Emulsifying Properties of Oxidatively Stressed Myofibrillar Protein

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Emulsifying Properties of Oxidatively Stressed Myo#brillar Protein Emulsion Gels Prepared with (-)-Epigallocatechin-3-gallate and NaCl Lin Chen, Na Lei, Shuangxi Wang, Xing-lian Xu, Guanghong Zhou, Zhixi Li, and Xianchao Feng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05517 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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Journal of Agricultural and Food Chemistry

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Emulsifying Properties of Oxidatively Stressed Myofibrillar Protein Emulsion Gels

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Prepared with (-)-Epigallocatechin-3-gallate and NaCl

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Lin Chen,‡, Na Lei, Shuangxi Wang, Xinglian Xu‡, Guanghong Zhou‡, Zhixi Li, Xianchao

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Feng *,

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Yangling, Shaanxi 712100, China

7

‡

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Synergetic Innovation Center of Food Safety and Nutrition, Nanjing Agricultural University,

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Nanjing, Jiangsu 210095, China

College of Food Science and Engineering, Northwest A&F University, No. 22 Xinong Road,

Lab of Meat Processing and Quality Control of EDU, College of Food Science and Technology,

10 11 12

*Corresponding author-Xianchao Feng

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Associate Professor, College of Food Science and Engineering, Northwest A&F University, No.

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22 Xinong Road, Yangling, Shaanxi, China 712100. Email address: [email protected]

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Tel/Fax: 86029-87092486.

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ACS Paragon Plus Environment


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ABSTRACT: The dose-dependent effects of (-)-epigallocatechin-3-gallate (EGCG; 0, 100 or

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1000 ppm) on the textural properties and stability of a myofibrillar protein (MP) emulsion gel

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were investigated. EGCG addition significantly inhibited formation of carbonyl but promoted the

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loss of both thiol and free amine groups. Addition of EGCG, particularly at 1000 ppm, initiated

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irreversible protein modifications, as evidenced by surface hydrophobicity changes, patterns in

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SDS-PAGE, and differential scanning calorimetry (DSC). These results indicated that MP was

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modified by additive reactions between the quinone of EGCG and thiols and free amines of

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proteins. These adducts increased cooking loss and destabilized the texture, especially at a high

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dose of EGCG. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy

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(SEM) images clearly indicated the damage to the emulsifying properties and the collapse of the

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internal structure when the MP emulsion gel was treated with a high dose of EGCG. A high

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concentration of NaCl (0.6 M) improved modification of MP and increased deterioration of

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internal structure, especially at the high dose of EGCG (1000 ppm), resulting in extremely

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unstable emulsifying properties of MP emulsion gel.

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KEYWORDS: emulsion gel; myofibrillar protein; EGCG; chemical properties; microstructural

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properties; confocal laser scanning microscopy

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INTRODUCTION

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Myofibrillar proteins (MPs) are the main constituent of total muscle protein (55% to 60%).1

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The quality of MP is mainly responsible for the properties of processed meat products.2 It is

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necessary to start with high-quality MP to produce a well-formed, uniformly textured,

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physicochemically stable meat product, as the MP serves to stabilize fat globules and entrap

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water.1, 3 MP is prone to attack by the reactive oxygen species that are generated during food

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processing (i.e., cooking, drying and storing), which results in direct conversion of amino acid

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residues to carbonyls and cross-links.4 These oxidative modifications can improve the

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polymerization and aggregation of proteins, but can also alter the secondary and tertiary structure

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of proteins, leading to changes in the physical properties of proteins, such as hydrophobicity.5

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Consequently, protein oxidation has been linked to deteriorated quality of meat products, such as

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loss in juiciness, increase in cooking loss, and toughness of meat.6 There is an increasing volume

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of research on using antioxidants to prevent protein oxidation in the meat product industry.7

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There is a growing focus on using natural preservatives due to their safety and their perceived

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non-toxicity compared to synthetic additives in the meat industry.7 Extracts from herbs and spices

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are widely used as natural alternatives. These extracts provide promising antioxidant effects that

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are attributed to phenolic compounds, due to their hydrogen-donating capacity and

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metal-chelating potential. Polyphenols are of upmost interest due to their health-beneficial

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effects.8 Phenolic derivatives have been widely and commonly used to inhibit oxidative

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modification in meat products.9

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However, aside from the positive effect on scavenging free radicals, some researchers found

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that polyphenols had negative effects on the quality of processed meat products. The addition of a 3 / 35



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phenolic-rich extract from dog-rose to beef patties had an antioxidant effect on lipids and proteins

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but increased cooking loss and storage loss.10 The addition of 1000 ppm green tea extract

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decreased the content of thiobarbituric acid reactive substances (TBARS) and carbonyls but

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deteriorated the emulsion properties of meat products, resulting in increased cooking loss and

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texture instability.11 The addition of 500 ppm white grape extract reduced the TBARS and protein

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carbonyls but also reduced the desired thiols in beef patties.12

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In recent years, it has been found that polyphenols can be oxidized and converted to quinines,

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which can irreversibly react with the thiol and amino moieties of proteins.8 Polyphenols react with

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a cysteinyl thiol derivative (N-benzoylcysteine methyl ester) though formation of thiol-quinone

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adducts under radical oxidation conditions.13 When 4-Methylcatechol was used to inhibit protein

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oxidation, the formation of thiol-quinone adducts led to an additional loss of thiol groups.14

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Reactive quinones, derived from rosmarinic acid, will form adducts with thiol compounds, such

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as cysteine, glutathione, and peptides, derived from myosin.15 Moreover, quinones can also

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irreversibly react with the anime groups of proteins.8, 16 Intermolecular disulphide (S-S) linkages

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due to oxidation of thiol groups are one of the principal forces for gelation of muscle protein

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during heating. A significant decrease in the storage modulus (G'), gel strength, and cook yield of

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MP emulsion gels occurs when thiols of MP were modified by N-ethylmaleimide before

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heating.17 Despite all these recent reports, the effects of thiol-quinone and amino-quinone adducts

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between polyphenol and meat proteins on the quality of food products in processing are relatively

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unreported.10-12 Furthermore, it is well known that high NaCl concentrations result in more

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soluble MP with a swollen appearance, which enhances protein-protein interactions. However,

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effects of NaCl concentrations on the adducts of thiol-quinone and amino-quinone have yet to be 4 / 35


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thoroughly investigated.

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Tea is the second-most widely consumed beverage worldwide, second only to water.18 Green

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tea extract contains polyphenolic compounds known as catechins, which may constitute up to 30%

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of the dry weight of tea leaves.18-19 The most abundant catechin is (-)-epigallocatechin-3-gallate

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(EGCG), which has been reported to be responsible for many of the effects of green tea.20 Green

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tea extracts are broadly used to inhibit lipid oxidation in emulsion-type meat products, e.g.

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various bologna, wieners, and frankfurters, which generally contain at least 30% fat.21 However,

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the influence of thiol-quinone and amino-quinone adducts between catechins and protein in these

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meat products on the emulsifying properties has not been investigated.

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In this work, the effects of EGCG addition on the emulsifying properties of pork MP emulsion

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gel under controlled oxidizing conditions (H2O2) were investigated. Cooking loss and textural

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profiles of MP emulsion gel were observed. Chemical, structural, thermal and rheological

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properties of EGCG-modified MP were analyzed to discern the internal mechanism. The effects

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of salt concentration on adducts between EGCG and MP were also studied.

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MATERIALS AND METHODS

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Materials. Fresh pork from the Longissimus muscle of pig carcasses (large white crossbred,

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within 2 days post mortem) was bought from a local supermarket (Haoyouduo, Yangling, Shaanxi,

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China). Chemicals used in present study were of reagent grade and purchased from Aladdin

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Industrial Corporation (Fengxian, Shanghai, China) or Sigma-Aldrich Co. (St. Louis, MO, USA).

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Myofibrillar Protein (MP) Preparation. MP was prepared according to published methods,

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with some modifications.5, 22 Briefly, 200 g of fresh pork was firstly homogenized with a blender

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(Joyung Co., Ltd. Jinan, China) in 1 L isolation buffer [20 mM PBS, 150 mM NaCl, 25 mM KCl, 5 / 35



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3 mM MgCl2, and 4 mM EDTA at pH 7.0]. The suspension was further homogenized with an

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Ultra Turrax dispersing instrument (IKA T18-Digital, Staufen, Germany) in an ice bath. After

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filtratration through gauze, the homogenate was spun twice at 2000 × g for 15 min at 4 °C. Then,

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the pellets were washed twice with 4 L of 0.1 M NaCl solution. The collected pellets were washed

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again with 4 L of 20 mM PBS pH 7.0. The protein concentration was measured using the Biuret

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method.23

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Oxidative Treatments with EGCG. MP suspensions were prepared with 20 mM PBS buffer

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(pH 7.0). Nine different reaction mixtures (final protein concentration, 40 mg/mL) were made

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with various EGCG (0, 100 and 1000 mg/kg protein) and NaCl (0, 0.2, and 0.6 M) concentrations

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in five replicates according to a total factorial design. Mixtures were oxidized at 4 °C for 12 h

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using 5 mM H2O2, 10 mM FeCl3, and 100 mM ascorbic acid. Sodium azide (final concentration,

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0.02% by wt) was used to prevent microbial growth in the mixtures. Addition of Trolox

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C/butylated hydroxytoluene (BHT)/ EDTA (each at 1 mM final concentration) was applied to stop

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the oxidation reaction. A non-oxidized, EGCG-free MP suspension was used as the control.

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Carbonyl Analysis. The carbonyl levels in the treated MP samples were analyzed according to

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the method of Oliver et al.24 Briefly, carbonyl levels, in the form of hydrazones, were detected by

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2,4-dinitrophenylhydrazine (DNPH). Absorbance was measured at 370 nm with an absorption

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coefficient of 22000 M−1 cm−1 to calculate the carbonyl content.

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Free Amines. Free anime levels in the treated MP samples were analyzed as described by Liu

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et al.25 Briefly, 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used to measure free anime levels

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with absorbance at 420 nm.

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Total Sulfhydryl Content. Total sulfhydryl content in the treated MP was measured using 6 / 35


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absorbance at 412 nm with the indicator 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB).26-27

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Absorption coefficients of 13600 M-1 cm-1 were used for calculating the total sulfhydryl content.

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Surface

Hydrophobicity.

Surface

hydrophobicity

was

determined

using

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1-anilino-8-naphthalenesulfonate (ANS) as hydrophobic fluorescent probe. A series of MP

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solutions with a protein concentration ranging from 0.05 to 2.0 mg/mL were prepared. An aliquot

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of 4 mL of each MP solution was mixed by vortex with 20 µL of 8 mM ANS solution. The

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fluorescence intensity of all samples was measured after static reaction for 2 min using a

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PerkinElmer LS-55 spectrofluorometer (Waltham, MA, USA). The excitation and emission

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wavelengths were 390 and 470 nm, respectively. Both excitation slit width and emission slit width

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were set at 10 nm. Protein surface hydrophobicity was expressed by linear regression slope on a

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curve plotting fluorescence intensity against protein concentration.

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Electrophoresis. Polymerization of protein was investigated using sodium dodecyl sulfate

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polyacrylamide gel electrophoresis (SDS−PAGE) with a 4% polyacrylamide stacking gel and 12.5%

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separating gel.5, 28 The MP solution (2 mg/mL) was mixed with 4-fold sample buffer containing

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10% β-mercaptoethanol (βME) or 0% βME, and then boiled for 5 min. After electrophoresis using

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10 µL of each sample, 0.1% Coomassie Brilliant Blue was used to show protein bands on the gel.

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After eliminating the free dye, the band profile on each gel was recorded with a Gel Doc XRTM

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System (Bio-Rad Laboratories, Hercules, CA).

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Thermal Analysis by Differential Scanning Calorimetry (DSC). DSC was performed on a

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TA Instruments Q2000 DSC (TA Instruments, New Castle, DE, USA). Raw samples (40 mg/mL)

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were accurately weighed (approximately 15 mg) and sealed in hermetic aluminum pans. An

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empty pan was used as control. Thermal scan was performed from 30 to 80 °C at a 5 °C /min rate. 7 / 35



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The temperature maximum (Tm) for protein transition was estimated using Universal Analysis

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software (TA Instruments, Ver. 4.3A).

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Emulsion Gel Preparation. Oil-in-water (O/W) emulsions were prepared by dispersing

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soybean oil in MP suspension (v:v = 1:4) using an Ultra Turrax dispersing instrument (IKA

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T18-Digital, Staufen, Germany) operating at 11000 rpm for 1 min. Five grams of each emulsion

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were added into glass vials (2.5 cm i.d., 5 cm height) and incubated from room temperature to

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74°C, and stayed at 74°C for 10 min.

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Cooking Loss. Raw emulsions were weighed before cooked. The emulsion gels were chilled to

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room temperature after cooking to a core temperature of 74 °C. The weight of the samples was

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recorded again after removing the juice from the surface of the emulsion gels. Cooking loss was

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calculated as: ୑బ ି୑భ

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Cooking loss (%) =

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where M0 is the weight of the sample prior to cooking and M1 is the weight of the sample after

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୑భ

× 100%

cooking and chilling.

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Gel Strength. The gel strength of the MP emulsion gels was measured using a TA-XT plus

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texture analyzer (Stable Micro Systems Co. Ltd., Surrey, UK) fitted with a cylindrical probe

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(P/0.5). Analysis was performed using the following conditions: pre-speed, 1.00 mm/s; trigger

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force, 5 g; test speed, 1.00 mm/s; and post-speed, 1.00 mm/s. The data acquisition rate was 200

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pps.

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Dynamic Rheological Testing During Gelation. Viscoelastic characteristics of emulsion were

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measured during the heat-induced gelation with a Model AR1000 rheometer (TA Instruments,

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West Sussex, UK) in an oscillatory mode equipped with parallel plates geometry (40 mm 8 / 35


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diameter). Five gram of each sample was added between parallel plates (1.0 mm gap) and silicon

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oil was used to cover the exposed rim to prevent dehydration. Gelation was induced by heating

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the protein emulsion from 30 to 80 °C at a 2 °C/min rate. Shear force of samples was measured

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with a sinusoidal strain at a 2% amplitude and an oscillating frequency of 0.1 Hz during heating.

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Changes in the storage modulus (G′) were monitored continuously.29

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Confocal Laser Scanning Microscopy (CLSM). The microstructure of the MP emulsion gels

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was imaged with a confocal laser scanning microscope (A1R, Nikon Inc., Tokyo, Japan). A Fast

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Green and Nile Red mixture (0.038%, w/v) was used to stain the protein and oil droplets,

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respectively. The images were taken with a 10 x magnification lens by CLSM.

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Scanning Electron Microscopy (SEM). The morphology of the gels was observed using a

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Hitachi-S-4800 field emission scanning electron microscope (Hitachi High Technologies Corp.,

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Tokyo, Japan). Cubic samples (1 × 0.8 × 0.5 cm3) obtained from gels were fixed using 2.5%

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glutaraldehyde in 0.1 M PBS (pH 7.4) for 24 h. Samples were then washed using 0.1 M PBS (pH

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7.4) for 20 min, and then fixed in 1% osmium tetraoxid prepared using PBS buffer (pH 7.4) for 5

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h. After washing three times with 0.1 M PBS (pH 7.4), fixed samples were then dehydrated in

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gradient ethanol solutions (50, 60, 70, 80, 90, and 95%, and three times with 100%).

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Statistical Analysis. All measurements were repeated five times. Data from the analysis were

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collected and subjected to statistical analyses. Two-way analysis of variance (ANOVA) was

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applied to assess the effect of the different concentrations of EGCG and NaCl (SPSS 20.0,

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Chicago, IL, USA). A LSD was applied when ANOVA found significant differences between

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different treatments. The statistical significance was set at P < 0.05.

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RESULTS AND DISCUSSION 9 / 35



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Results of this work showed that the addition of EGCG and NaCl significantly affected all

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measured parameters, such as the chemical and emulsifying properties of MP under oxidative

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stress. Cooking loss, gel strength and microstructure of the modified MP emulsion gels were also

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influenced by addition of EGCG and NaCl.

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Effect of EGCG and NaCl on the Chemical and Structural Properties of MP. Reactive

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oxygen species (ROS) readily oxidize the lysine, arginine, proline and threonine residues within

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proteins into carbonyls (aldehydes and ketones). Carbonyl levels are a well-known index of the

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level of protein oxidation in meat products during processing.6 Application of oxidation stress to

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pork MP significantly increased carbonyl content compared to the non-oxidized MP (Figure 1 A).

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The addition of EGCG reduced the carbonyl content, especially for MP treated with a high dose

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of EGCG (Figure 1 A). This result was in accordance with previous studies showing that

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polyphenols prevented the formation of carbonyls in protein.15, 30

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Free amine content is another indicator of protein oxidation,5 since the ε-NH2 groups of some

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amino residues can be readily converted into carbonyls through a deamination process under

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oxidation stress.22 Under oxidative stress in the present study, the ε-NH2 group content was higher

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in the absence of EGCG (Figure 1 B). In the presence of EGCG, the ε-NH2 groups showed

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additional loss that was EGCG-dose dependent (Figure 1 B). This could be due to the formation

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of amine–quinone adducts between the quinone of EGCG and the free amines of protein under

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oxidative stress, since quinones can irreversibly react with the anime groups of proteins.8, 16

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The free amine and carbonyl contents of the oxidatively stressed MP were also significantly

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affected by NaCl addition (Table 1, Figure 1A & B). More soluble MP was available at higher

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NaCl concentration.31 Consequently, the ε-NH2 groups of lysine residues on the protein surface 10 / 35


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were more prone to be attacked by the quinone groups of EGCG at high NaCl concentration (0.6

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M NaCl).5

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MP is rich in thiol groups, which are susceptible to attack by ROS and subsequent conversion

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to intra- and intermolecular disulfide bond linkages.5 Compared to the non-oxidized MP (control),

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the thiol group content of oxidized MP was significantly lower in the absence of EGCG (Figure 1

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C). Furthermore, the thiol group levels showed an EGCG-dose-dependent decrease. This could be

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caused by the formation of thiol–quinone adducts.14-15 These results were in accordance with

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previous studies.11, 30 The thiol group levels in oxidized MP were also significantly decreased with

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the increases in NaCl concentration (Table 1). This could also be explained by the same changes

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in protein status that affected the ε-NH2 groups.31

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Protein surface hydrophobicity is a marker of protein unfolding. The surface hydrophobicity of

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the oxidized MP, in the absence of EGCG, was significantly higher (P < 0.05) than the

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non-oxidized MP (Control, Figure 1 D). The surface hydrophobicity of oxidized MP significantly

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decreased in the presence of EGCG, especially at the high dose of EGCG (1000 ppm) (Figure 1

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D). This decrease in surface hydrophobicity with increased EGCG may be due to protein

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aggregation caused by EGCG addition, which would partially shield the effect of unfolding in

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oxidized MP. Protein aggregation has been proposed to be the reason for loss of surface

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hydrophobicity in oxidized MP samples.3 A combination of high NaCl and high EGCG

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concentrations seems to result in greater aggregation of oxidized MP (Table 1 & Figure 1 D).

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Consequently, the oxidized MP treated with 1000 ppm EGCG had the lowest surface

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hydrophobicity with 0.6 M NaCl (Figure 1 D).

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The interaction between EGCG and NaCl had significant effects on these chemical and 11 / 35



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structural markers of the protein (carbonyl, free amine, thiol group and surface hydrophobicity;

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Table 1). The MP gained a swollen appearance at high NaCl concentration, which would promote

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modification by quinone of EGCG and the ·OH generated by the hydroxyl-radical-generating

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system.31

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Effect of EGCG and NaCl on the DSC Thermal Properties of MP. Non-oxidized MP

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(control) showed two clear endothermic peaks with Tmax values at 60.7 and 69.6 °C (Figure 2); the

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first was attributed to myosin, and the second to actin.32 Oxidative stress at 5 mM H2O2 tended to

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diminish the second peak, which was consistent with previous studies.3 The addition of EGCG

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remarkably shifted all the peaks to lower temperatures, especially for the MP treated with the

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highest dose of EGCG (Figure 2). The total heat of denaturation (∆H) was remarkably reduced

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upon oxidation, and EGCG addition further exacerbated the decrease in ∆H, especially for the MP

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treated with the high dose of EGCG (Figure 2). The high concentration of NaCl (0.6 M) lowered

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the Tmax and ∆H of MP compared to MP with low concentration of NaCl (0 and 0.2 M) (Figure 2).

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These results indicated that the higher EGCG dose led to significant MP denaturation, which

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could be accelerated by high NaCl concentration.

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Effect of EGCG and NaCl on the SDS−PAGE Patterns of MP. Oxidative stress can result in

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intra- and inter-molecular cross-links between proteins.4-5 Compared to the non-oxidized MP

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(control), the band intensities in the oxidized MP for both myosin heavy chain (MHC) and actin

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decreased. In oxidized MP, the intensities of both bands decreased with increasing EGCG,

271

resulting in formation of polymers appearing at the top of the stacking gel (Figure 3 A). This

272

indicated that addition of EGCG improved the polymerization of MP, resulting in the reduction of

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MHC and actin bands (Figure 3 A). However, most MHC and actin bands were recovered when 12 / 35


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treated with βME except for the MP with high dose of EGCG (Figure 3 B). This indicated that

275

these polymers were largely caused by disulfide bonds.5, 33 In the reduced samples, oxidized MP

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treated with EGCG clearly had more polymer bands on the top of both the stacking and separating

277

gels compared to the oxidized MP without EGCG. This indicated that more covalent bonds that

278

were not disulfides might be formed due to the addition of EGCG. Combined with the data on the

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EGCG-dose dependent decrease of thiol groups and free amines, this indicates that quinone-thiol

280

adducts and amine-quinone adducts likely formed. Jongberg et al. found that thiol-quinone

281

adducts were formed between quinone of 4-methylcatechol and protein thiol groups.14 Tang et al.

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also found that quinone of rosmarinic acid can react with compounds containing thiol groups

283

(R−SH), forming thiol−quinone adducts.15 Moreover, quinone of polyphenol can react with amino

284

side chains of polypeptides.8,34 Hence, amine-quinone adducts can contribute to the formation of

285

non-disulfide polymerizations. Cysteine and lysine residues of protein have nucleophilic side

286

chains that may form covalent bonds with the quinone formed by oxidation of the phenol by

287

nucleophilic addition. The proposed reaction mechanism in Figure 4 explains how the

288

thiol−quinone adducts and the amine-quinone adducts could form.

289

Effect of EGCG and NaCl on the Cooking Loss and Gel Strength of MP Emulsion Gel.

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Cooking loss was analyzed to access the emulsifying stability of different MP emulsion gels

291

(Figure 5). EGCG addition had significant effects on cooking loss of the MP emulsion gel (Table

292

1). At each NaCl concentration, the MP emulsion gel containing EGCG had significantly higher

293

cooking loss than the MP emulsion gel without EGCG, especially for 1000 ppm EGCG (0 M

294

NaCl, 36.96%; 0.2 M NaCl, 41.88%; 0.6 M NaCl, 39.75%) (Figure 5). At lower NaCl

295

concentrations, the MP emulsion gel treated with 1000 ppm EGCG had a 1.88- to 2.16-fold higher 13 / 35



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cooking loss than MP emulsion gel without EGCG (Figure 5). NaCl addition significantly

297

increased cooking loss of the MP emulsion gel (Table 1). With 0.6 M NaCl, the cooking loss of

298

the MP emulsion gel treated with 1000 ppm EGCG was 9.46-fold higher compared to the MP

299

emulsion gel without EGCG (Figure 5). Covalent bonds induced by heating have important

300

contributions to the homogeneous, three-dimensional network of the gel, especially disulfide

301

bonds. The formation of thiol−quinone and amine-quinone adducts blocked the formation of

302

covalent bonds, such as disulfide bonds and active carbonyl−NH2 interactions.5 This resulted in a

303

poor protein network, and consequently, higher cooking loss of MP emulsion gel treated with

304

1000 ppm EGCG, especially with 0.6 M NaCl (Figure 5).

305

Gel strength was also measured to evaluate the properties of the MP emulsion gels. Both

306

EGCG and NaCl had significant effects on gel strength of the MP emulsion gel (Table 1). At 0 M

307

NaCl and 0.2 M NaCl, the high dose of EGCG significantly decreased the gel strength (Figure 5).

308

The high dose of EGCG modified the thiol groups and free amines by additive reaction, hence

309

blocking the cross-linking during heat-induced emulsion gel processing. Consequently, the high

310

dose of EGCG decreased the force needed to compress the sample to reach a given deformation

311

(gel strength). Jongberg et al. found that a high dose of green tea extract increased the cooking

312

loss and decreased the firmness/hardness of a meat emulsion gel, indicating that a high dose of

313

EGCG damaged the emulsifying stability of the emulsion gels.11 However, the combination of

314

high EGCG (1000 ppm) and high NaCl (0.6 M) significantly increased the gel strength of the MP

315

emulsion (Figure 5). At high ionic strength (0.6 M NaCl), it is possible that the MP could have a

316

higher degree of modification, resulting in highly denatured MP and high level of aggregation.

317

Consequently, the MP emulsion gel treated with high EGCG and high NaCl was more compact 14 / 35


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due to protein aggregation caused by the high hydrophobic force. Moreover, the molecular size of

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the MP could become larger in the presence of 1000 ppm EGCG at 0.6 M NaCl, which might

320

hinder relative motion between protein molecules. As a result, at 0.6 M NaCl, the MP emulsion

321

gel treated with 1000 ppm EGCG had higher gel strength compared to MP emulsion gel treated

322

with 0 or 100 ppm EGCG (Figure 5). The negative charges on the surface of MP could be

323

neutralized by the positive sodium ions at higher NaCl concentration, which may also cause

324

aggregation of proteins.35 This could also account for the higher gel strength of the MP emulsion

325

gel treated with 1000 ppm EGCG at 0.6 M NaCl compared with that at 0 and 0.2 M NaCl.

326

Dynamic Rheological Properties of MP Emulsion Gel. Dynamic oscillatory analyses are

327

commonly used to assess the viscoelastic characteristics of emulsion gels.36 The storage modulus

328

(G′), which represents the amount of recoverable energy stored in the elastic gel, of the MP

329

emulsion gel was analyzed and plotted as G′ versus temperature (Figure 6). The oxidized sample

330

had significantly higher final G′ compared to the control (Figure 6), suggesting that interactions

331

between MP were promoted by oxidation. 16 The G′ of the MP emulsion gel showed an EGCG

332

dose-dependent decrease at 0 and 0.2 M NaCl, especially in the sample prepared with 1000 ppm

333

EGCG (Figure 6 A & B). It is possible that the high dose of EGCG promoted the formation of

334

thiol-quinone and amino-quinone adducts, which blocked the formation of intermolecular

335

covalent bonds between proteins and prevented formation of ordered, heat-induced,

336

three-dimensional protein network. As a result, G′ was reduced in the MP emulsion gel treated

337

with a high dose of EGCG at a low NaCl concentration. The G′ of the MP emulsion gel treated

338

with a low dose of EGCG (100 ppm) was higher than the control (Figure 6), suggesting that

339

addition of 100 ppm EGCG did not significantly prevent the cross-linking induced by heating 15 / 35



340

under oxidative stress nor the formation of other chemical interactions/bonds between MP and did

341

not inhibit the gelation of MP compared to the control.37 Previous studies have shown that the

342

structure and function of MP can be affected by ionic strength,31 and that high ionic strength (>

343

0.4 M NaCl) can improve the transverse expansion of MP.38 The swollen MP enhanced the

344

protein−protein interactions and the formation of a thicker layer surrounding the lipid droplet to

345

improve the stability of the MP emulsion gel treated with high NaCl concentration.31, 39 It is

346

surprising that the MP treated with a high dose of EGCG (1000 ppm) had significantly higher G′

347

at 0.6 M NaCl (Figure 6 C). At high NaCl concentration (0.6 M NaCl), the MP was in an evenly

348

swollen state, which would aggravate unfolding of protein due to interaction with EGCG. The

349

significantly higher hydrophobic force initiated by the high surface hydrophobicity might cause

350

the shrinking of the MP gel with a compact structure during heating, resulting in higher G′ (Figure

351

6 C).31, 40 Moreover, the molecular size of the swollen MP would become larger due to covalent

352

and non-covalent interactions with high EGCG (1000 ppm), which might increase entanglements

353

between protein chains. This could further improve the G′ of MP emulsion gel. Therefore, at low

354

NaCl concentrations, the EGCG-mediated prevention of covalent interactions between proteins

355

caused a decrease of G' (Figure 6 A & B). On the other hand, at high NaCl concentration,

356

hydrophobic forces induced by unfolding of protein caused by high level of EGCG contributed to

357

the increase of G' (Figure 6 C).

358

Effect of EGCG and NaCl on the Microstructure of MP Emulsion Gel. CLSM imaging was

359

used to reveal distribution of lipid droplets in the MP emulsion gels (Figure 7), especially since

360

cooking loss of emulsion gels is largely dependent upon gel microstructure.31 The protein network

361

is formed by cross-links upon heating, which can bind and embed water and oil droplets.41 Oil 16 / 35


Page 16 of 35

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362

droplets had uniform size and even distribution in the control MP emulsion gel (Figure 7 A). The

363

oxidized MP emulsion gel treated with 0 or 100 ppm EGCG showed distributions of oil droplets

364

similar to control (Figure 7). These images indicated that oxidation (5 mM H2O2) and low level of

365

EGCG did not significantly jeopardize the internal structure of the MP emulsion gels. Stability of

366

MP emulsion gels was evenly matched compared to the control, as evidenced by the similar

367

cooking loss and gel strength values (Figure 5). In the presence of NaCl, the sizes of the oil

368

droplets coated with protein become smaller and more evenly dispersed in the oxidized MP

369

emulsion gel treated with 0 or 100 ppm EGCG, especially at high NaCl concentration (0.6 M)

370

(Figure 7). When the concentration of EGCG was at 1000 ppm and NaCl levels increased, oil was

371

coalesced into larger droplets (Figure 7). The oil droplets in the MP emulsion gels were likely

372

stabilized by an interfacial protein film and the gel matrix that restricted their movement.31 At

373

1000 ppm EGCG, adducts between quinone of EGCG and –SH and –NH2 groups of the amino

374

acid residues might prevent interaction covalent bonds, such as S–S and carbonyl–NH2 covalent

375

bonds, between interfacial proteins and proteins in the gel matrix, resulting in coalescence of and

376

larger emulsion droplets (Figure 4 & 7).

377

The microstructure of the protein matrix was investigated using SEM images as well (Figure 8).

378

The unoxidized MP emulsion gel (control) exhibited a continuous protein network structure

379

(Figure 8). With 0 or 0.2 M NaCl, oxidation contributed to light protein aggregation compared to

380

control (Figure 8). Moreover, the higher dose of EGCG (1000 ppm) resulted in further MP

381

aggregation and the more compact gel structure, which might be caused by the higher

382

hydrophobic forces that come with higher levels of modification. The MP emulsion gel had a

383

more porous three-dimensional network at 0.6 M NaCl concentration compared to MP emulsion 17 / 35



384

gels at low NaCl concentrations in absence of EGCG (Figure 8). However, addition of 1000 ppm

385

EGCG and 0.6 M NaCl led to severe protein aggregation, shrinking the gel matrix (Figure 8). The

386

shrinking of the matrix could lead to the higher cooking loss and gel strength seen in the MP

387

emulsion gel treated with 1000 ppm EGCG and 0.6 M NaCl (Figure 5).

388

Interfacial protein interactions and protein in continuous phase can prevent the coalescence of

389

lipid droplets, by forming a three-dimensional network in meat products.17 Addition of 1000 ppm

390

EGCG significantly increased cooking loss but deteriorated the gel strength of the MP emulsion

391

gel. At low NaCl concentration (≤0.2 M NaCl), high dose of EGCG decreased the gel strength

392

and increased cooking loss (Figure 5). However, at high NaCl concentration (0.6 M NaCl), a high

393

dose of EGCG increased both the gel strength and the cooking loss (Figure 5). Together, these

394

results indicated that addition of high dose of EGCG jeopardized the stability of the MP emulsion

395

gel. A schematic representation of the formation of MP emulsion gels with or without EGCG is

396

shown in Figure 9. The loss of thiol groups and free amines and the appearance of polymers in the

397

reducing SDS-PAGE images indicated that thiol-quinone and amino-quinone adducts were

398

formed. These adducts blocked the formation of covalent bonds between the proteins absorbed on

399

the surface of lipid droplets and the proteins in the continuous phase (non-absorbed proteins),

400

resulting in a poor three-dimensional gel network. Consequently, the cooking loss of the

401

EGCG-treated emulsion gel significantly increased and the texture became softer with

402

significantly lower gel strength (Figure 5). Moreover, a high dose of EGCG promoted further

403

unfolding of the protein compared to the oxidized MP.30 Hydrophobic forces caused protein

404

aggregation and shrunk the emulsion gel, which further increased the cooking loss. Shrinking and

405

aggregation of the emulsion gel was shown by the compact microstructure of the emulsion gel 18 / 35


Page 18 of 35

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406

treated with the high dose of EGCG (Figure 8). However, high NaCl concentration (0.6 M NaCl)

407

resulted in evenly swollen MP, which aggravated unfolding of protein due to interaction with

408

EGCG. The hydrophobic forces might be too high to mask the effect of covalent adducts on the

409

texture of the emulsion gel. Consequently, the emulsion gel treated with the high dose of EGCG

410

had much more compact microstructure (Figure 8) and higher gel strength compared to other

411

samples (Figure 5) at high NaCl concentration (0.6 M NaCl). Other interactions, such as hydrogen

412

bonds or ionic bonds, between protein molecules were not depicted in Figure 9.

413

In conclusion, EGCG can be oxidized and converted to quinine, which can irreversibly react

414

with the thiol and amino moieties in protein under oxidative stress. These irreversible adducts are

415

more prevalent at higher EGCG doses and prevent interfacial proteins and gel matrix proteins

416

from interacting through covalent bonds, such as S–S and carbonyl–NH2 covalent bonds. In

417

addition, the high dose of EGCG induced protein aggregation. Consequently, the MP emulsion gel

418

structure was jeopardized, and its emulsifying properties became unstable, resulting in

419

significantly higher cooking loss and texture deterioration. Notably, high NaCl concentration

420

magnified the modification of MP by the high dose of EGCG, contributing to the poorer quality

421

of the MP emulsion gel. The results of present paper may help the meat processing industry to

422

fully understand how antioxidants such as EGCG interact with NaCl and alter emulsion-type meat

423

products. This understanding can lead to optimized formulations designed to produce high quality

424

muscle-based foods.

425

ACKNOWLEDGMENTS

426

We would like to thank Dr Anita K. Snyder, Donald Danforth Plant Science Center, America

427

for her helpful advices and assistance with the English language. This work was supported by the 19 / 35



428

National Natural Science Fund for Young Scholars (Grant No.: 31601497; 31401515), the China

429

Postdoctoral Science Foundation Project (Grant No.: 2016M591857). All authors read,

430

commented on, and approved the final manuscript.

431

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432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467

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Lund, M. N.; Heinonen, M.; Baron, C. P.; Estevez, M., Protein oxidation in muscle foods: A review. Mol. Nutr.

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Li, C.; Xiong, Y. L.; Chen, J., Oxidation-induced unfolding facilitates myosin cross-linking in myofibrillar protein by

microbial transglutaminase. J. Agr. Food Chem. 2012, 60 (32), 8020-8027. 4.

Chen, L.; Hackman, R. M.; Li, C.; Xu, X.; Zhou, G.; Feng, X., Different Physicochemical, Structural and Digestibility

Characteristics of Myofibrillar Protein from PSE and Normal Pork before and after Oxidation. Meat Sci. 2016, 121, 228-237. 5.

Feng, X.; Li, C.; Ullah, N.; Hackman, R. M.; Chen, L.; Zhou, G., Potential Biomarker of Myofibrillar Protein

Oxidation in Raw and Cooked Ham: 3-Nitrotyrosine Formed by Nitrosation. J. Agr. Food Chem. 2015, 63 (51), 10957-10964. 6.

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Karre, L.; Lopez, K.; Getty, K. J. K., Natural antioxidants in meat and poultry products. Meat Sci. 2013, 94 (2),

220-7. 10. Utrera, M.; Morcuende, D.; Rui, G.; Estévez, M., Role of Phenolics Extracting from Rosa canina L. on Meat Protein Oxidation During Frozen Storage and Beef Patties Processing. Food Bioprocess Technol. 2015, 8 (4), 854-864. 11. Jongberg, S.; Terkelsen, L. d. S.; Miklos, R.; Lund, M. N., Green tea extract impairs meat emulsion properties by disturbing protein disulfide cross-linking. Meat Sci. 2015, 100, 2-9. 12. Jongberg, S.; Skov, S. H.; Tørngren, M. A.; Skibsted, L. H.; Lund, M. N., Effect of white grape extract and modified atmosphere packaging on lipid and protein oxidation in chill stored beef patties. Food Chem. 2011, 128 (2), 276-283. 13. Fujimoto, A.; Masuda, T., Chemical Interaction between Polyphenols and a Cysteinyl Thiol under Radical Oxidation Conditions. J. Agr. Food Chem. 2012, 60 (20), 5142-5151. 14. Jongberg, S.; Lund, M. N.; Waterhouse, A. L.; Skibsted, L. H., 4-methylcatechol inhibits protein oxidation in meat but not disulfide formation. J. Agr. Food Chem. 2011, 59 (18), 10329-35. 15. Tang, C. B.; Zhang, W. G.; Dai, C.; Li, H. X.; Xu, X. L.; Zhou, G. H., Identification and Quantification of Adducts between Oxidized Rosmarinic Acid and Thiol Compounds by UHPLC-LTQ-Orbitrap and MALDI-TOF/TOF Tandem Mass Spectrometry. J. Agr. Food Chem. 2014, 63 (3), 902-911. 16. Cao, Y.; Xiong, Y. L., Chlorogenic acid-mediated gel formation of oxidatively stressed myofibrillar protein. Food Chem. 2015, 180, 235-243. 17. Wu, M.; Xiong, Y. L.; Chen, J., Role Of Disulphide Linkages Between Protein-Coated Lipid Droplets And The Protein Matrix In The Rheological Properties Of Porcine Myofibrillar Protein–Peanut Oil Emulsion Composite Gels. 20 / 35


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Meat Sci. 2011, 88 (3), 384–390. 18. Graham, H. N., Green tea composition, consumption, and polyphenol chemistry. Prev. Med. 1992, 21 (3), 334-50. 19. Chan, E. W. C.; Lim, Y. Y.; Chew, Y. L., Antioxidant activity of Camellia sinensis leaves and tea from a lowland plantation in Malaysia. Food Chem. 2007, 102 (4), 1214-1222. 20. Bose, M.; Lambert, J. D.; Ju, J.; Reuhl, K. R.; Shapses, S. A.; Yang, C. S., The major green tea polyphenol,(-)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat–fed mice. J. Nutr. 2008, 138 (9), 1677-1683. 21. Jiang, J.; Xiong, Y. L., Role of interfacial protein membrane in oxidative stability of vegetable oil substitution emulsions applicable to nutritionally modified sausage. Meat Sci. 2015, 109, 56-65. 22. Feng, X.; Li, C.; Jia, X.; Guo, Y.; Lei, N.; Hackman, R. M.; Chen, L.; Zhou, G., Influence of sodium nitrite on protein oxidation and nitrosation of sausages subjected to processing and storage. Meat Sci. 2016, (116), 260-267. 23. Gornall, A. G.; Bardawill, C. J.; David, M. M., Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 1949, 177 (2), 751-766. 24. Oliver, C. N.; Ahn, B.-W.; Moerman, E. J.; Goldstein, S.; Stadtman, E. R., Age-related changes in oxidized proteins. J. Biol. Chem. 1987, 262 (12), 5488-5491. 25. Liu, G.; Xiong, Y.; Butterfield, D., Chemical, Physical, and Gel‐forming Properties of Oxidized Myofibrils and Whey- and Soy-protein Isolates. J. Food Sci. 2000, 65 (5), 811-818. 26. Ellman, G. L., Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82 (1), 70-77. 27. Martinaud, A.; Mercier, Y.; Marinova, P.; Tassy, C.; Gatellier, P.; Renerre, M., Comparison of oxidative processes on myofibrillar proteins from beef during maturation and by different model oxidation systems. J. Agr. Food Chem. 1997, 45 (7), 2481-2487. 28. Chen, L.; Feng, X. C.; Zhang, Y. Y.; Liu, X. B.; Zhang, W. G.; Li, C. B.; Ullah, N.; Xu, X. L.; Zhou, G. H., Effects of ultrasonic processing on caspase-3, calpain expression and myofibrillar structure of chicken during post-mortem ageing. Food Chem. 2015, 17 (7), 280-287. 29. Martinez-Alvarenga, M.; Martinez-Rodriguez, E.; Garcia-Amezquita, L.; Olivas, G.; Zamudio-Flores, P.; Acosta-Muniz, C.; Sepulveda, D., Effect of Maillard reaction conditions on the degree of glycation and functional properties of whey protein isolate–Maltodextrin conjugates. Food Hydrocolloid. 2014, 38, 110-118. 30. Cao, Y.; Xiong, Y. L., Chlorogenic acid-mediated gel formation of oxidatively stressed myofibrillar protein. Food Chem. 2015, 180, 235-243. 31. Zhou, F.; Sun, W.; Zhao, M., Controlled Formation of Emulsion Gels Stabilized by Salted Myofibrillar Protein under Malondialdehyde (MDA)-Induced Oxidative Stress. J. Agr. Food Chem.2015, 63 (14), 3766–3777. 32. Sun, J.; Wu, Z.; Xu, X.; Li, P., Effect of peanut protein isolate on functional properties of chicken salt-soluble proteins from breast and thigh muscles during heat-induced gelation. Meat Sci. 2012, 91 (1), 88-92. 33. Li, C.; Xiong, Y. L.; Chen, J., Protein Oxidation at Different Salt Concentrations Affects the Cross‐Linking and Gelation of Pork Myofibrillar Protein Catalyzed by Microbial Transglutaminase. J. Food Sci. 2013, 78 (6), C823-C831. 34. Strauss, G.; Gibson, S. M., Plant phenolics as cross-linkers of gelatin gels and gelatin-based coacervates for use as food ingredients. Food Hydrocolloid. 2004, 18 (1), 81-89. 35. Wu, J.; Shi, M.; Li, W.; Zhao, L.; Wang, Z.; Yan, X.; Norde, W.; Li, Y., Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking. Colloid. Surface. B 2015, 127, 96-104. 36. Jimenez-Colmenero, F.; Cofrades, S.; Herrero, A. M.; Solas, M. T.; Ruiz-Capillas, C., Konjac gel for use as potential fat analogue for healthier meat product development: Effect of chilled and frozen storage. Food Hydrocolloid. 2013, 30 (1), 351-357. 37. Wang, H.; Pato, M.; Pietrasik, Z.; Shand, P., Biochemical and physicochemical properties of thermally treated 21 / 35



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natural actomyosin extracted from normal and PSE pork Longissimus muscle. Food Chem. 2009, 113 (1), 21-27. 38. Offer, G.; Trinick, J., On the mechanism of water holding in meat: The swelling and shrinking of myofibrils. Meat Sci. 1983, 8 (8), 245-81. 39. Li, C.; Xiong, Y. L.; Chen, J., Protein Oxidation at Different Salt Concentrations Affects the Cross-Linking and Gelation of Pork Myofibrillar Protein Catalyzed by Microbial Transglutaminase. J. Food Sci. 2013, 78 (6), C823–C831. 40. Moreno, H. M.; Bargiela, V.; Tovar, C. A.; Cando, D.; Borderias, A. J.; Herranz, B., High pressure applied to frozen flying fish ( Parexocoetus brachyterus ) surimi: Effect on physicochemical and rheological properties of gels. Food Hydrocolloid. 2015, 48 (4), 127-134. 41. Gibis, M.; Schuh, V.; Weiss, J., Effects of carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) as fat replacers on the microstructure and sensory characteristics of fried beef patties. Food Hydrocolloid. 2015, 45, 236-246.

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523

Figure and Table Captions:

524

Figure 1. Effect of EGCG on the physicochemical and structural characteristics of MP at

525

different NaCl concentrations. EGCG concentrations were 0, 100 and 1000 ppm. Salt

526

concentrations were 0, 0.2 and 0.6 M. Control represents unoxidized MP. Values are mean ± SD.

527

Different lowercase letters indicate significant differences (P < 0.05).

528

Figure 2. Representative DSC thermal curves of MP treated with EGCG at different NaCl

529

concentrations. EGCG concentrations were 0, 100 and 1000 ppm. Salt concentrations were 0, 0.2

530

and 0.6 M. Control represents unoxidized MP.

531

Figure 3. Images after SDS–PAGE of pork Myofibrillar Protein (MP) treated with EGCG and/or

532

NaCl. Native (A, - βME) and reducing (B, + βME) SDS-PAGE conditions. EGCG concentrations

533

were 0, 100 and 1000 ppm. Salt concentrations were 0, 0.2 and 0.6 M. Control represents

534

unoxidized MP.

535

Figure 4. Proposed reactions of EGCG quinone derivatives with MP 8, 15.

536

Figure 5. Effect of EGCG addition on cooking loss (A) and gel strength (B) of MP emulsion gels

537

at different NaCl concentrations. EGCG concentrations were 0, 100 and 1000 ppm. Salt

538

concentrations were 0, 0.2 and 0.6 M. Control represents unoxidized MP. Values are mean ± SD.

539

Different lowercase letters indicate significant differences (P < 0.05).

540

Figure 6. Storage modulus (G′) of MP emulsion gels prepared with A, 0 M NaCl; B, 0.2 M NaCl;

541

C, 0.6 M NaCl and different dosages of EGCG (0, 100 or 1000 ppm). Control represents

542

unoxidized MP.

543

Figure 7. Representative CLSM images of MP emulsion gels prepared with EGCG (0, 100 and

544

1000 ppm) and NaCl (0, 0.2, and 0.6 M). Control represents unoxidized MP. 23 / 35



545

Figure 8. SEM images of the microstructure of MP emulsion gels prepared with EGCG (0, 100

546

and 1000 ppm) and NaCl (0, 0.2, and 0.6 M). Control represents unoxidized MP.

547

Figure 9. Schematic representation of the proposed interactions of oil droplets and MP in

548

emulsion gels prepared with EGCG and different NaCl concentrations, emphasizing the

549

importance of protein disulfides and hydrophobic forces for texture.

550

Table 1. Significance values for the main effects of EGCG, NaCl, and their interaction on protein

551

chemical and structural markers, cooking loss and gel strength in MP Emulsion Gels.

552

24 / 35


Page 24 of 35

Page 25 of 35


90

f

A

Free amine (nM/mg protein)

Carbonyl (nM/mg protein)

2.0

e 1.5

b cg

1.0

bc

g d

d

h a

0.5

0.0

B

a

80 b

b

70

d

60

c

c 50

c

c

e

40 30

f

20 10 0

0MNaCl

0.2MNaCl

Control

0ppm

0.6MNaCl

100ppm

0MNaCl

1000ppm

0.2MNaCl

Control

0ppm

0.6MNaCl

100ppm

1000ppm

553

50

1500

C

a

d b

b

D b

Surface hydrophobicity

Thiol groups (nM/mg protein)

60

b

b

f

40 30 c e

20

g

1000

ad

d ae

e

e

500

c

c

g

10 0

0 0MNaCl Control

0.2MNaCl 0ppm

100ppm

0.6MNaCl

0MNaCl

1000ppm

Control

554 555

b

Figure 1 25 / 35


0.2MNaCl 0ppm

100ppm

0.6MNaCl 1000ppm


556 557

Figure 2 26 / 35


Page 26 of 35

Page 27 of 35


558

559 560

Figure 3

27 / 35



561 562

Figure 4

563 564 565 566 567 568 569 570 571 572 573 574

28 / 35


Page 28 of 35

Page 29 of 35


A

2.16 folds

1.88 folds 50%

9.46 folds

d

Cooking loss (%)

cd

c

40%

30%

b a

b

a

a

20%

10% e

f

0% 0M NaCl

0.2M NaCl

Control

575

0ppm

100ppm

0.6M NaCl 1000ppm

0.7

B

c

0.6

Gel strength (N)

a 0.5

a

a

a

a

a

a

0.4 b

b

0.3 0.2 0.1 0.0 0M NaCl Control

0.2M NaCl 0ppm

100ppm

0.6M NaCl 1000ppm

576 577

Figure 5

29 / 35



578

579 580

Figure 6 30 / 35


Page 30 of 35

Page 31 of 35


581 582

Figure 7 31 / 35



583 584

Figure 8

32 / 35


Page 32 of 35

Page 33 of 35


585 586

Figure 9

33 / 35



587

Table 1 carbonyls thiol groups free amines Surface Hydrophobicity Cooking loss Gel strength EGCG

Emulsifying Properties of Oxidatively Stressed Myofibrillar Protein

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