM

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1,4-piperazinediethanesulfonic acid (PIPES) buffer, pH 6.25, 0.6 M NaCl]. Iron (Fe) was

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included to produce •OH from H2O2 formed by the GluOx-catalyzed oxidation of glucose. The

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mixtures were incubated for 12 h at 4 °C to allow oxidative modification. To investigate TGase-

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induced protein cross-linking, control and oxidized MP samples were treated with TGase (10 µg

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per mg MP) in the same PIPES buffer (pH 6.25) with 0.6 M NaCl at 4 °C for 2 h.

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Measurement of Structure-Related Changes and Aggregation of MP. Samples of control

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and GluOx-oxidized MP (40 mg/mL) with or without TGase treatment were diluted to 2 or 4

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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

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diluted to 0.25 mg/mL with the same PIPES buffer solution.

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Free Sulfhydryls. Total free sulfhydryl content (4 mg/mL MP solution) was determined as

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described by Liu et al.19 A molar extinction coefficient of 13,600 M−1 cm−1 was used to calculate

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the sulfhydryl concentration.

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ANS Fluorescent Spectra. Fluorescence spectra were acquired immediately after addition of

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20 µL 8-anilinonaphthalene-1-sulfonic acid (ANS, 8.0 mM in 0.01 M phosphate buffer, pH 7.0)

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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

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recorded, using a FluoroMax-3 spectrofluorometer (Horiba Jobin Yvon Inc., Edison, NJ, USA).

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Protein Solubility. MP sample solutions (2 mg/mL) were centrifuged at 5000 g and 4 °C for

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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.

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Electrophoresis. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)

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was conducted on control and GluOx/TGase-modified MP samples according to the method of

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Laemmli20 with a 4% polyacrylamide stacking gel and a 12% polyacrylamide resolving gel. For

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all MP samples, 1 mM N-ethylmaleimide (NEM, a thiol-blocking agent) was added before

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heating to prevent possible formation of disulfide artifacts during sample preparation. Each

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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

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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

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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

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homogenizing 20 mg/mL MP suspension and canola oil (25%, w/w) with a Polytron PT 10-

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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

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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

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composites were continuously monitored during the heat-induced gelation with a Bohlin CVO

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100 rheometer (Malvern Instruments, Westborough, MA, USA). Sols were heated between

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parallel plates (upper plate, 30 mm diameter) from 20 to 75 °C at a 1 °C/min rate with a fixed

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frequency of 0.1 Hz and a controlled maximum strain of 0.02. To prevent dehydration, a thin

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layer of silicone oil was applied to the exposed edge of samples. Changes in the storage modulus

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(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

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transferred into glass vials (16.5 mm diameter), then heated from 20 to 75 °C at a rate of 1 °C

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/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

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are, respectively, the weight of the emulsion gel after heating and the weight of the original sol

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before heating: Cooking loss (%) =

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Wsol – Wgel Wsol

×100.

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Microstructure of MP–Lipid Emulsion Composite Gels. To observe the effect of GluOx

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oxidation/TGase induced MP modification on the gel structure, the microstructure of MP–lipid

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emulsion composite gels was examined.

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Light Microscopy. MP–lipid emulsion gel samples were prepared for light microscopy using

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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

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overnight. The samples were dehydrated by immersion in a series of ethanol (50, 70, 90, and

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100%), fixed in paraffin, and sectioned at a thickness of 8 µm using a microtome. After removal

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of paraffin with xylene, the slides were stained with Ehrlich's hematoxylin, then imaged using a

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MICROPHOT-FXA Nikon photomicroscope equipped with a built-in digital camera (Nikon Inc.,

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Garden City, NY, USA).

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Scanning Electron Microscopy (SEM). Samples for SEM examination were prepared as

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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%

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paraformaldehyde and 1% glutaraldehyde. Fixed samples were washed with 0.1 M phosphate

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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

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were sputter-coated with gold and examined under a FEI Quanta 250 (FEI Inc., Hillsboro, OR,

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USA) with 2 kV accelerating voltage.

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Statistical Analysis. Data obtained from two independent trials (n = 2), each employing a

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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

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Software, Tallahassee, FL, USA). Significant (P < 0.05) differences between means were

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identified by the least significance difference (LSD) all-pairwise multiple comparisons.

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RESULTS AND DISCUSSION

163 164

Protein Structural Changes. Thiol oxidation, with disulfide bonds being the most common

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end products, affects protein structure and protein–protein interactions.22 Reduction of the

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sulfhydryl content and solubility of MP caused by oxidation with GluOx followed by cross-

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linking with TGase is shown in Figure 1. The total free sulfhydryl (SH) content of non-oxidized

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MP without TGase was 64.9 nmol/mg protein. After GluOx oxidation, the SH content decreased

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significantly with increasing GluOx concentrations to 57.8 nmol/mg protein (P < 0.05),

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suggesting the formation of disulfide bonds (S–S). The results were consistent with our previous

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observation12 that high concentrations of GluOx in the presence of glucose and Fe substantially

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modified MP, promoted S–S bond and polymer formation, and decreased the solubility (Figure

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1B). The SH content was reduced after TGase treatment for 2 h, probably caused by reburying of

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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,

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solubility, aggregation tendency, as well as adsorption behavior at the oil/water interface, which

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affects its functionality.23 ANS, as an extrinsic fluorescent dye with an aromatic structure and a

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negatively charged sulfonate group, can bind to the hydrophobic pockets on protein through

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aromatic–aromatic interactions and to positively charged amino acids, e.g., histidine, lysine, and

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arginine, via electrostatic interactions.23 Therefore, an increased ANS fluorescence intensity is

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indicative of the exposure of hydrophobic groups of MP. The surface hydrophobicity of MP due

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to oxidation as shown by the ANS spectra was GluOx-dose-dependent, where the fluorescence

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intensity decreased with increasing GluOx concentrations (Figure 2A). A similar phenomenon

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was observed previously,24 which was attributed to intramolecular S–S bonds that stabilized the

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secondary structure of myosin.

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With subsequent TGase treatments (Oxidation + TGase), the surface hydrophobicity

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decreased further (Figure 2B). The steady drop in intensity of fluorescence suggests that GluOx

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oxidation and TGase cross-linking caused MP to aggregate, resulting in a partial burial of the

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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

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electrostatic interactions between the negatively charged sulfonate group in ANS and the

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positively charged amino group in lysine, leading to an additional fluorescence attenuation. A

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slight increase of surface hydrophobicity in oxidized MP was reported by Li et al.16, where mild

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oxidation predominantly induced protein unfolding over aggregation, which may initiate a

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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

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inset displays the relative content (%) of soluble polymers (SP) formed and the amount of

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myosin heavy chain (MHC) and actin lost over non-oxidized MP without TGase. In oxidized MP,

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the intensity of MHC and actin bands attenuated with increasing the GluOx concentration: up to

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25.3% reductions for MHC and 16.9% for actin, and a 112% increase in SP, compared to non-

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oxidized MP samples. To elucidate the effect of GluOx oxidation on TGase cross-linking, the

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relative changes (∆) in band intensity (pixel) attributed to TGase were calculated. The loss of

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actin due to TGase increased from 9.3% in non-oxidized to 14.5–21.7% in oxidized MP.

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However, the loss of MHC due to TGase decreased from 21.7% in non-oxidized to 7.2% in

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oxidized MP (treated with 16 µg/mg GluOx). Interestingly, the TGase treatment produced a 34.7%

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increase of SP in non-oxidized MP, contrasting to a net loss (up to 18.3%) in the SP content in

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GluOx-oxidized samples. This may be due to the further aggregation of SP in the latter samples

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forming exceedingly large polymers that were unable to enter the stacking gel.

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Under oxidative stress, protein unfolding and aggregation coexist, and the effectiveness of

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TGase will depend on the preponderance of these specific processes. When unfolding was

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caused in a chemically-produced •OH system, myosin cross-linking by TGase was favored.25 In

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the present study with GluOx, protein aggregation (evidenced by the substantial loss of MHC)

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rather than unfolding appeared to be dominant process. Enhanced intramolecular disulfide

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bridging and occlusions of accessible glutamine and lysine residues in MP under the relatively

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slow H2O2 production condition would render TGase less productive. When electrophoresed

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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

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solubility due to TGase treatment was also influenced by the oxidative status of MP. As shown

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in Figure 1B, 88% of the protein was soluble in non-oxidized MP samples, and the solubility

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decreased sharply to 65% (P < 0.05) following TGase treatment. While the solubility generally

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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.

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Rheological Behavior of MP–Lipid Emulsion Composite Gels. It is widely accepted that

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in comminuted meat, hydrophobic regions of MP associate with fat globules, producing an

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interfacial protein membrane that continuously interacts with the surrounding three-dimensional

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protein network to form an interconnected cage-like gel structure during thermal processing.1

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Such composite gel matrixes impart a viscoelastic physical barrier to hold both water and lipid in

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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

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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

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emulsion sols made from GluOx/TGase modified MP during thermal gelation was investigated.

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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

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attributable to the sequential unfolding of the myosin head (heavy meromyosin, HMM) followed

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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

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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

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41 °C, demonstrating a significant GluOx-dose-dependent effect on the thermal elastic behavior

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of emulsion gels. The facile gel formation of oxidized MP samples as indicated by the earlier

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elastic rheological response can be explained, because pre-formed polymers in the 12-h

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oxidation with GluOx (Figure 3) can readily aggregate into a gel network upon subsequent

249

heating.28,

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enthalpy (determined by differential scanning calorimetry), Li et al.16 deduced an increased

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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

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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

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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

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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

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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,

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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.

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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).



310

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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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


Page 17 of 30


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.



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.


Page 18 of 30

Page 19 of 30


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.

ACS Paragon Plus Environment


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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.


Page 23 of 30


Table of content



Page 24 of 30

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).


Page 25 of 30


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).



Page 26 of 30

8

6 RFI (c.p.s., ×10 )

0 µg GluOx/mg MP

6

4 µg GluOx/mg MP 0 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.


Page 27 of 30


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).



1200

Page 28 of 30

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


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.


Page 29 of 30


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.



Glucose

Myosin Fe3+

Fe2+

GluOx

Page 30 of 30

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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