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Controlled formation of emulsion gels stabilized by salted myofibrilalr protein under malondialdehyde (MDA)-induced oxidative stress Feibai Zhou, Weizheng Sun, and Mouming Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf505916f • Publication Date (Web): 09 Mar 2015 Downloaded from http://pubs.acs.org on March 29, 2015

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Journal of Agricultural and Food Chemistry

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Controlled Formation of emulsion gels stabilized by salted Myofibrilalr protein under

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malondialdehyde (MDA)-induced oxidative stress

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Feibai Zhou,† Weizheng Sun,† and Mouming Zhao*,†,‡

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† College of Light Industry and Food Science, South China University of Technology, Guangzhou

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510640, China

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*Corresponding author:

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Mouming Zhao, Professor

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Tel/Fax: +86 20 87113914

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E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: This study presented the cold-set gelation of emulsions stabilized by salted

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myofibrillar protein (MP) under oxidative stress originated from malondialdehyde (MDA). Gel

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properties were compared in range of MDA/NaCl concentrations by of gel viscoelastic properties,

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strength, water-holding capacity (WHC), amount of protein entrapped, and microstructure. The

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oxidative stability of emulsion gels as indicated by lipid hydroperoxide was further determined and

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compared. Results indicated that emulsion stabilized by MP at swollen state under certain ionic

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strength (0.2 – 0.6 M) was the premise of gel formation under MDA. In the presence of intermediate

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MDA concentrations (2.5 – 10 mM), the emulsion gels showed an improved elasticity, strength,

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WHC, and oxidative stability. This improvement should be mainly attributed to the enhanced

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protein-protein cross-linkings via MDA, which were homogeneously formed among absorbed and/or

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unabsorbed proteins, entrapping more amount and fractions of protein within network. Therefore, the

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oil droplets were better adherent to gel matrix. Nevertheless, addition of high MDA concentrations

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(25 – 50 mM) led to the formation of excessive covalent bonds, which might break protein-protein

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bonds and trigger the desorption of protein from interface. This ultimately caused “oil leak”

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phenomena as well as the collapse of gel structure, and thus overall decreased gel properties and

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oxidative stability.

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KEYWORDS: Emulsion gel, Myofibrillar protein, Malondialdehyde, Protein oxidation, Cold-set

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INTRODUCTIO

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Many food products could be categorized as protein-based emulsion gels, such as cheese, yoghurt,

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sauces, mayonnaise and processed meats.1 Specifically, in the manufacture of comminuted muscle

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foods, fats are chopped into small globules and further stabilized largely by meat proteins.2 Therefore,

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in many cases, processed meats products are emulsion gels in nature.2-4 To date, however, due to the

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relatively complex structure of meat protein, investigations of protein-stabilized emulsion gels have

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been performed mainly on milk proteins, whey proteins and soy bean proteins,5-11 whereas limited

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work has been done on that of meat proteins.12,13 Considering the increasing demand for meat and

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meat products due to its high biological importance, a better understanding of the structuring of

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related emulsion gel is of significance not only for texture modulation, but also to improve the

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sensory properties of meat products.

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Myofibrillar proteins (MP), contributing to 55 ~ 65% of total muscle protein, are known as

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excellent gelling agents largely responsible for the textural and structural characteristics of meat

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products,14 essentially including those in forms of emulsion gels. Recent research on porcine muscle

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protein emulsion gels has indicated that long-term stability and structural reinforcement are enhanced

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by heat-induced formation of disulfide bridges between the adsorbed MP proteins and the MP in the

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continuous phase of gel matrix.13 Reports have also confirmed that changes in protein secondary

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structure could lead to changes in the protein absorbed and therefore the interactions with oil phase

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or the neighboring adsorbed proteins.11 These previous works demonstrated that the structure and

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physico-chemical properties of proteins could influence the interactions of absorbed protein with

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proteins in the aqueous phase, thus playing a critical role in controlling the formation and stability of

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emulsions gels and therefore the texture of meat products.2.15

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With the increasing understanding that food proteins are sources and targets for reactive oxygen

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species (ROS), numerous studies have now dealt with the occurrence of protein oxidation in muscle

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foods and have tried to shed light on the influence of protein structure and physico-chemical



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properties.16,17 Results indicated that oxidation could alter the secondary and tertiary structure of MP,

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leading to the unfolding of protein structure under mild conditions, which further increased the

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accessibility to effectively initiate protein-protein interactions, and thus enhanced the MP gelling

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and/or emulsifying properties.18-21 Accordingly, these oxidative modifications could definitely

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influence the properties of MP-stabilized emulsion gels. Moreover, to the best of our knowledge, no

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report about the influence of structural changes caused by oxidation other than heating on the

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properties of MP-stabilized emulsion gel has been found to date.12,13

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Considering the coexistence of protein and lipid in meat products, protein oxidation would be

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inevitable in meat industry due to the close interaction between protein and oxidizable substrates (i.e.,

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unsaturated lipids).22-24 More recently, we reported that malondialdehyde (MDA), the most abundant

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individual aldehyde resulting from lipid peroxidation, could lead to cold-set gelation of MP in the

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presence of certain ionic strength. 25 MDA is naturally generated under meat processing conditions

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and could oxidatively modify side chains and polypeptide backbone of protein, resulting in its

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conformational changes.26 Meanwhile, MDA can react with amino groups of proteins, producing

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strong intermolecular cross-links of the Schiff base type.27 In our previous study,25 the novel cold-set

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MP gel could be formed on the premise of the swelling of MP under 0.6 M NaCl and the

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non-disulfide covalent bond induced by MDA. The gelation mechanism of salted MP mainly

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involving simultaneous protein oxidation and internal cross-linking via MDA was also proposed.25

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On the basis of our previous findings, we thus postulated that incubation with MDA in MP-based

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emulsion systems may result in the formation of a new type of cold-set emulsion gel stabilized by

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MP. This speculation would provide foundational information for further exploring the potential

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impact of MDA in real meat emulsion during manufacturing and storage process.

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The objective of this study was to test the hypothesis that MDA affect the gelation of MP-stablized

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emulsion under different ionic strength conditions. The consequential changes in emulsion gel in

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terms of the amount/fraction of protein entrapped/non-entrapped as well as mechanical properties


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(gel elasticity, strength, water-holding capacity), microstructure and oxidative stability were all

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investigated to elucidate the structure-modifying effect of MDA.

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MATERIALS AND METHODS

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Materials. Longissimus muscle from three pork carcasses (48 h postmortem) was purchased from

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a local commercial abattoir (Zhongshan, China) and the pigs were slaughtered about 6 months of age

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following standard industrial procedures. Fat was trimmed away and muscle was cut into cubes,

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minced, vacuum packaged (ca. 100 g) and frozen at -80 °C until use (used within 3 days).

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Commercially available Jinlongyu soybean oil (pure) produced by Yihai Kerry Food Company

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(Shanghai, China) was purchased from a local supermarket. MDA solution was prepared by acid

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treatment of 1,1,3,3-tetramethoxypropane obtained from Sigma-Aldrich (Chemical Co. St Louis, MO,

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USA). Nile Blue A and Nile Red were obtained from Sigma Aldrich (Sigma Chemical Co., St. Louis,

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MO, USA). All other chemicals were of analytical reagent grade.

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Extraction of Myofibrillar Proteins (MP). Three batches of MP were prepared from different

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bags of raw muscle thawed at 4 °C according to the method of Park, Xiong, Alderton, and Ooizumi24

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with slight modifications. The pH of MP suspension at 0.1 M NaCl in the last wash was adjusted to

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6.25 before centrifugation. The pellet was finally suspended in 20 mM sodium phosphate buffer (pH

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6.0) containing NaCl at different levels (0, 0.2, 0.4 and 0.6 M), and the protein concentration was

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determined by the Biuret method using BSA as standard.

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Emulsion Preparation. O/W emulsions was prepared by dispersing 20 wt % soybean oil in 20

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mM phosphate buffer (pH 6.0) containing 3% (w/v) MP at different NaCl levels (0, 0.2, 0.4 and 0.6

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M). Coarse emulsions were prepared using a high-speed blender operating at 11000 rpm for 3 min

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(Ultra-Turrax, IKA-Labortechnik, Staufen, Germany). The coarse emulsions were further

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homogenized using a 2-stage single-piston homogenizer (APV-1000, Albertslund, Denmark) at 30

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MPa. Sodium azide (0.02 wt %) was then added to the emulsions to prevent microbial growth.

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Emulsions were placed in lightly sealed screw-cap vials or 2-ml centrifuge tubes and stored in a dark



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oven at 25 ° C for 30 days.

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Evaluation of Emulsions. Particle Size Distribution. The mean particle size and size distribution

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of emulsions were measured by a Mastersizer 2000 (Malvern Instruments Co. Ltd., Worcestershire,

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UK) at 25 °C. The refractive indices of soybean oil and phosphate buffer were taken as 1.456 and

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1.330, respectively. The absorption index was 0.001. The particle sizes measured are reported as the

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volume-weighted mean diameter d4,3 (d4,3= ∑ nidi4/∑ nidi3), where ni is the number of particles with

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diameter di.

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Rheological Ccharacterization. The rheological properties of the emulsions stabilized by MP at

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different ionic levels were characterized using a HAAKE MARS III Rheometer (Thermo Electron

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GmbH, Karlsruhe, Germany) with a parallel plate (diameter 35 mm), at 25 °C. The gap between two

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plates was set to 1.0 mm. For determination of steady shear viscosities, shear rate was ramped from 1

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to 200 s-1. Shear stress, shear rate, and steady shear (apparent) viscosity (η) were recorded by

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RheoWin 4 Data Manager. Dynamic viscoelastic properties were characterized using small

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amplitude oscillatory frequency sweep mode. The frequency was oscillated from 0.1 to 100 rad/s and

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all measurements were performed within the identified linear viscoelastic region and made at 0.5%

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strain. The elastic modulus (G′), loss modulus (G′′), and loss tangent (tan δ) were recorded.

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Preparation of MDA-modified MP Emulsion Gels. MDA stock solution was freshly prepared

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by hydrolyzing 1,1,3,3- tetramethoxypropane according to the method described by Wu et al.28 with

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minor modifications.25 The concentration of MDA was determined by spectrophotometric

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measurements of the dilution 10-5 at 267 nm (ε= 31500).

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MP emulsion in the presence of 0.6 M NaCl was mixed with MDA at different concentrations (0,

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0.5, 2.5, 5, 10, 25, 50 mM, final). The resulting mixtures were immediately transferred into tightly

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sealed glass vials (2.5 cm inner dia 5 cm height) and 2-ml centrifuge tubes and incubated at 25 °C in

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the dark for 24 h. After incubation, a series of treated samples with different states were obtained. To

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further investigate the influence of ionic strength, MP emulsions at different NaCl levels (0, 0.2, 0.4


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and 0.6 M) were mixed with 10 mM MDA. The incubation condition used was the same as

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mentioned above. MP emulsion in the presence of 0.6 M NaCl treated without MDA was selected as

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control.

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Evaluation of Gel Characteristics. Low-amplitude Dynamic Oscillatory Measurements. Small

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deformation rheological measurements using parallel plates (diameter 35 mm, 1 mm gap) were

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investigated in a HAAKE MARS III Rheometer (Thermo Electron GmbH, Karlsruhe, Germany).

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Frequency sweep analysis between 0.1 and 100 rad/s (at 25 °C and 0.5% strain) was applied to

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investigate the viscoelastic properties.

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Gel Strength. The gel strength of emulsion gels was measured using a cylinder measuring probe (P

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0.5SS) attached to a TA.TX2 texture analyzer (Stable Micro System, Surrey, UK) at a constant probe

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speed of 1.0 mm/min at room temperature (25 ± 1°C). Gel strength is defined as the initial force

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required to disrupt the gels. All samples were prepared in triplicate.

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Water Holding Capacity (WHC). WHC of the emulsion gels were determined according to the

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method of Wu et al.,12 with slight modifications. Briefly, gels (3 g) were centrifuged at 8000 × g for

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30 min at 4 °C. WHC (%) was expressed as the final weight as a percentage of the weight before

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centrifugation.

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Amount of Protein Entrapped. The amount of proteins entrapped was evaluated according to that

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of Chanyongvorakul et al.29 with modifications. After incubation, the emulsion gel in 2-ml centrifuge

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tubes were centrifuged (10,000 × g, 30 min, 25 °C) and used to determine the amount of proteins

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entrapped. The protein concentration of separated aqueous layer, filtered through a 0.45 µm filter

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(Millipore Corp.), was determined by the Bradford assay, using BSA as standard. The percent of

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entrapped proteins was calculated as (1-protein concentration in separated aqueous layer/initial

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protein concentration in the aqueous phase) × 100

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SDS-PAGE Analysis. SDS–PAGE was performed on entrapped and non-entrapped proteins of the

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emulsion gels according to the method described Flores, et al.,30 under either non-reducing or



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reducing conditions. Both aqueous and creamed layer after centrifuging (10,000 × g, 30 min, 4 °C)

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were collected and mixed with certain volume of sample buffer (with and without 5%

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β-mercaptoethanol (β-ME)) to obtain a theoretical concentration of 1 mg mL−1 protein. Samples were

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vortexed, incubated at room temperature overnight. The ones with β-ME were boiled for 5 min

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before centrifugation (10,000 × g, 10 min). Then, 24 µL of samples were loaded onto the

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polyacrylamide gel made of 12% running gel and 5% stacking gel and subjected to electrophoresis at

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a constant current of 25 mA per gel using a Mini-PROTEAN 3 Cell apparatus (Bio-Rad Laboratories,

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Hercules, CA, USA).

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Confocal Laser Scanning Microscopy (CLSM). The microstructure of the MDA-modified

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emulsion gels was determined in a confocal laser scanning microscope (Zeiss, LSM 710, Germany),

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using Nile red and Nile blue A as fluorescence dyes for the protein and oil phases, respectively.8

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Each freshly prepared MDA-treated emulsion sample (1 mL) was stained with about 40 µL of

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fluorescence dye mixtures of Nile blue A (0.1%, w/v) and Nile red (0.1%, w/v) in 1, 2 -propanediol.

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The emulsion gels containing the dyes were directly formed in concave confocal micro-scope slides

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(Sail; Sailing Medical-Lab Industries Co. Ltd., Suzhou, China), covered with glycerol-coated

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coverslips (further sealed with tin foil), according to the same conditions mentioned above. Finally,

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the CLSM images were examined with a 100 magnification lens and an argon/ krypton laser having

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an excitation line of 514 nm and a Helium Neon laser (HeNe) with excitation at 633 nm for Nile blue

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A and 488 nm foe Nile red, respectively. Factually, most of the protein-coated oil droplets would be

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predominantly yellow in appearance of the overlay image (green + red = yellow)

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Lipid Hydroperoxide Measurement. Lipid hydroperoxide was measured according to the

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method described by Tong et al.31 with some modifications. Emulsions/emulsion gels were diluted in

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4 volumes (w/v) of distilled water before homogenization (11000 rpm, 1min). Aliquots (0.2 mL) of

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the diluted were mixed with 1.5 mL of isooctane/2-propanol (3:1, v/v) and vortexed (10 s, three

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times). After centrifugation at 3400 g for 2 min, the organic solvent phase (200 µL) was added to 2.8


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mL of methanol/1-butanol (2:1, v/v), followed by 15µL of 3.94 M ammonium thiocyanate and 15 µ L

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of ferrous iron solution (prepared by mixing 0.132 M BaCl2 and 0.144 M FeSO4). 20 mins later, the

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absorbance was measured at 510 nm using a UV − vis spectrophotometer (Genesys 10, Thermo

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Scientific, USA). The distilled water was used instead of diluted emulsion as a reagent blank.

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Hydroperoxide concentrations were determined using a standard curve made from hydrogen

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peroxide.

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Statistical Analysis. Three batches of MP were prepared on different days, each used for

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independent trials (i.e., replicate preparation of MP, emulsion and emulsion gel), and all the

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experiments were repeated with three different batches of MP. Statistical calculation was investigated

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using the statistical package SPSS 11.5 (SPSS Inc., Chicago, IL) for one-way ANOVA.

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Student-Newman-Keuls test was used for comparison of mean values among determinations using a

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level of significance of 5%. Data were expressed as means ± standard deviations (SD) of triplicate

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determinations unless specifically mentioned.

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RESULTS AND DISCUSSION

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Emulsion Characteristics. Particle Size Distribution and Physical Stability. The particle size

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distributions and changes in the mean droplet diameter (d4,3) for MP-stabilized emulsions at different

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NaCl levels were used to assess emulsifying ability and physical stability, as shown in Figure 1. The

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fresh emulsion stabilized by MP without addition of NaCl almost had a bimodal distribution with

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relatively small d4,3 (Figures 1A and B). The increase of NaCl concentration increased d4,3 and

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caused a gradual shift of the size distribution peak to the right, indicating a decreased emulsifying

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capacity.20 It has been reported that MP began to expand transversely when exposed to about 0.4 to

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0.5 M NaCl, and swell profusely at about 0.6 M NaCl due to enhanced electrostatic repulsion by

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Cl-binding to actomyosin.25,32 Thus, the swollen MP in the presence of NaCl on one hand decreased

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the protein adsorption rate on the droplets surface due to increased steric hindrance, on the other

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hand, it increased protein-protein interactions,18,25 resulting in the formation of a thicker layer



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surrounding the droplet (Figure 1A, inset), and thus the larger d4,3 (Figure 1).

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To evaluate the physical stability of emulsions, the changes in the d4,3 of emulsions were followed

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during storage (Figure 1B). As shown in Figure 1B, for the emulsion without NaCl, a marked

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increase in d4,3 was observed with increasing storage time, reaching 20.16 µm at day 30, almost twice

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larger than that at day 0. Meanwhile, the emulsion exhibited an obvious phase separation (data not

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shown) after 30 days. These results indicated that MP-stabilized emulsion without NaCl was less

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stable after 30 days due to the coalescence of oil droplets, which were consistent with our previous

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report.20 Compared to the emulsion without NaCl, the stability of emulsions in the presence of NaCl

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was obviously improved (Figure 1B). Specifically, when 0.6 M NaCl were added, the d4,3 showed

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only a slight increase during the storage (Figure 1B) and no visible phase separation was found (data

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not shown), suggesting the enhanced stability of emulsions. As has been previously reported, more

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soluble MP with swollen appearance were available at high NaCl concentrations.18,32 Therefore, the

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more hydrated absorbed protein layer with higher thickness around the droplets could be formed

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(Figure 1A, inset) due to enhanced protein-protein interactions, thus slowing down the coalescence

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of oil droplets and improving the emulsion stability (Figure 1B).1,10

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Rheological Characterization. The rheological properties, including flow and dynamic

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viscoelastic properties of emulsions stabilized by MP at different NaCl levels were characterized, as

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shown in Figure 2A and 2B, respectively. All the emulsions exhibited shear-thinning behaviors at the

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tested shear rates in the range of 0.1 to 100 s-1, with apparent viscosity (η) progressively decreasing

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upon increasing shear rate (Figure 2A). This phenomena is supposed to be associated with the

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flocculation of fat droplets, as has been well addressed in previous studies.10,33 Noticeably, at any test

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shear rate, η progressively increased with increasing NaCl from 0 to 0.6 M (Figure 2A). As has been

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aforementioned, the swelling of MP with increasing NaCl concentrations could enhance

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protein-protein interactions,32 opposing the free flow of the emulsion in a shear field, thus increasing

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its apparent viscosity (Figure 2A). The increased system viscosity in turn retarded the migration of


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oil droplets, and thus enhanced the emulsion stability (Figure 1B).

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As to emulsion viscoelastic properties, in all cases, the storage modulus (G′) was higher than the

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loss modulus (G′′) in the test linear viscoelastic range (Figure 2B), suggesting the potential to form

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elastic gel or gel-like structure. The G′ progressively increased with increasing oscillatory frequency

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(Figure 2B), as has also been observed in some other gel-like emulsions.10,33 Furthermore, as

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expected, at any oscillatory frequency, the G′ of emulsions stabilized by MP continuously and

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significantly increased with increasing NaCl concentration from 0 to 0.6 M (Figure 2B), indicating

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gradual increase in stiffness of emulsions. These results completely reproduced the general trends

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observed in the data from emulsion stability and steady viscosity (Figures 1B and 2A), further

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suggesting enhanced inter-droplet interactions in the presence of 0.6 M NaCl, which contributed to

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an improvement in the emulsion stability.

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Evaluation of MDA-modified MP Emulsion Gels. Visual Observation. Figure 3 shows the

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images of MDA-modified MP emulsion either at various MDA concentrations (Figure 3A) or at

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different NaCl levels (Figure 3B). After incubation, vials containing samples were kept upside down

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for 10 min and pictured. As shown in Figure 3A, in the presence of 0.6 M NaCl, with the increasing

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addition of MDA, a transition from sol to gel was observed, and emulsion gel samples were obtained

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when MDA concentration was above 2.5 mM. Nevertheless, an “oil leak” phenomenon was also

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observed when samples were treated with MDA above 25 mM (Figure 3A, bottom). These results

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indicated that MDA played a critical role in emulsion gel formation. Additionally, necessity of NaCl

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on emulsion gel formation was further confirmed, as shown in Figure 3B. After being upside down

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for 10 min, the emulsion gel sample with 0.2 M NaCl collapsed (Figure 3B), indicating a weak gel

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structure. Thus, these results indicated that both MDA and NaCl played an important role in

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controlling gel formation and quality.

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Low-Amplitude Dynamic Oscillatory Measurements. For better understanding the influence of

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MDA and NaCl on viscoelastic properties of emulsion gels, variations of G′ and tan δ with frequency



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were recorded, using small strain measurement, as shown in Figure 4. For MDA-modified MP

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emulsion with 0.6 M NaCl, as can be seen from Figure 4A, G’ in magnitude in the test linear

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viscoelastic range firstly continuously increased with increasing MDA concentration from 0 to 10

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mM. At certain frequency (1 rad/s), G’ of the sample treated with 10 mM MDA reached 1986 Pa,

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representing an evident increase in G′ approximately 6.0 times compared with that of control, which

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indicated the elastic property of the sample was markedly enhanced and was well corresponding to

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the formation of gel structure as shown in Figure 3A. This might be due to the development of

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protein-protein crosslinks under MDA-induced oxidative stress,25,27 resulting in an entanglement

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network between adsorbed and non-adsorbed proteins.10 Nevertheless, further addition of MDA (25 –

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50 mM) sharply declined the G’, suggesting a weakened gel structure, which might be related to the

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oil leakage (Figure 3A).13 As has been extensively literatured,6,34 incorporated oil could functionalize

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as active fillers, leading to a gel network with an enhanced rigidity, and thus the reduced oil

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incorporated would decrease the gel elasticity (25 – 50 mM MDA). In addition, the loss factor, tan δ,

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indicative of whether elastic or viscous properties were predominant, was further measured, as

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shown in Figure 4C. In all cases, it can be clearly seen that, tan δ was almost independent of the

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frequency throughout the tested range and the tan δ values were lower than 1 (Figure 4C), indicating

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all the samples exhibited a predominant elastic behavior with frequency independency.10

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Furthermore, with increasing MDA concentrations from 0 to 10 mM, an evident decrease in the tan δ

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values was observed, suggesting enhanced elastic properties. However, when MDA concentration

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was above 10 mM, the tan δ values increased. These results were in good agreement with previous

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analysis (Figures 3A and 4A).

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As to the influence of NaCl, changes in G’ and tan δ with frequency were also recorded, as

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presented in Figures 4B and 4D, respectively. With increasing NaCl concentrations from 0 to 0.6 M,

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a dramatic increase in G′ (Figure 4B) and overall decrease in tan δ values (Figure 4D) were observed,

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respectively, indicating improved elastic properties of the emulsion gel. These results were again in


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line with visual observation (Figure 3B). As mentioned above, emulsions with thicker interfacial

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layer were obtained in the presence of 0.6 M NaCl (Figure 1A, inset) due to profusely swelling of

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MP.25,32 The thicker protein layer surrounding the oil droplets would increase the inter-droplet

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interactions via several bridges, including ionic interaction and the stronger covalent bonds upon the

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presence of MDA,27,35 which thus could promote the formation of emulsion gels with high elasticity

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(Figure 3B and 4). Nevertheless, in the absence of NaCl, protein fails to swell and disperses

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unevenly due to its low solubility.25,32 Thus, the lack of effective molecular interaction between

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absorbed/non-absorbed MP and MDA caused protein coated oil droplets clusters, rather than a

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network,25 thus resulting in the failure of gel formation and the low elasticity (Figures 3B and 4).

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Gel Strength and WHC. To further evaluate the properties of formed emulsion gels, the gel

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strength and WHC were further measured and compared, as shown in the A and B of Figure 5. In the

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presence of 0.6 M NaCl, the formed emulsion gel could be obtained when MDA concentration

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reached 2.5 mM (see Figure 3A). As can be seen from Figure 5A, with increasing MDA

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concentrations, emulsion gel strength remarkably increased, reaching a maximum of 1.09 N at 10

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mM. This should be mainly attributed to the enhanced protein-protein interactions due to the

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swelling of MP and thus the increased inter- and intra-molecular interaction within protein absorbed

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or unabsorbed under MDA-induced oxidative stress.10,25,27,32 These analyses were in good agreement

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with the G′ and tan δ data of emulsion gels (Figures 4A and 4C), which was related to the gel

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strength.19 Nevertheless, with further addition of MDA (25 – 50 mM), a drastic decrease in gel

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strength was observed (Figure 5A), implying a collapse of gel structure. This might be mainly due to

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the occurrence of separated oil phase (Figure 3A, bottom) and was again in good agreement with gel

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elasticity (Figures 4A and 4C). In addition, the influence NaCl concentration on gel the strength in

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the presence of 10 mM MDA was also investigated as shown in Figure 5A. As expected, gel strength

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increased along with increasing NaCl levels (Figure 5A), which was largely due to the swollen

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appearance of MP promoting gel formation, and is in well consistent with results from G’ (Figure



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4B).

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Furthermore, the gel WHC, which could reflect spatial structure of emulsion gel, showed a very

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similar trend as compared to gel strength, as shown in Figure 5B. This was also in good agreement

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with G′ and tan δ data (Figure 4). As has been reported, the gel strength or stiffness played a

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prominent role in the water-holding property of gels,8,12 thus, the progressive increase/decrease in

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WHC can be attributed to compact/loose structure and strengthened/weakened gel structure with

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MDA/ NaCl at different levels.

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Amount of Protein Entrapped. The MDA-induced gelation of MP emulsions is closely associated

328

with the ionic and covalent interactions between adsorbed and/or unadsorbed proteins, as mentioned

329

above. Once the emulsion gel network is formed, content of the proteins entrapped, including those

330

adsorbed to the surface of oil droplets and some of the unabsorbed, can be approximately determined

331

using centrifugation of the emulsions or emulsion gels.9,29 Figure 5C shows the influence of MDA

332

and NaCl on the amount of protein entrapped within the emulsion gel matrix. We could clearly see

333

that in the presence of 0.6 M NaCl, the amount of entrapped proteins slightly but progressively

334

increased as the MDA concentration increased from 2.5 to 10 mM, indicating more proteins

335

participating in the gel network formation. This should be mainly due to the enhanced

336

protein-protein interactions under MDA-induced oxidative stress, which reinforced the gel structure

337

(Figures 4A, 4C, 5A, and 5B), thus increasing the amount of protein entrapped.

338

further addition in MDA (25 – 50 mM) caused a significant (p < 0.05) decrease in the amount of

339

protein entrapped. As aforementioned, emulsion gel with separated oil phase and decreased gel

340

elasticity, strength were observed when MDA concentration was added above 25 mM (Figures 3A,

341

4A, and 5A). Therefore, we could speculated that excessive covalent bond might be formed when

342

addition of MDA exceeding 25 mM,25 which could break protein-protein bonds or drag protein away

343

from interface. Accordingly, the separated oil drops would lead to the protein separated from the gel

344

matrix, no longer staying in the upper layer after centrifugation due to enhanced density, and thus


Nevertheless,

Page 15 of 36

345


resulting in the decreased amount of protein entrapped (Figure 5C).

346

As for the influence of NaCl levels, the amount of protein entrapped decreased with increasing

347

NaCl levels (Figure 5C), which seems to be inconsistent with the visual observation of emulsions

348

after centrifugation (Figure 1A, inset). However, in fact, the amount of interfacial protein of

349

emulsions (Figure 1A, inset) also decreased with increasing NaCl concentrations (data not shown).

350

This could be explained by the fact that MP swell profusely at around 0.6 M NaCl, which decreased

351

the protein adsorption rate on droplets surface, thus decreasing the amount of interfacial protein of

352

emulsions (data not shown). Furthermore, it is worth noting that, in the presence of 0.6 M NaCl, the

353

amount of protein entrapped within the gel matrix (Figure 5C) was higher than that of interfacial

354

protein of the corresponding emulsion (data not shown), suggesting that the unadsorbed proteins in

355

aqueous phase of emulsion was involved in gel matrix formation.34,36

356

SDS-PAGE. Protein patterns of non-entrapped and entrapped proteins in the MDA-induced

357

emulsion gels were examined by performing SDS-PAGE in either non-reducing or reducing

358

conditions, as shown in Figure 6. Additionally, the control emulsion stabilized by MP in the presence

359

of 0.6 M NaCl without MDA was also recorded for better comparison. As can be seen from Figure

360

6A, for non-entrapped proteins, in the absence of β- ME, basically 4 MP components—α-actinin,

361

light meromyosin (LMM), troponin T (TnT) and tropomyosin (Tm) were observed in the control

362

sample (0 mM), within which TnT and Tm were two predominates. Previous studies have already

363

indicated that TnT and Tm were uncoagulable and did not contribute to muscle protein gel network

364

formation.12,37 With increasing concentration of MDA, the band intensities of four MP components

365

progressively decreased or vanished (Figure 6A). Concomitantly, the band intensity of high

366

molecular weight polymers (HMWP), which accumulated on the top part of stacking gel or barely

367

entered, increased accordingly (Figure 6A). However, in the presence of β-ME, most of these HMWP,

368

but not reduced/lost band, could be recovered, especially within the MDA range from 0 to 10 mM

369

(Figure 6B). These results implied that HMWP formed were partially through disulfide bonds, rather



370

than mainly non-disulfide covalent bonds as observed in other cases,25,27 indicating that

371

non-entrapped protein were less influenced by MDA-induced oxidative modification, similar to the

372

previous report.38 Moreover, when concentration of MDA reached 50 mM, diffuse bands with

373

molecular weight similar to Tm or around occurred again (Figure 6B), which might be related to the

374

collapse of gel structure(Figures 4A and 4C).

375

For entrapped proteins, in the absence of β-ME, essentially myosin heavy chain (MHC) and actin

376

were observed in the control sample (0 mM), and no clear bands corresponding to TnT or Tm, which

377

have been discovered in the non-entrapped ( Figure 6A), were obtained (Figure 6C). Nevertheless,

378

MHC in all the gel samples vanished and all bands were lost with excessive addition of MDA

379

(>25mM), besides actin remaining faint (Figure 6C). However, no HMWP were detected with

380

increasing MDA concentration (25 – 50 mM). This should be due to the over cross-linking of

381

proteins, leading to the formation of very high molecular weight polymers, which could not penetrate

382

into the gel.25,39 In the presence of β-ME, MHC in all gel samples could not be recovered (Figure 6D),

383

suggesting that most cross-linking originated from myosin was not through disulfide bonds and the

384

covalent bond in gel was too strong to break, which are similar to the previous reports that MDA can

385

react with amino groups of proteins, producing strong intermolecular cross-links of the Schiff base

386

type.25-27 In addition, these results also indicated that oxidative modifications concerned more

387

specifically the proteins adsorbed or entrapped, in agreement with previous report.38 Interestingly,

388

TnT and Tm, which have just been observed in the non-entrapped, though progressively reduced or

389

lost with increasing addition of MDA, actually were obtained in the entrapped protein upon

390

incubation with MDA (Figure 6C). These results, together with that obtained from changes in the

391

non-entrapped, implied that within MDA range of 2.5 to 10 mM, the reduced/lost protein

392

composition (TnT and Tm) in non-entrapped might participate in the gel network formation under

393

MDA-induced oxidative stress, thus turning into the entrapped proteins. Herein, the progressive

394

entrapment of proteins with strong covalent bonds would provide structure reinforcement which


Page 16 of 36

Page 17 of 36


395

resulted in gel matrix capable of sustaining higher stresses and therefore the higher gel strength

396

(Figure 4). These analyses are also in good agreement with the increased amount of protein

397

entrapped as previously stated (Figures 5).

398

As for the influence of NaCl level, increase in NaCl level did not change the protein pattern of

399

both the non-entrapped and the entrapped (Figure 6), but slightly decreased the stability of actin

400

(Figure 6D), as has been previously reported.25,40

401

Microstructure. The gel strength and WHC of the formed emulsion gels are, to a large degree,

402

related to their gel microstructure. Therefore, the microstructure of the MDA-induced MP emulsion

403

gels, formed at various MDA concentrations (2.5 – 50 mM) or at different NaCl levels ( 0.2, 0.4 and

404

0.6 M), was further characterized using CLSM, as displayed in Figure 7A and 7B, respectively. In

405

addition, the control emulsion in the presence of 0.6 M NaCl without MDA was also recorded and

406

compared. As shown in Figure 7A, for the control emulsion, most of the oil droplets with similar

407

sizes were evenly dispersed in the emulsion, indicating a relative stable state, which was in

408

agreement with the d4,3 data of emulsion (Figure 1B). In the presence of 0.6 M NaCl, when the

409

concentration of MDA increased from 2.5 to 10 mM, the dispersed oil droplets with protein coated

410

gradually got close and the adjacent droplets even became tightly packed when addition of MDA

411

reached 10 mM. This might be attributed to enhanced protein-protein covalent binding among

412

absorbed and/ or non-absorbed (Figure 6), forming compact and homogenous gel network with oil

413

droplets perfectly adherent to the gel matrix (Figure 7A). These analyses were well consistent with

414

the gel strength and WHC (Figure 5), and were also in good agreement with the data from

415

low-amplitude dynamic oscillatory measurements (Figure 4), for it has been well accepted that the

416

samples that have more compact and homogenous microstructure possess the higher elastic modulus

417

and lower tan δ.10,19 Nevertheless, further increase in MDA concentration (25 – 50 mM) greatly

418

changed the gel morphology, and a gel microstructure with obvious phase separation, accumulated

419

protein aggregation and oil droplets with larger size was observed. These results indicated the



420

collapse of the gel microstructure, which was well consistent with the “oil leak” phenomenon

421

observed (Figure 3A) as well as the sharp decrease in gel elasticity/strength and WHC (Figures 4 and

422

5).

423

As for the influence of NaCl, a more homogenous gel microstructure was observed with

424

increasing NaCl concentration (Figure 7B). It has been confirmed that the network of emulsion gels

425

was closely related to the state of oil droplets in the original emulsions.9 Thus, at low NaCl level (i.e.

426

0.2 M), the relative low emulsion apparent viscosity and small steric hindrance (Figures 1 and 2)

427

easily led to the flocculation of oil droplets. Meanwhile, the protein absorbed or unabsorbed failed to

428

swell profusely,32,41 which resulted in the insufficient interaction between MP and MDA,25 thus

429

leading to the resultant discontinuously poor gel microstructure (Figure 7B). However, when the

430

addition of NaCl increased, more stable oil droplets coated with MP were obtained, in a more

431

swollen state, forming a more interactive gel network (Figure 7B).

432

Oxidative Stability of Emulsion Gels. With the fact of improved emulsion gel structure within

433

certain MDA range (2.5 – 10 mM) taken into consideration, the oxidative stability of MDA-modified

434

MP emulsions was further studied, as shown in Figure 8. Lipid hydroperoxides, generally accepted

435

as the first oxidation products, is the most used analysis for determining the initial oxidation rate.42,43

436

Compared to the control emulsion, it is interesting to note that hydroperoxides of MDA-modified MP

437

emulsion firstly decreased along with MDA from 0 to 10 mM, reaching minimum at 10 mM MDA

438

level, before increasing sharply to a maximum at 50 mM (Figure 8A). These results indicated that

439

MDA-induced gelation of MP emulsion obtained higher oxidative stability, depending on the MDA

440

concentration. As aforementioned, the emulsion gels with enhanced gel quality (elasticity, strength,

441

WHC) and better microstructure were obtained within MDA ranging from 2.5 to 10 mM (Figures 4,

442

5 and 7). Thus, the reinforced gel network between absorbed and/or unabsorbed protein might work

443

as physical barrier, separating the lipid substrates from the pro-oxidants present in the aqueous phase

444

or retarding the oxygen diffusion within the systems, which thus could well protect the oil droplets


Page 18 of 36

Page 19 of 36


445

(Figure 8A). Similar protective effect of gelation on emulsion oxidative stability has also been

446

reported.34 Nevertheless, the collapsed gel microstructure due to formation of excessive covalent

447

bond led to the “oil leak” (Figures 3 and 6), favoring the direct contact of oil with oxidizer, which

448

thus decreased oxidative stability of the system (25 – 50 mM MDA) (Figure 8A). As for the

449

influence of NaCl, similarly, lower amount of hydroperoxides at higher levels of NaCl was obtained

450

(Figure 8B), which could also be largely attributed to the protective effect of improved gel properties

451

(Figures 3, 4 and 5). Additionally, the higher interfacial area of emulsions/emulsion gels at lower

452

NaCl level (Figure1) might also promote interactions between lipids and pro-oxidants in the aqueous

453

phase, and thus the lower oxidative stability (Figure 8B).34,44

454

General Discussion. Based on the analyses above, we can conclude that addition of MDA could

455

lead to the formation of emulsion gel stabilized by MP in the presence of NaCl. Meanwhile, the gel

456

properties as well as the gel oxidative stability were enhanced at certain concentration range of MDA

457

(Figures 4, 5 and 8). To clarify a reasonable understanding of the either improved or deteriorated gel

458

quality, a schematic illustration of the formation process of the MDA-induced MP emulsion gels is

459

proposed and shown in Figure 9. In the absence of NaCl, MP fails to swell,32 and thus emulsions

460

with smaller size were obtained due to relatively quick absorption of protein to the oil droplet surface

461

(Figure 1A). Nevertheless, the small steric hindrance of the protein at interface as well as the

462

unevenly dispersed protein in aqueous phase due to its low solubility made the droplet free to flow

463

and easier to flocculate (Figures 1B and 2),32 which further limited the interaction between MP

464

(absorbed or unabsorbed) and MDA, leading to coagulation, rather than gelation of emulsion (Figure

465

3B). However, in the presence of 0.6 M NaCl, MP swell profusely,18,32,45 and the swollen

466

MP-stabilized emulsions with larger size possessed a thicker absorbed protein layer due to enhanced

467

protein-protein interaction (Figure 1A),18,25 which thus increased the physical stability of emulsions

468

(Figure 1B). Upon incubation with MDA, the stretching structure of MP at 0.6 M NaCl firstly

469

facilitates the interactions among absorbed and/or unabsorbed proteins,38 forming strong covalent



470

bonds (Figure 6), which thus promoted the gel formation (Figure 3A). Specifically, within MDA

471

concentrations ranging from 2.5 to 10 mM, enhanced protein-protein cross-links were observed with

472

more amounts of protein, even the unabsorbed protein fractions (i.e. Tm), being progressively

473

entrapped (Figures 5C and 6), contributing to a more homogeneous and compact gel structure with

474

higher gel elasticity, strength, and WHC (Figures 4, 5 and 7). Interestingly, with oil droplets perfectly

475

adherent to the homogeneous gel matrix (Figure 7), the enhanced oxidative stability of gelled

476

emulsion was also obtained (Figure 8). Nevertheless, further increase in MDA concentration (above

477

25 mM) led to the formation of excessive covalent bonds (Figure 6), breaking the protein–protein

478

bonds and/or triggering the separation of protein-oil interface, which thus resulted in the resultant

479

“oil leak” as well as the collapse of gel structure (Figures 3, 4, 5, and 7). Accordingly, the gel

480

oxidative stability was also remarkably decreased (Figure 8).

481

In conclusion, this work demonstrated an interesting discovery of the formation of a cold-set,

482

MDA-induced emulsion gels stabilized by salted MP, and the possible mechanism involved was also

483

proposed. The gel was formed on the premise of the swelling of MP under certain ionic strength and

484

the non-disulfide covalent bonds formed via MDA. Within certain MDA ranges (2.5 – 10 mM), the

485

oil droplets were perfectly adherent to the gel matrix due to the moderate amount of covalent bonds

486

formed between absorbed and/or unabsorbed proteins, endowing the emulsion gel with a better

487

quality. Meanwhile, the reinforced gel network was also responsible for enhanced oxidative stability

488

of emulsion gels. Considering the real meat manufacturing or storage process, the link between

489

MDA-induced protein oxidation and MP stabilized emulsion gel is far from straightforward.

490

However, the results presented in this paper could provide a new aspect for better understanding the

491

effect of oxidation in meat protein/oil composite system.

492

ACKNOWLEDGEMENTS

493

This work was supported by the National Natural Science Foundation of China (No.31201387), the

494

Natural Science Foundation of Guangdong Province (No. S2012040007533) and the Doctoral Fund


Page 20 of 36

Page 21 of 36


495

of Ministry of Education of China (No. 20120172120017). The assistance provided by Dr. Zhi-Li

496

Wan is gratefully acknowledged and appreciated.

497

REFERENCES

498

(1) Dickinson, E. Emulsion gels: The structuring of soft solids with protein-stabilized oil droplets.

499

Food Hydrocolloids 2012, 28, 224–241.

500

(2) Gordon, A.; Barbut, S. Mechanisms of meat batter stabilization: a review. Crit. Rev. Food. Sci.

501

Nutr. 1992, 32, 299–332.

502

(3) Xiong, Y. L. Meat binding: emulsions and batters. In: Osburn WN, Sebranek JG,Schultz Kaster

503

CM, editors. Meat processing technology series. Savoy, Ill.: American Meat Science Assn. 2007,

504

1–28.

505

(4) Ziegler, G. R.; Foegeding, E. A. The gelation of proteins. Adv. Food. Nutr. Res. 1990, 34,

506

203–298.

507

(5) Line, V. L. S.; Remondetto, G. E.; Subirade, M. Cold gelation of β-lactoglobulin oil-in-water

508

emulsions. Food Hydrocolloids 2005, 19, 269–278.

509

(6) Chen, J.; Dickinson, E. Effect of surface character of filler particles on rheology of heat-set whey

510

protein emulsion gels. Colloid Surface B. 1999, 12, 373–381.

511

(7) Sala, G.; Van Aken, G. A.; Stuart, M. A. C.; Van De Velde, F. Effect of droplet-matrix interactions

512

on large deformation properties of emulsion-filled gels. J. Texture Stud. 2007, 38, 511–535.

513

(8) Tang, C.-H.; Chen, L.; Foegeding, E. A., Mechanical and water-holding properties and

514

microstructures of soy protein isolate emulsion gels induced by CaCl2, glucono-δ-lactone (GDL),

515

and transglutaminase: Influence of thermal treatments before and/or after emulsification. J. Agric.

516

Food Chem. 2011, 59, 4071–4077.

517

(9) Tang, C.-H.; Luo, L.-J.; Liu, F.; Chen, Z., Transglutaminase-set soy globulin-stabilized emulsion

518

gels: Influence of soyβ-conglycinin/glycinin ratio on properties, microstructure and gelling

519

mechanism. Food Res. Int. 2013, 51, 804–812.



520

(10) Manoi, K.; Rizvi, S. S. Emulsification mechanisms and characterizations of cold, gel-like

521

emulsions produced from texturized whey protein concentrate. Food Hydrocolloids 2009, 23,

522

1837–1847.

523

(11) Lee, S.-H.; Lefevre, T.; Subirade, M.; Paquin, P., Changes and roles of secondary structures of

524

whey protein for the formation of protein membrane at soy oil/water interface under high-pressure

525

homogenization. J. Agric. Food Chem. 2007, 55, 10924–10931.

526

(12) Wu, M.; Xiong, Y. L.; Chen, J.; Tang, X.; Zhou, G. Rheological and microstructural properties

527

of porcine myofibrillar protein-lipid emulsion composite gels. J. Food Sci. 2009, 74, E207–E217.

528

(13) Wu, M.; Xiong, Y. L.; Chen, J., Role of disulphide linkages between protein-coated lipid

529

droplets and the protein matrix in the rheological properties of porcine myofibrillar protein–peanut

530

oil emulsion composite gels. Meat Sci. 2011, 88, 384–390.

531

(14) Acton, J. C.; Ziegler, G. R.; Burge Jr, D. L.; Froning, G. W. Functionality of muscle constituents

532

in the processing of comminuted meat products. Crit. Rev. Food. Sci. Nutr. 1982, 18, 99–121.

533

(15) Asghar, A.; Samejima, K.; Yasui, T. Functionality of muscle proteins in gelation mechanisms of

534

structured meat products. Crit. Rev. Food. Sci. Nutr. 1985, 22, 27–106.

535

(16) Lund, M. N.; Heinonen, M.; Baron, C. P.; Estevez, M. Protein oxidation in muscle foods: A

536

review. Mol. Nutr. Food. Res. 2011, 55, 83–95.

537

(17) Zhang, W.; Xiao, S.; Ahn, D. U. Protein oxidation: basic principles and implications for meat

538

quality. Crit. Rev. Food. Sci. Nutr. 2013, 53, 1191–1201.

539

(18) Li, C.; Xiong, Y. L; Chen, J. Protein oxidation at different salt concentrations affects the

540

cross-linking and gelation of pork myofibrillar protein catalyzed by microbial transglutaminase. J.

541

Food Sci. 2013, 78, 823–831.

542

(19) Xiong, Y. L.; Blanchard, S. P.; Ooizumi, T.; Ma, Y. Hydroxyl radical and ferryl‐generating

543

systems promote gel network formation of myofibrillar protein. J. Food Sci. 2010, 75, C215–C221.

544

(20) Sun, W.; Zhou, F.; Sun, D.-W.; Zhao, M. Effect of oxidation on the emulsifying properties of


Page 22 of 36

Page 23 of 36


545

myofibrillar proteins.Food Bioprocess Tech. 2012, 6, 1703–1712.

546

(21) Zhou, F.; Zhao, M.; Zhao, H.; Sun, W.; Cui, C. Effects of oxidative modification on gel

547

properties of isolated porcine myofibrillar protein by peroxyl radicals. Meat Sci. 2013,

548

96,1432–1439.

549

(22) Estévez, M.; Kylli, P.; Puolanne, E.; Kivikari, R.; Heinonen, M. Oxidation of skeletal muscle

550

myofibrillar proteins in oil-in-water emulsions: interaction with lipids and effect of selected phenolic

551

compounds. J. Agric. Food Chem. 2008, 56, 10933–10940.

552

(23) Koutina, G.; Jongberg, S.; Skibsted, L. H. Protein and lipid oxidation in parma ham during

553

production. J. Agric. Food Chem. 2012, 60, 9737–9745.

554

(24) Park, D.; Xiong, Y. L.; Alderton, A. L.; Ooizumi, T. Biochemical changes in myofibrillar protein

555

isolates exposed to three oxidizing systems. J. Agric. Food Chem. 2006, 54, 4445–4451.

556

(25) Zhou, F.; Zhao, M.; Su, G.; Cui, C.; Sun, W. Gelation of salted myofibrillar protein under

557

malonaldehyde-induced oxidative stress. Food Hydrocolloids 2014,40, 153–162.

558

(26) Xiong, Y. L.; Park, D.; Ooizumi, T. Variation in the cross-linking pattern of porcine myofibrillar

559

protein exposed to three oxidative environments. J. Agric. Food Chem. 2008, 57, 153–159.

560

(27) Buttkus, H., The reaction of myosin with malonaldehyde. J. Food Sci. 1967, 32, 432–434.

561

(28) Wu, W.; Zhang, C.; Hua, Y. Structural modification of soy protein by the lipid peroxidation

562

product malondialdehyde. J. Sci. Food Agr. 2009, 89, 1416–1423.

563

(29) Chanyongvorakul, Y.; Matsumura, Y.; Sawa, A.; Nio, N.; Mori, T. Polymerization of

564

β-lactoglobulin and bovine serum albumin at oil-water interfaces in emulsions by transglutaminase.

565

Food Hydrocolloids 1997, 11, 449–455.

566

(30) Flores, M.; Barat, J. M.; Aristoy, M.; Peris, M. M.; Grau, R.; Toldrá, F. Accelerated processing

567

of dry-cured ham. Part 2. Influence of brine thawing/salting operation on proteolysis and sensory

568

acceptability. Meat Sci. 2006, 72, 766–772.

569

(31) Tong, L. M.; Sasaki, S.; McClements, D. J.; Decker, E. A. Antioxidant activity of whey in a



570

salmon oil emulsion. J. Food Sci. 2000, 65, 1325–1329.

571

(32) Offer, G.; Trinick, J. On the mechanism of water holding in meat: the swelling and shrinking of

572

myofibrils. Meat Sci. 1983, 8, 245–281.

573

(33) Tang, C.-H.; Liu, F., Cold, gel-like soy protein emulsions by microfluidization: Emulsion

574

characteristics, rheological and microstructural properties, and gelling mechanism. Food

575

Hydrocolloids 2013, 30, 61–72.

576

(34) Sato, A.; Moraes, K.; Cunha, R. Development of gelled emulsions with improved oxidative and

577

pH stability. Food Hydrocolloids 2014, 34, 184–192.

578

(35) Intarasirisawat, R.; Benjakul, S.; Visessanguan, W. Stability of emulsion containing skipjack roe

579

protein hydrolysate modified by oxidised tannic acid. Food Hydrocolloids 2014, 41, 146–155.

580

(36) Kim, K.-H.; Renkema, J.; Van Vliet, T. Rheological properties of soybean protein isolate gels

581

containing emulsion droplets. Food Hydrocolloids 2001, 15, 295–302.

582

(37) Samejima, K.; Ishioroshi, M.; Yasui, T. Heat induced gelling properties of actomyosin: effect of

583

tropomyosin and troponin. Agric. Biol. Chem. 1982.46,535−540.

584

(38) Berton, C.; Ropers, M.-H.; Guibert, D.; Sole, V.; Genot, C. Modifications of interfacial proteins

585

in oil-in-water emulsions prior to and during lipid oxidation. J. Agric. Food Chem. 2012, 60,

586

8659–8671.

587

(39) Coupland, J. N.; McClements, D. J. Lipid oxidation in food emulsions. Trends Food Sci. Tech.

588

1996, 7, 83–91.

589

(40) Thorarinsdottir, K. A.; Arason, S.; Geirsdottir, M.; Bogason, S. G.; Kristbergsson, K. Changes in

590

myofibrillar proteins during processing of salted cod (Gadus morhua) as determined by

591

electrophoresis and differential scanning calorimetry. Food Chem. 2002, 77, 377–385.

592

(41) Liu, Z.; Xiong, Y. L.; Chen, J. Identification of restricting factors that inhibit swelling of

593

oxidized myofibrils during brine irrigation. J. Agric. Food Chem. 2009, 57, 10999–11007.

594

(42) Wan, Z.-L.; Wang, J.-M.; Wang, L.-Y.; Yang, X.-Q.; Yuan, Y. Enhanced physical and oxidative


Page 24 of 36

Page 25 of 36


595

stabilities of soy protein-based emulsions by incorporation of a water-soluble stevioside–resveratrol

596

complex. J. Agric. Food Chem. 2013, 61, 4433–4440.

597

(43) Sun-Waterhouse, D.; Zhou, J.; Miskelly, G. M.; Wibisono, R.; Wadhwa, S. S., Stability of

598

encapsulated olive oil in the presence of caffeic acid. Food Chem. 2011, 126, 1049–1056.

599

(44) Waraho, T.; McClements, D. J.; Decker, E. A., Mechanisms of lipid oxidation in food

600

dispersions. Trends Food Sci. Tech. 2011, 22, 3–13.

601

(45) Xiong, Y. L., Role of myofibrillar proteins in water-binding in brine-enhanced meats. Food Res.

602

Int. 2005, 38, 281–287.

603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623



624

Figure Captions:

625

Figure 1. (A) Particle size distributions of fresh emulsions stabilized by MP at different NaCl levels

626

(0−0.6M). Inset: visual observation of fresh emulsions after centrifugation (10000 × g, 30 min). (B)

627

Changes in mean particle diameter d4,3 of emulsions during storage for 30 days.

628

Figure 2. Variation of apparent viscosity (η) with shear rate (A) and of storage modulus (G′) / loss

629

modulus (G′′) with frequency (B) for MP stabilized emulsions at different NaCl levels (0−0.6M).

630

Figure 3. Visual observations of MDA-modified MP emulsions with various MDA concentrations

631

(0−50 mM MDA, 0.6 M NaCl) (A) or at different NaCl levels (0−0.6M NaCl, 10 mM MDA) (B).

632

Figure 4. The typical dependence profiles of storage modulus (G′) (A, B) and tan δ (C, D) on

633

frequency, for MDA-modified MP-stabilized emulsions at various MDA concentrations (A, C) or at

634

different NaCl levels (B, D).

635

Figure 5. Gel strength (A), WHC (B) and protein entrapped (C) of MDA-modified emulsion gels at

636

various MDA concentrations (black columns) or at different NaCl levels (gray columns). Columns

637

with different letters are significantly different (p < 0.05).

638

Figure 6. Representative SDS−PAGE patterns of the non-entrapped (A, B) and entrapped (C, D)

639

protein in MDA-induced MP emulsion gels in the absence (non-reducing, A andC) or presence

640

(reducing, B and D) of 5% β-ME. LMM, light meromyosin; MHC, myosin heavy chain; TnT,

641

troponin T; Tm, tropomyosin.

642

Figure 7. Typical CLSM images of MDA-induced MP emulsion gels at various MDA concentrations

643

(A) or at different NaCl levels (B). All of the images were obtained with the dyes Nile red (protein)

644

and Nile blue (oil phase), excited at 488 and 633 nm, respectively.

645

Figure 8. Changes in lipid hydroperoxides of emulsions gels stabilized by MP either at various MDA

646

concentrations (A) or at different NaCl levels (B) during storage at 25 ° C for 21 days.

647

Figure 9. Schematic illustration of formation process of the MDA-induced emulsion gels stabilized

648

by salted MP.


Page 26 of 36

Page 27 of 36

649


Figure 1.

650

651 652 653 654 655 656 657 658 659



660 661

Figure 2.

662

663 664 665


Page 28 of 36

Page 29 of 36


Figure 3.



Figure 4.


Page 30 of 36

Page 31 of 36


Figure 5.



Figure 6.


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Figure 7.



Figure 8.


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Figure 9.



TABLE OF CONTENTS GRAPHIC


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Controlled Formation of Emulsion Gels Stabilized ... - ACS Publications

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