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Controlled Cross-linking with Glucose Oxidase for the Enhancement of Gelling Potential of Pork Myofibrillar Protein Xu Wang, Youling L. Xiong, Hiroaki Sato, and Yoshiyuki Kumazawa J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03934 • Publication Date (Web): 30 Nov 2016 Downloaded from http://pubs.acs.org on December 2, 2016

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Journal of Agricultural and Food Chemistry

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Controlled Cross-linking with Glucose Oxidase for the Enhancement of Gelling Potential of Pork Myofibrillar Protein

Xu Wang, † Youling L. Xiong, *,† Hiroaki Sato, ‡ and Yoshiyuki Kumazawa, ‡

†

Department of Animal and Food Sciences, University of Kentucky, Lexington, Kentucky 40546,

United States

‡

Institute of Food Sciences and Technologies, Food Products Division, Ajinomoto CO., INC.,

Kawasaki 210-8681, Japan

(Submitted to: J. Agric. Food Chem.)

ACS Paragon Plus Environment


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ABSTRACT: Differential oxidative modifications of myofibrillar protein (MP) by hydroxyl

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radicals generated in an enzymatic system with glucose oxidase (GluOx) in the presence of

3

glucose/FeSO4 versus a Fenton system (H2O2/FeSO4) were investigated. Pork MP was modified

4

at 4 °C and pH 6.25 with hydroxyl radicals produced from 1 mg/mL glucose in the presence of

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80, 160, or 320 µg/mL GluOx and 10 µM FeSO4. Total sulfhydryl content, solubility, cross-

6

linking pattern, and gelation properties of MP were measured. The H2O2 production proceeded

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linearly with the concentration of GluOx and increased with reaction time. GluOx- and H2O2-

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dose dependent protein polymerization, evidenced by faded myosin heavy chain and actin in

9

SDS−PAGE as well as significant decreases in sulfhydryls, coincided with protein solubility loss.

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Firmer and more elastic MP gels were produced by GluOx than by the Fenton system at

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comparable H2O2 levels due to an altered radical reaction pathway.

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KEYWORDS: myofibrillar protein; glucose oxidase; oxidation; gelation; meat


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INTRODUCTION

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Gel-forming capacity of muscle protein plays an essential role in texture-forming properties of

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processed meats.1 Thermally-induced protein gelation in meat products can stabilize the colloidal

17

matrix of lipid, water, and salt with its interconnected cage-like unit structure.2 Myofibrillar

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protein (MP), predominantly myosin, is the main gelling component in processed muscle foods,

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therefore, has been extensively studied in model systems for its functional role in the rheology

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and texture of comminuted and restructured meat products.3

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In the process of gel formation, native protein molecules are unfolded upon heating, which

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leads to progressive aggregation to form a highly viscoelastic network of polymers capable of

23

withholding water, namely, a gel.4 The latter involves hydrophobic and electrostatic interactions,

24

hydrogen bonds, van der Waal's interactions, and covalent bonds, including disulfide.5 In a meat

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emulsion (batter), these physicochemical forces impart attraction between myosin in the

26

continuous phase and myosin comprising the fat globule interfacial membrane,6 contributing to

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the stabilization of lipid and immobilization of water in cooked comminuted meat.7

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Intermolecular disulfide cross-linkages via oxidation of protein thiol groups have been found to

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directly influence hardness of cooked meat.8 As reviewed by Lund et al.9, oxidative structural

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modification can enable characteristic protein networks, therefore, may provide an interesting

31

means to control the texture and rheological properties of muscle foods for optimal palatability

32

and stability.

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Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), superoxide anion (O2-),

34

and hydroxyl radical (•OH), have been investigated as oxidants to modify the structure of muscle

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proteins and their functionality.10-12 Furthermore, oxidized lipids are implicated in protein



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oxidation during meat processing, thereby affecting the textural properties of meat products.13

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Free radicals of lipid peroxidation and lipid oxidation products, for example, malondialdehyde

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(MDA), have been shown to promote MP gelation.14, 15 Phenolic compounds, either as natural

39

constituents of herbs and spices or as purified extracts, are also capable of modifying MP

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gelation through oxidative mechanisms. For example, chlorogenic acid has been reported to

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promote MP oxidation, mediating gel formation of oxidatively stressed MP.16, 17 On the other

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hand, in packaged meat, high-oxygen modified atmospheres promote protein disulfide cross-

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linking and increase toughness of pork, beef, and lamb.8, 18-20

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Exposures of MP to •OH generated from H2O2/Fe2+ induce remarkable structural changes in

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a H2O2-dose-dependent manner, and mild oxidation by •OH has been shown to promote the gel

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network formation of MP.12 The Fenton oxidative modification also can significantly enhance

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hydration of muscle although this does not necessarily improve water holding.21 In addition,

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structural unfolding induced by oxidation facilitates myosin cross-linking by transglutaminase.22

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Moreover, the extent of protein modification required to elicit significant MP functionality

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improvement can occur at very low oxidant concentrations, for example, < 1 mM H2O2,22 or

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before the onset of lipid oxidation.23 For these reasons, a controllable method to achieve

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deliberate mild oxidation could be beneficial to meat textural modification and potentially useful

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in industrial applications.

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Glucose oxidase (GluOx; EC number 1.1.3.4), a commercial food industry enzyme, catalyzes

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the specific oxidation of glucose into gluconic acid while producing H2O2.24 It can be used as a

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substitute of H2O2 for different food applications, for example, in baking to introduce

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intermolecular disulfide cross-linking for an improved dough performance.25 We hypothesize

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that by slowly generating H2O2 which is then converted to •OH, GluOx may allow controllable


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oxidative modification of MP in such a way that is conducive to gelation (Figure 1). Hence, the

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objective of this study was to investigate differential oxidative modifications of MP by •OH

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generated with the GluOx method compared to a Fenton system (H2O2/FeSO4). The

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consequential cross-linking and gelling properties of MP were assessed to investigate the

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efficacy of GluOx as an alternative gelation promoter.

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MATERIALS AND METHODS

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Materials. Porcine Longissimus lumborum (48 h post-mortem) muscle was collected at 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, stored in a –30 °C freezer, and used within 6 months. No measurable

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lipid and protein oxidation due to storage was noted in the samples in our preliminary

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evaluations. Glucose oxidase was donated by Ajinomoto Co., Inc. (Kawasaki, Japan). MP was

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isolated from muscle thawed at 4 °C by washing with a buffer of 10 mM sodium phosphate, 0.1

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M NaCl, 2 mM MgCl2, and 1 mM EGTA at pH 7.0.26 The MP pellet was washed 2 more times

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with 0.1 M NaCl, and the protein concentration was measured by the Biuret method using bovine

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serum albumin as a standard.27

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H2O2 Generation Via GluOx Pathway. The amount of hydrogen peroxide, produced from

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glucose by the catalysis of GluOx, was measured on the principle of iodine oxidation in the

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presence of ammonium molybdate.28 Briefly, mixtures of 1 mg/mL glucose and 0, 5, 10, 20, 40,

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80, 160, or 320 µg/mL GluOx (all final concentrations) were prepared in pre-chilled (4 °C) 15

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mM piperazine–N,N–bis(2–ethanesulfonic acid) (PIPES) buffer with 0.6 M NaCl at pH 6.25.

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The mixtures were incubated at 4 °C for 2, 12, and 24 h. At the end of each incubation time, 100

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µL of reaction solution was sampled and mixed into 2 mL of 50 mM HCl, followed by the



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addition of 0.2 mL of 1 M KI then 0.2 mL of 1 mM ammonium molybdate in 0.5 M H2SO4.

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After setting for 20 min, 0.2 mL of 50 mg/mL starch solution was added, and the absorbance at

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570 nm was measured after 20 min. A hydrogen peroxide standard curve (0–30 mg/mL H2O2)

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was established.

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Oxidative Modification of MP. Enzymatic •OH-producing reagents (GluOx/glucose/FeSO4)

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comprised of final concentrations of 80, 160, and 320 µg/mL GluOx, 1 mg/mL glucose, and 10

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µM FeSO4 were added to a 20 mg/mL MP suspension in 15 mM PIPES buffer (pH 6.25) with 0.6

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M NaCl at 4 °C. For direct chemical oxidation, •OH-producing reagents with 0.25, 0.5, 1, and 2

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mM H2O2 and 10 µM FeSO4 (all final concentrations) were added to the same 20 mg/mL MP

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suspension as above. For both oxidation systems, MP samples were oxidized up to 24 h, and the

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oxidation was terminated by the addition of propyl gallate/Trolox/EDTA (1 mM each). As a

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separate control, the same 20 mg/mL MP suspension without oxidation was incubated with 0–2

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mM gluconic acid for up to 24 h. All control and treated MP samples were kept in an ice slurry

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before analysis within 2 h.

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Measurement of Oxidative Changes in MP. Control and oxidatively modified MP samples

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were diluted to 2 or 4 mg/mL with 25 mM PIPES (pH 6.25) containing 0.6 M NaCl and then

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subjected to thiol content and solubility measurement. In addition, SDS–PAGE was performed to

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determine the type and extent of covalent cross-linking induced by oxidants.

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Total Sulfhydryls. Total free sulfhydryl content (in a 4 mg/mL MP solution) was measured

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using the 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) reagent as detailed by Liu et al.29 A molar

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extinction coefficient of 13600 M–1cm–1 was used for sulfhydryl calculation.


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Solubility. MP sample solutions (2 mg/mL) were centrifuged at 5000g for 15 min at 4 °C.

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Protein solubility was defined as the protein concentration of the supernatant divided by that of

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the original myofibril suspension.

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Electrophoresis. SDS–PAGE was run according to Laemmli30 with a 4% polyacrylamide

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stacking gel and a 12% polyacrylamide separating gel to observe original myofibrillar

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constituents and cross-linked protein polymers. MP samples (4 mg/mL protein) were mixed with

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an equal volume of SDS–PAGE sample buffer with or without 10% β–mercaptoethanol (βME)

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and then boiled for 3 min. To each sample well, 50 µg protein was loaded. After running, the

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gels were stained for 1 h with 0.1% Coomassie brilliant blue R250 in 50% methanol and 6.8%

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acetic acid, and subsequently destained with 5% methanol and 7.5% acetic acid. Pixel intensity

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of myosin heavy chain (MHC) and actin was measured with the UN-SCAN-IT software (Silk

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Scientific, Orem, UT). Relative reduction of MHC and actin bands in samples after treatment

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with Fenton-H2O2 or GluOx-H2O2 was calculated using the following formula: Pixel intensity in MP control – Pixel intensity in sample

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Relative reduction (%) =

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Evaluation of Gelation Properties. Oxidized and non-oxidized MP sols containing 20

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mg/mL protein in 15 mM PIPES buffer (pH 6.25) with 0.6 M NaCl were deaerated by

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centrifugation at 2000g for 2 min then set overnight at 4 ºC to allow a protein solubility

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equilibrium before heating to initiate gelation.31 A dynamic non-disruptive rheological

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measurement with a small-strain oscillation mode was performed. Moreover, a large-strain

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extrusion test was conducted to analyze the fracture properties of the MP gels.

Pixel intensity in MP control

×100.

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Dynamic Rheology Measurement. MP sols (20 mg/mL protein) were subjected to oscillatory

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shear analysis with a Model CVO rheometer (Malvern Instruments, Westborough, MA) to

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investigate gel structure formation during the sol-to-gel transformation upon heating. An



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appropriate amount of protein sol was loaded between 2 parallel plates (upper plate: 30 mm

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diameter) and heated from 20 to 75 °C at a 1 °C/min heating rate. The shear force of heated

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samples was measured using a fixed frequency of 0.1 Hz and a controlled maximum strain of

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0.02. In order to prevent dehydration, a thin layer of silicone oil was applied to the exposed edge

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of the gelling samples. Changes in the storage modulus (G′, an elastic force) and loss modulus

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(G", a viscous force) were recorded.

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Extrusion for Gel Strength Testing. Aliquots of 5 g of MP sols (20 mg/mL protein) were

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carefully transferred into glass vials (16 mm diameter) then heated from 20 °C to a final

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temperature of 75 °C at a rate of 1 °C/min in a water bath to form gels. After heating, gels in the

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vials were immediately chilled in an ice slurry for 12 h. Afterwards, the vials were allowed to

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equilibrate at room temperature (22 °C) for at least 1 h. A flat-surface aluminum probe (14.8 mm

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diameter) was used to slowly (20 mm/min) penetrate into the gel. The initial force required to

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disrupt the gel was designated as gel strength.

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Statistical Analysis. Experiments were conducted with two independent trials (n = 2, except

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for gel strength, n = 3) each with a new batch of isolated MP. Triplicate samples were analyzed.

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Data were subjected to the analysis of variance (ANOVA) using Statistix software 9.0

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(Analytical Software, Tallahassee, FL) in a general linear model’s procedure. Significant

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differences (P < 0.05) between means were identified by LSD all pair-wise multiple comparisons.

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RESULTS AND DISCUSSION

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Enzymatic H2O2 Production. The catalytic production of H2O2 from glucose by GluOx is a

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time-dependent process. As shown in Figure 2, formation of H2O2 generally increased with the


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reaction time, exhibiting a generally linear trend with the addition of GluOx at above 50 µg/mL

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for reactions times of 12 and 24 h. However, with 2 h of reaction, the amount of H2O2 produced

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by 80–320 µg/mL GluOx from 1 mg/mL glucose leveled off at approximately 0.2 mM. As

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expected, the concentration of H2O2 increased with the reaction time, i.e., 24 h > 12 h > 2 h.

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Based on these H2O2 production results and the stoichiometry of substrate-to-product conversion

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(Figure 1), a chemical oxidation system with the direct application of 0–2 mM H2O2 and a

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control system with 0–2 mM gluconic acid were designed as equivalent chemical systems for

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effect comparison with enzymatically produced GluOx-H2O2 in subsequent MP modification

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experiments.

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Oxidation-induced Changes in Thiol Groups and Protein Solubility. Of the amino acid

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residue side chain groups in muscle foods, sulfhydryls (SH) from cysteine residues are the most

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sensitive group to oxidative modification with disulfide (S–S) bonds being the most common end

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product.32 In the chemical oxidation solutions with 0–2 mM H2O2/FeSO4 (Fenton-H2O2

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treatment), total free SH content in MP deceased progressively (P < 0.05) from 51 to 46

163

nmol/mg protein (Figure 3A). This oxidation system has been shown to cause significant MP

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tertiary structural changes with simultaneous formation of carbonyl and disulfide derivatives.26

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In a similar but more pronounced fashion, the GluOx oxidizing system (with glucose/FeSO4)

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that generated comparable amounts of H2O2 to the Fenton system reduced total free SH in MP

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from 51 to 37 nmol/mg protein within 24 h (P < 0.05). As expected, the addition of gluconic acid,

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one of the end products of GluOx catalysis, to non-oxidant MP solution confirmed to have no

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lowering effect on SH content (P > 0.05). Therefore, the more pronounced SH decrease by

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GluOx was caused by a more sustainable supply of •OH. Liu et al.33 have reported that in the

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Fenton reaction, the concentration of H2O2 added in a single dose gradually attenuates with



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reaction time. Thus, the sudden exposure to the initial high concentration of H2O2 makes MP

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vulnerable to attack by •OH and HO2•. Therefore, continuous feeding of smaller quantities of

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H2O2 generated in the GluOx oxidizing system would ensure a sustained •OH supply thereby

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enabling a steady oxidative SH modification. It is noteworthy that high concentrations of H2O2

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produced in enzyme aqueous systems could oxidatively damage GluOx leading to decreased

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enzyme activity and oxidation efficacy.34 Krishnaswamy and Kittrell35 have estimated that the

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inactivation constant of GluOx due to the presence of oxygen (which converts reduced GluOx to

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the less active form, oxidized GluOx) increases from 0.024 to 0.039 h-1 when 1.0 mM H2O2 is

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added to the solution. It is noted that in the present study, the H2O2 production corresponding to

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the GluOx dosage (Figure 2) was obtained without the presence of MP. Because proteins will

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consume both H2O2 and iron-converted •OH, in the MP samples, myosin and other myofibrillar

183

components would conceivably protect the enzyme, thereby allowing more sustainable

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enzymatic H2O2 production than the system without MP. Therefore, the 80–320 µg/mL GluOx-

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H2O2 oxidizing systems in Figure 3 would probably produce more than 0.7–1.0 mM H2O2 and

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oxidize MP more effectively.

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Between two myosin heavy chains (MHC), both intermolecular and intramolecular S–S

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bonds can be formed since cysteine residues are transversely positioned in the tail of MHC, and

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several others located in the global head are quite accessible.36 The formation of intermolecular

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S–S bonds has received much attention due to their intimate involvements in protein polymer

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and entanglement network formation and in the textural characteristics of processed muscle

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foods.37, 38 Extremely large protein polymers and aggregates formed via excessive S–S linkages

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and intramolecular reactions resulting from quick and strong Fenton-H2O2 would bury reactive

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thiol groups or prevent •OH attack due to steric hindrance.


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Covalent interactions through disulfide bond formation have been shown to lead to protein

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aggregation affecting protein structural stability and solubility.39 No significant difference in

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solubility was detected between control and gluconic acid treatment (P > 0.05), while a solubility

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decrease in GluOx-H2O2 treatment was more noticeable (P < 0.05) than that in Fenton-H2O2

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treatment (Figure 3B). The solubility loss appeared to be well correlated with the formation of

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disulfides by both Fenton and GluOx, deduced from the decreased SH content that was displayed

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in Figure 3A.

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Electrophoretic Evidence of Differential Cross-linking. Protein polymerization upon

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exposure to Fenton or GluOx •OH-generating oxidants was evaluated by SDS–PAGE (Figure 4).

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MP samples treated with >0.5 mM H2O2 and 80–320 µg/mL GluOx without β–mercaptoethanol

205

(βME) contained considerable amounts of cross-linked polymers that occupied the top of the

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stacking gel. These oxidation-induced aggregates were quantitatively related to H2O2 and GluOx

207

concentrations. Simultaneously, the gradual disappearance of MHC and actin became

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accentuated as compared to control MP (Figure 4A and 4C). The relative reduction of MHC in

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samples without βME rose from 11.5% to 27.9% with increasing the concentration of H2O2 in

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Fenton, and from 11.3% to 32.9% with increasing the concentration of GluOx that produced

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comparable amounts of H2O2 as in Fenton. Meanwhile, the loss of actin increased from 12.0% to

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24% in Fenton-H2O2 treatments and from 17.1% to 32.1% in the GluOx-H2O2 counterpart

213

treatments. MHC and actin in the samples were mostly recovered by reaction with the disulfide-

214

breaking agent βME with which the relative loss of MHC and actin decreased to 3.4–10.2% and

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3.7–13.1% in these chemical and enzymatic •OH-oxidizing systems, respectively (Figure 4B and

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4D). The dominant role of S–S in the oxidant-induced polymerization of MP as evidenced by the



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SDS–PAGE confirms that disulfide was the primary product of SH oxidation shown in Figure

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3A.

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Except for S–S bonds, the carbonylation of MP in oxidation could contribute to cross–links

220

via Schiff base formation; Dityrosine was also involved in the cross-linking of oxidized

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proteins.40, 41 The relative reduction of MHC and actin in samples treated with βME (Figure 4B

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and 4D) provided evidence of other covalent bonds than S–S linkages. After subtracting the

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effect from the “other covalent bonds” in the samples with βME, the contribution of S–S in the

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relative reduction of MHC and actin can be calculated, which was expressed as ∆MHC and

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∆actin. The much reduced ∆MHC and ∆actin values with βME quantitatively indicate S–S cross-

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linking was a predominant force in the polymers generated from oxidation (Figure 4A and 4C).

227

The intensity of the newly formed polymers on the top of stacking gel with 0.7–1.0 mM H2O2

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produced by the oxidative enzyme (80–320 µg/mL GluOx, Figure 4C) appeared to be similar to

229

that induced by 2 mM H2O2 in the direct Fenton system (Figure 4A). This finding was

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compatible with the relative reduction of MHC, namely, no significant difference in effect was

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found between 80–320 µg/mL GluOx and 2 mM H2O2 treatments.

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Thermorheological Behavior of Oxidatively Modified MP. The association of

233

polypeptides through covalent linkages influences the hydrodynamic characteristics of the

234

protein system, which can have a profound effect on the functional properties of muscle food.

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The rheological profiles of MP oxidatively stressed under Fenton chemical H2O2 and GluOx-

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H2O2 are compared for elastic behavior (G') (Figure 5A vs. Figure 5C) and viscous

237

characteristics (G") (Figure 6A vs. Figure 6C). The plots are from representative samples of 24 h

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oxidation that were subjected to thermal sol-to-gel transformation. Because gluconic acid is an


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end product of glucose oxidation, its potential influence on MP gelation was also tested, and the

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results are displayed in Figure 5B (G') and Figure 6B (G").

241

During heating from 20 to 75 °C, all MP samples showed two major G' peaks (around 45 and

242

55 °C) providing evidence of dynamic formation of an elastic gel network involving different

243

polypeptides (Figure 5). The G" curve rapidly ascends at approximately 38 °C (Figure 6)

244

producing a single transition with a maximum (peak) temperature of 1–2 °C less than that of G'

245

(first peak) of the corresponding samples. Because G" is a measure of hydrodynamic bulkiness

246

of a polymer, the increased value signifies protein denaturation (unfolding). The exposure of

247

hydrophobic groups promotes the interaction of myosin resulting in an enhanced elasticity of the

248

protein semi-gel and gel system.42 The peak temperature for G" was reduced from 44.8 °C

249

(control) to 42.8 °C with 320 µg/mL GluOx (or 1.08 mM H2O2 produced) (Figure 6C), but there

250

was no G" transition shift due to Fenton-H2O2 treatment (Figure 6A). Covalent bonds that

251

formed during GluOx-H2O2 modification with a slower reaction rate might cause gradual

252

exposures of hydrophobic patches to form non-covalent aggregation and earlier denaturation

253

during thermal treatment.

254

The MP samples treated with GluOx, which produced 0.72–1.08 mM H2O2, displayed higher

255

magnitude G' and G", both at the transitions and end of heating (75 °C), than those treated with

256

Fenton-H2O2 at the same concentration range. Egelandsdal et al.43 attributed the first G' peak to

257

the aggregation of heavy meromyosin, and the second peak to the formation of permanent,

258

irreversible myosin filaments or complex that involves light meromyosin and all other MP

259

components. Yongsawatdigul and Park44 also suggested that as the temperature continuously

260

increased to 46 °C, entanglements of unfolded actomyosin occurred to facilitate gel network

261

formation, resulting in an overall higher G' value. With an increasing level of GluOx treatment,



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both rheological attributes became more pronounced. Furthermore, the higher the dosage of

263

GluOx, the earlier the unfolding and network development began (shown by the downward shift

264

of the initial G" and G' peaks from about 44.8 to 42.7 °C, P < 0.05), and a more viscoelastically

265

distinctive gel at the end heating temperature.

266

In contrast, although the G' value of all Fenton-H2O2-treated MP samples rose significantly

267

(P < 0.05) when compared with nonoxidized control MP (Figure 5A), the effect attenuated rather

268

quickly with increasing the concentration of H2O2 from 0.25 to 2 mM. The results confirmed a

269

previous observation that mild oxidation was promotive of protein gelation, whereas strong

270

oxidation hampered the ability of MP to form an ordered gel network.45 The discrepant response

271

of MP to •OH generated from the enzyme pathway (GluOx-H2O2) compared to the chemical

272

(Fenton-H2O2) system may be explained, as described earlier, because H2O2 was gradually

273

produced with GluOx, while in Fenton, H2O2 was mixed into MP samples in a bust. The slow

274

release of H2O2 in the GluOx system would allow a more gentle oxidative modification of MP

275

than in the Fenton chemical reaction that is considerably much faster. It has long been proposed

276

that the initial step of the MP gelation process, i.e., structural unfolding, must be sufficiently

277

slow to allow exposed reactive groups from different protein molecules to orient and align with

278

each other if a well-structured gel matrix is to be built.46 This seems to apply to the GluOx

279

treatment in the present study.

280

Intermolecular disulfide bonds during heat treatment are considered to be vital for textural

281

development that occurs in comminuted meat during cooking.47 Nieto, Jongberg, Andersen and

282

Skibsted32 reported that free sulfhydryls may only decrease in quantity to a certain level in

283

oxidation and not all free cysteine residues in MP possessed similar reactivity. They suggested

284

that some of the free SH groups were occluded inside the core of the protein, hence, protected


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from oxidation. Moreover, reburying of cysteine residues within the MP aggregates, which

286

occurred in the strong Fenton-H2O2 oxidation system, would further prevent reactive SH from

287

participating in cross-linking and gel formation. An extensive blockage of thiol groups during the

288

final stage of gelation is believed to be a primary factor for reduced stress-at-fracture of protein

289

gels.48 This is because SH/S–S interchanges are important for protein structural rearrangements

290

on the submicron level, without which it may be difficult if not impossible to form a filamentous

291

gel network conducive to a high viscoelasticity.49

292

Interestingly, gluconic acid at 1 and 2 mM levels promoted MP gelation (Figure 5B). Its

293

effect can be assigned to the polar polyols (hydroxyls) and the anionic carboxyl group; both can

294

mediate protein–protein interactions. Notwithstanding, neither gluconic acid nor Fenton-H2O2

295

was able to produce as much gel-reinforcement effect as GluOx-H2O2. For example, the

296

maximum gain (∆) in G' at 75 °C for gluconic acid (2 mM) and Fenton-H2O2 (0.25 mM) was,

297

respectively, 51.1 Pa (Figure 5B) and 42.3 Pa (Figure 5A), whereas GluOx-H2O2 (1.08 mM)

298

oxidative modification achieved the greater improvement for G' at 75 °C (∆GluOx = 106.0 Pa)

299

(Figure 5C). As aforementioned, some ordering of protein filaments is beneficial for the

300

formation of a strong gel.50 The new disulfide bonds formation in sols by the slower GluOx-

301

H2O2 oxidation appeared to induce sufficient unfolding and proper structural alignments for an

302

initial ordered aggregate matrix, and this would allow redistributions of intermolecular forces to

303

contribute to the viscoelastic enhancement of the MP gel.

304

Gel Strength. Newly formed disulfide bonds, which tether the aggregates that lower the

305

solubility, could potentially facilitate structural formation of entangled protein matrix for

306

gelation.48 To test the structural properties of formed MP gels, ‘set gels’ (cooked then chilled)

307

were subjected to extrusion and the initial rupture force recorded. As shown in Figure 7A, gels



Page 16 of 31

16

308

prepared from oxidized MP by Fenton-H2O2 exhibited no detectable difference in structural

309

strength (P > 0.05). However, upon GluOx-H2O2 oxidative treatments, MP gel strength

310

increased substantially with oxidation time from 12 to 24 h and enzyme dosage from 80 to 320

311

µg/mL when more H2O2 was produced (see data points corresponding to rising H2O2

312

concentration, 0.5–1.1 mM) (Figure 7B). The maximum increase was 2.3 fold of the control MP

313

sample. There was no obvious change for 2 h oxidation (P > 0.05). A nominal gain (0.05 N) in

314

gel strength with the maximum level of gluconic acid addition was noted, but the effect was

315

negligible. In the GluOx-H2O2 system, the concentrations of H2O2 produced over 0–24 h

316

corresponding to the specific enzyme levels were plotted to distribute the enzymatically

317

produced H2O2 concentration effect. The results indicate a good agreement with the rheological

318

properties of the same MP samples with GluOx-H2O2 treatments (Figure 6C) showing an overall

319

benefit of an enzymatic approach to enhance the gelling potential of MP. Even though it is not

320

entirely clear what specific forces were involved, S–S bridges ostensibly played a major role in

321

improving the gelation properties in the GluOx-H2O2 system. Enhancing the gelation properties

322

of MP through controllable oxidative modification can therefore be achieved through

323

manipulation of the reaction time and GluOx application level.

324

In conclusion, the advantage of progressive oxidation via the GluOx-mediated H2O2 pathway

325

over Fenton-H2O2 oxidation is demonstrated and explainable by the mechanism of slow and

326

controllable reactions. Such oxidative modification has potential application in muscle food

327

processing where deliberate protein structural changes conducive to optimal functionality are

328

desirable. The GluOx/glucose/Fe oxidizing system can induce appropriate structural changes,

329

increase the coordination of protein–protein associates and aggregates, therefore, facilitate the

330

formation of ordered protein matrices for elastic gel networks. It remains unclear whether


Page 17 of 31


17

331

disulfide bonds produced in different oxidizing systems are formed in the junction zone in the

332

gel network, or whether they simply contribute to the increase in effective chain length in the MP

333

sols that subsequently further aggregate to reinforce the gel structure during thermal treatment.

334

Further research is warranted to explore the roles of disulfide bonds in the variation of size and

335

shape of MP aggregates from deliberate mild oxidation by GluOx-H2O2 for enhanced gelation

336

properties.

337 338

AUTHOR INFORMATION

339

Corresponding Author

340

*(Y.L.X) Phone: (859) 257-3822. Fax: (859) 257-5318. E-mail: [email protected].

341

Funding

342

This research was supported by the USDA National Institute of Food and Agriculture (Hatch

343

project 1005724), Ajinomoto Co., Inc., Japan, and an Oversea Study Fellowship from the China

344

Scholarship Council (to X.W.). Approved for publication as journal article number 16-07-040 by

345

the Director of the Kentucky Agricultural Experiment Station.

346

Notes

347

The authors declare no competing financial interest.

348 349

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Figure 1. Schematic representation for the proposed mechanism of glucose oxidase-induced cross-linking and gelation of myofibrillar protein. LH: lipid molecule; P–SH: protein sulfhydryl; P–S–S–P: cross-linked proteins with disulfide bond.


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25

1.2 2h 12 h 24 h

H2O2 (mM) produced

1.0 0.8 0.6 0.4 0.2 0.0

0

50

100

150

200

250

300

350

Glucose oxidase (µg/mL)

Figure 2. Production of hydrogen peroxide (H2O2) from 1 mg/mL glucose in the presence of different concentrations of glucose oxidase as a function of reaction time (2, 12, and 24 h).



Page 26 of 31

26

50

a

a

a

ab

(A)

800

ab

720 ab

b

c

640

c

45

d

e

40 35

a

Gluconic acid Fenton-H2O2 GluOx-H2O2

560

LSD0.05 = 0.50

f

480

g

400 320

30

240 25 160 H2O2 produced by GluOx

20 15

80 0

0.0

0.5

1.0

1.5

2.0

Glucose Glucose oxidase (µg/mL) oxidase (µg/mL)

Free sulfhydryl (nmol/mg protein)

55

2.5

H2O2/Gluconic acid (mM)

40

A

A

Solubility (%)

(B)

800 720

B C CD

35 D

25

Gluconic acid Fenton-H2O2 GluOx-H2O2

DE

640

LSD0.05 = 1.03

DE

30

20

A

A

A

560

D EF

480

F

400 320

15

240

10

160 H2O2 produced by GluOx

5 0

80 0

0.0

0.5

1.0

1.5

2.0

Glucose Glucose oxidase (µg/mL) oxidase (µg/mL)

45

2.5

H2O2/Gluconic acid (mM)

Figure 3. Changes in the sulfhydryl content (A) and protein solubility (B) of myofibrillar protein samples after treatment with Fenton-H2O2 (0–2 mM H2O2/10 µM FeSO4), enzymatically generated GluOx-H2O2 (0–320 µg/mL glucose oxidase/1 mg/mL glucose/10 µM FeSO4), or 0–2 mM gluconic acid. Means without a common letter (a–g; A–F) differ significantly (P < 0.05).


Page 27 of 31


27

Figure 4. SDS–PAGE patterns of myofibrillar protein after treatments with Fenton-H2O2 (0–2 mM H2O2/10 µM FeSO4, A and B) and enzymatically generated GluOx-H2O2 (0–320 µg/mL glucose oxidase/1 mg/mL glucose/10 µM FeSO4, C and D). Protein samples were prepared in presence (+βME) or absence (–βME) of 10% β–mercaptoethanol. Lane MW = molecular weight (kDa) marker; MHC: myosin heavy chain. ∆= Relative reduction of pixel intensity (sample without βME – sample with βME). Means without a common letter (a–g; A–K) differ significantly (P < 0.05).



Page 28 of 31

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140

(A) Fenton-H2O2 Control 0 mM 0.25 mM 0.5 mM 1 mM 2 mM

120

G' (Pa)

100 80

0.25 mM H2O2 0.5 mM H2O2 1 mM H2O2 2 mM H2O2 Control 0 mM H2O2

60 40

∆ H O = 42.3 Pa 2 2 20 0 20

30

40

50

60

70

80

Temperature (°C) 140

(B) Gluconic acid

120 100

G' (Pa)

2 mM GA 1 mM GA

0 mM 0.25 mM 0.5 mM 1 mM 2 mM

0.5 mM GA 0 mM GA

80

0.25 mM GA

60 40

∆ GA = 51.1 Pa 20 0 20

30

40

50

60

70

80

Temperature (°C) 200 180

Control Control 00 µg/mL µg/mL 80 80 µg/mL µg/mL 160 160 µg/mL µg/mL 320 320 µg/mL µg/mL

160 140

G' (Pa) (Pa) G'

320 µg/mL GluOx (1.08 mM H2O2)

(C) GluOx-H2O2

120

160 µg/mL GluOx (0.87 mM H2O2) 80 µg/mL GluOx (0.72 mM H2O2) 0 µg/mL GluOx (0 mM H2O2)

100 100

Control

80 80 60 60 40 40

∆∆GluOx ==106.0 Pa GluOx 106.0 Pa

20 20 0 0 20 20

30 30

40 40

50 50

60 60

70 70

80 80

Temperature Temperature (°C) (°C)

Figure 5. Representative storage modulus (G') development of myofibrillar protein during thermal gelation. Prior to gelation, protein samples were treated 24 h with Fenton-H2O2 (A: 0–2 mM H2O2/10 µM FeSO4), 0–2 mM gluconic acid (B), or enzymatically generated GluOx-H2O2 (C: 0–320 µg/mL glucose oxidase/1 mg/mL glucose/10 µM FeSO4). Calculated differences (∆): between highest G' and the control for each treatment; GA: gluconic acid.


Page 29 of 31


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15

Control 0 mM 0.25 mM 0.5 mM 1 mM 2 mM

12

G" (Pa)

T max shift

(A) Fenton-H2O 2

H2O2 (mM) Control 0 0.25 0.5 1 2

9

∆ (°C) 0 0 0 0 0 0

6

3

20

30

40

50

60

70

80


(B) Gluconic acid

12

G" (Pa)

T max shift

0 mM 0.25 mM 0.5 mM 1 mM 2 mM

GA (mM) 0 0.25 0.5 1 2

9

∆ (°C) 0 0 0 0 -1

6

3

20

30

40

50

60

70

80


(C) GluOx-H2O2

12

G" (Pa)

T max shift

Control 0 µg/mL 80 µg/mL 160 µg/mL 320 µg/mL

14

GluOx (µg/mL) Control 0 80 160 320

10

∆ (°C) 0 0 -1 -1 -2

8 6 4 2 20

30

40

50

60

70

80

Temperature (°C)

Figure 6. Loss modulus (G") development of myofibrillar protein during thermal gelation. Prior to gelation, protein samples were treated with Fenton-H2O2 (A: 0–2 mM H2O2/10 µM FeSO4), 0–2 mM gluconic acid (B), or enzymatically generated GluOx-H2O2 (C: 0–320 µg/mL glucose oxidase/1 mg/mL glucose/10 µM FeSO4). Tmax (temperature of maximum G" peak) shift ∆ = Tmax of treatment – Tmax of control; GA: gluconic acid.



Page 30 of 31

30

0.6

(A) 2h 12 h 24 h

Gel strength (N)

0.5

∆H2O2 = 0.04

0.4

P > 0.05

0.3

0.2

0.1

0.0 MP

0

0.25

0.5

1

2

H2O2(mM) Fenton 0.5

(B)

a ab b

Gel strength (N)

0.4

GluOx-H2O2

c

c

c

0.3

0.2

d d

d

d

d

d

Gluconic acid ∆ GA = 0.05 N ∆GluOx = 0.26 N ∆ net = 0.21 N

0.1

0.0 0.0

0.2

0.4

0.6

0.8

1.0

1.2

H2O2 (mM) produced by GluOx, Gluconic acid (mM)

Figure 7. Gel strength of myofibrillar protein subjected to treatments with Fenton-H2O2 (A: 0–2 mM H2O2/10 µM FeSO4), enzymatically generated GluOx-H2O2 (B: 0–320 µg/mL glucose oxidase/1 mg/mL glucose/10 µM FeSO4, 0–24 h) in a composite plot where the effect of 0–2 mM gluconic acid is also displayed. Means without a common letter (a–d) differ significantly (P < 0.05). Calculated differences (∆): between highest gel strength and the control.


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