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The Dual Role (Anti- and Pro-oxidant) of Gallic Acid in Mediating Myofibrillar Protein Gelation and Gel in Vitro Digestion Yungang Cao, Alma D True, Jie Chen, and Youling L. Xiong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00314 • Publication Date (Web): 22 Mar 2016 Downloaded from http://pubs.acs.org on March 23, 2016

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Journal of Agricultural and Food Chemistry

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The Dual Role (Anti- and Pro-oxidant) of Gallic Acid in Mediating Myofibrillar Protein

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Gelation and Gel in Vitro Digestion

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Yungang Cao,† Alma D. True, ‡ Jie Chen, † and Youling L. Xiong *,†,‡

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†

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Safety and Nutrition, and School of Food Science and Technology, Jiangnan University, Wuxi

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214122, People’s Republic of China

State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food

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‡

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United States

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

13 14 15 16 17 18 19

(Submitted to: Journal of Agricultural and Food Chemistry)

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1

ACS Paragon Plus Environment


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ABSTRACT

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The dose-dependent effects of gallic acid (GA, at 0, 6, 30 and 150 µmol/g protein) on

24

chemical changes and gelling properties of oxidatively-stressed porcine myofibrillar protein (MP)

25

and in vitro digestibility of the gels were investigated. The incorporation of GA suppressed lipid

26

oxidation and protein carbonyl formation but promoted the loss of thiol and amine groups,

27

destabilization of the tertiary structure, aggregation, and crosslinking. The gelling potential

28

(storage modulus) of MP was increased by nearly 50% with 6 and 30 µmol/g of GA,

29

corresponding

30

disulfide-dominant covalent bonds. However, GA at 150 µmol/g induced macroscopic

31

aggregations and insolubility of MP, resulting in poorly structured gels. Despite the oxidative

32

changes, MP gels did not show reduced susceptibility to digestive enzymes in vitro.

to

enhanced

protein

unfolding

and

aggregation

and

formation

of

33 34

KEYWORDS: gelation; digestibility; myofibrillar protein; covalent cross-linking; phenolic

35

antioxidants; gallic acid.

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INTRODUCTION

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Oxidation of lipids and proteins occurring during meat processing and storage has a major

40

impact on the functional, nutritional, and sensorial properties of meat products.1–3 Synthetic

41

antioxidants have traditionally been used to prevent oxidation; however, their potential role in

42

carcinogenicity has raised increasing concerns for consumer safety, leading to the current trend

43

of adopting natural antioxidants as an alternative meat quality control mechanism.4,5 Of various

44

natural antioxidants, plant-derived phenolic compounds are the most widely used.

45

Phenolic derivatives are abundantly found in spices, herbs, fruits, and vegetables; they also

46

exist in agri-industrial by-products, such as potato peels, fruit peels, and fruit pomaces.6 As

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natural antioxidants, phenolic compounds have been associated with the mitigation of a variety

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radical-mediated health issues due to their broad physiological activities identified in both in

49

vitro and in vivo tests, for example, antimicrobial, anti-inflammatory, anti-allergenic,

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anti-artherogenic, and anti-thrombotic properties. 6–9

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Plant extracts abundant in polyphenols have been widely incorporated into meat product

52

formulations to inhibit oxidative processes and extend products’ shelf-life.5 This antioxidant

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strategy has been shown to be effective for retarding lipid oxidation but not always for protein

54

oxidation. In fact, accelerated protein carbonylation and sulfhydryl loss caused by phenolic acids

55

have been observed in some cases. For example, green tea and rosemary extract were shown to

56

inhibit the formation of secondary lipid oxidation products and protein carbonyls in Bologna

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type sausages prepared from oxidatively stressed pork.10 However, green tea extract increased

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loss of thiol groups, myosin, and actin. Addition of polyphenol-rich Willowherb extract into beef

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patties resulted in reduced lipid oxidation but accelerated protein carbonylation.11 Rosemary and

3



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oregano essential oils prevented thiol loss, while garlic essential oil promoted thiol loss as well

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as myosin cross-linking in pork patties during chill storage.12

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In proteinaceous foods, plant phenolics can interact with proteins through both noncovalent

63

and covalent bonds to modify protein functional groups, structure stability, aggregation, and

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solubility, leading to functionality changes, such as gelation, which is the most important

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texture-forming property in processed muscle foods.13–16 However, existing reports are

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inconsistent and the underlying mechanisms have not been fully elucidated. Balange and

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Benjakul17 claimed that oxidized phenolic compounds induced protein cross-linking through

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amino groups or the induction of disulfide bond formation resulting in improved gel strength of

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bigeye snapper (Priacanthus tayenus) surimi. Jongberg et al.18 reported that covalent thiol–

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quinone adducts impaired the gel-forming potential of meat proteins by disturbing protein

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disulfide cross-linking. Despite these previous studies, there is very limited information on the

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potential influence of protein–polyphenol interactions on digestibility of protein. Also, there is no

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literature report on in vitro digestion behavior of muscle protein gels formed in the presence of

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phenolic compounds, although this type of composite protein gels with spice extract is a natural

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phenomenon in comminuted meat products.

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The objective of this study was to investigate dose-dependent effects of gallic acid (GA), a

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water-soluble phenolic antioxidant abundantly present in spices, herbs, and fruit extracts

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commonly used in meat processing, on the chemical and structural stability of myofibrillar

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protein (MP) when exposed to a radical-producing environment. Rheological properties of the

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protein sol during thermal gelation and the in vitro digestibility of the formed gels were

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subsequently investigated.

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MATERIALS AND METHODS

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Materials. Longissimus muscle was collected from pork carcasses (24 h postmortem)

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harvested at the University of Kentucky Meat Laboratory. Individual muscle samples (~100 g)

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were vacuum-packaged and stored in a −30 °C freezer until use. Gallic acid (97.5–102.5%,

87

titration) and porcine pepsin and pancreatin (8 × USP, United States Pharmacopeia specifications)

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were purchased from Sigma–Aldrich (St. Louis, MO). All other chemicals, at least analytical

89

grade, were acquired from Sigma–Aldrich or Thermo–Fisher Scientific (Altham, MA). Double

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deionized water (NANO pure Diamond, Barnstead, IA) was prepared in the lab and used

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throughout the study.

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Extraction of MP. Random frozen muscle samples were taken out from −30 °C freezer and

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tempered at 4 °C for 4 h. After chopping into small pieces they were used for MP extraction

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using an isolation buffer (10 mM sodium phosphate, 0.1 M NaCl, 2 mM MgCl, and 1 mM EGTA,

95

pH 7.0). 16 In the last washing step, MP was suspended in 0.1 M NaCl and the pH was adjusted to

96

6.25 using 1 M HCl before centrifugation (2000g for 15 min). The whole MP preparation was

97

conducted in a walk-in cooler (~4 °C). After the measurement of protein concentration by the

98

Biuret method,19 the MP pellet was stored on ice and used within 48 h.

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Oxidative Treatments with Gallic Acid (GA). MP suspensions (20 or 40 mg/mL, final

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protein concentration) were prepared by thorough dispersion with gently stirring of the MP pellet

101

into 15 mM piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) buffer containing 0.6 M NaCl,

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pH 6.25. GA at four final concentrations (0, 6, 30, and 150 µmol/g protein) was added to the

103

protein suspension through gently stirring. Samples were oxidatively stressed using a Fenton

104

system (10 µM FeCl3, 100 µM ascorbic acid, and 1 mM H2O2) by incubation at 4 °C for 12 h.

105

Oxidation was terminated by adding Trolox (1 mM). The non-oxidized, GA-free MP suspension

5



106

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was used as the control.

107

Determination of Lipid Oxidation. The oxidation of residual lipids in the MP isolate

108

(mostly membrane phospholipids, ~0.49% fat dry basis)20 was evaluated by thiobarbituric

109

acid-reactive substances (TBARS) according to Salih et al. 21 After MP samples were mixed with

110

TBA and TCA solutions, the reaction was initiated by heating at 95 °C and allowed to continue

111

for 30 min. The absorbance of the supernatant at 532 nm was read against a reagent blank, and

112

results were expressed as mg malonaldehyde (MDA) equivalent/kg protein.

113

Determination of Chemical and Structural Changes of MP. Oxidation-induced chemical

114

and structural changes in MP were analyzed by measurements of carbonyls, sulfhydryls, free

115

amines, intrinsic tryptophan fluorescence, and thermal stability. To eliminate the potential

116

influence of free GA on specific analyses (sulfhydryls and free amines), all treated samples were

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washed three times with pre-chilled deionized water then re-dissolved in 15 mM PIPES buffer

118

containing 0.6 M NaCl (pH 6.25).

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Carbonyls. Sample carbonyl content was determined using the 2,4-dinitrophenylhydrazine

120

(DNPH) colorimetric method of Levine et al. 22 Briefly, after reacting with DNPH under acidic

121

conditions, MP samples were precipitated using 20% TCA then recovered by centrifugation. The

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precipitated MP pellets were washed three times with ethanol/ethyl acetate (1:1, v/v) solution to

123

exhaustively remove unreacted DNPH, and then dissolved in 6 M guanidine hydrochloride (pH

124

2.3). The absorbance at 370 nm was read for carbonyl content and that at 280 nm was recorded

125

simultaneously for protein content through a standard curve of BSA. Carbonyl content was

126

calculated using a molar extinction coefficient of 22000 M−1cm−1.

127

Total Sulfhydryls. The total sulfhydryl content of the individual MP samples was determined

128

using the 5,5’-dithio-bis (2-nitrobenzoic acid) (DTNB) method.23 Briefly, MP samples were

6


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dissolved in a urea–SDS solution (8.0 M urea, 3% SDS, 0.1 M phosphate, pH 7.4) and incubated

130

with DTNB reagent at 25 °C for 15 min. The absorbance at 412 nm was read. Reagent blank and

131

sample blanks were run simultaneously. A molar extinction coefficient of 13600 M−1cm−1 was

132

used for sulfhydryl content calculation.

133

Free Amines. Free amines were determined according to Habeeb.24 Briefly, MP samples

134

were dissolved in 0.21 M sodium phosphate buffer (pH 8.2) containing 1% sodium dodecyl

135

sulfate (SDS) and then reacted with 2,4,6-trinitrobenzenesulfonic acid (TNBS) at 50 °C for 30

136

min in the dark. The absorbance at 420 nm was read, and a standard curve produced with

137

L-leucine (in 1% SDS) was used for free amine content calculation.

138

Intrinsic Tryptophan Fluorescence. This was determined using a FluoroMax-3

139

spectrofluorometer (Horiba Jobin Yvon Inc., Edison, NJ). MP suspensions (0.4 mg/mL in 15

140

mM PIPES buffer, 0.6 M NaCl, pH 6.25) were excited at 283 nm, and the emission spectra from

141

300 to 400 nm were recorded. The slit widths of both excitation and emission were set at 10 nm,

142

and the data were collected at a 500 nm/min rate. Fluorescence quenching induced by GA

143

binding under oxidizing conditions was evaluated according to the Sterne–Volmer equation: ⁄ = 1 + Q = 1 +

144

where and are the fluorescence intensities of MP in the absence and presence of GA,

145

respectively; is the bimolecular quenching rate constant; is the fluorescence lifetime in the

146

absence of a quencher (the value of for biopolymers is 10-8s-1);25 and is the quencher

147

concentration. is the Stern–Volmer quenching constant obtained by performing a linear

148

regression of a plot of ⁄ versus .

149

Differential Scanning Calorimetry. The conformational stability of MP samples was

150

measured using a Model 2920 differential scanning calorimeter (TA Instruments, Inc., New 7



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Castle, DE). MP samples (40 mg/mL, 16−18 mg) were hermetically sealed in aluminum pans

152

and subjected to thermal scan from 20 to 100 °C at a 5 °C/min rate. The temperature maximum

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(Tmax) for transition was measured using the Universal analysis software (Version 1.2 N)

154

supplied by the TA company.

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Detection of Protein Cross-linking. Sodium dodecyl sulfate–polyacrylamide gel

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electrophoresis (SDS–PAGE) was applied to examine protein patterns within the MP sols

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(unheated) and formed gels (heated) according to Laemmli26 with a 4% polyacrylamide stacking

158

gel and a 12% polyacrylamide resolving gel. To dissolve MP gels, 3 g crushed gels were

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homogenized in 27 mL of 5% SDS solution and heated at 80 °C for 1 h.27 The heated solutions

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were centrifuged at 3000g for 15 min and the supernatants were diluted to 2 mg/mL for SDS–

161

PAGE. Each sample well was loaded 30 µg protien.

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Evaluation of Gelation Properties. The gelling properties of MP samples were analyzed

163

with a small-strain dynamic rheological test (non-disruptive) and a large-strain extrusion test

164

(disruptive). Other properties of the heat-induced gels were evaluated at the same time.

165

Dynamic Rheological Measurement. MP sols (20 mg/mL in 15 mM PIPES, 0.6 M NaCl, pH

166

6.25) were deaerated by centrifuging at 1000g for 1 min then subjected to dynamic rheological

167

testing using a Model CVO rheometer (Malvern Instruments, Westborough, MA). Thermal

168

gelation was achieved by heating sols between the parallel plates (upper plate dia. 30 mm and

169

lower plate dia. 50 mm; gap 1 mm) from 20 to 72 °C at 1 °C/min (this final temperature was

170

chosen because it is a typical end cooking temperature for commercial gelling meat products

171

such as frankfurters). During heating, the force registered from shearing the sols in an oscillatory

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mode at a fixed frequency of 0.1 Hz and a maximum strain of 0.02 was recorded every 30 s. The

173

viscoelastic behavior of MP samples during the sol→gel transformation was described in terms 8


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174


of storage modulus (G′) and Tan δ values (the ratio of G′′/G′) where G′′ is loss modulus.

175

Extrusion Testing of Set Gels. Aliquots of MP sols (5 g, 40 mg/mL in 15 mM PIPES) were

176

deaerated by centrifuging at 1000g for 3 min then transferred into 16.5 mm (inner dia.) × 50 mm

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(length)

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temperature-programmable water bath (Boekel Scientific, Feasterville, PA) from 20 to 72 °C at a

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0.9 °C/min heating rate. After cooking, the heat-induced gels were immediately chilled in an ice

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slurry for 30 min then kept at 4 °C overnight to set. Prior to textural evaluation, gel samples were

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allowed to equilibrate at room temperature for 2 h. Gels in the glass vial were extruded with a

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flat-faced stainless steel probe (dia. 12.5 mm) attached to a Model 4301 Instron machine (Canton,

183

MA) at a crosshead speed of 50 mm/min. The penetration force, defined as the initial force (N)

184

required to disrupt the gel, was expressed as the gel strength.16

glass

vials,

loosely

covered

with

plastic

caps,

then

heated

in

a

185

Cooking Yield. Set gels were poured out from the glass vials, gently blotted then weighed.

186

Cooking yield was calculated using the following formula where and are,

187

respectively, the weight of the gel and the weight of the original sol. Cooking yield (%) =

× 100

188

Assessment of In Vitro Digestion. Protein digestibility of MP gels was determined to

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investigate the possible influence of GA treatment according to Ma and Xiong28 and

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Jongjareonrak et al.29 with some modifications. MP gel samples (mixture of gel and expelled

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liquid due to heating, if any, i.e., 5.0 g, 4% protein) were homogenized in 37 mL 10 mM HCl

192

solution, and 8 mL of pepsin solution (1 mg/mL in 10 mM HCl ) was then added. The mixed

193

solution (final protein concentration 4 mg/mL, pepsin 4% w/w protein basis, pH 2.0) was

194

incubated at 37 °C for 1 h for digestion under the gastric condition. This was followed by the pH

195

adjustment to 7.5 with 1 M NaOH to inactivate pepsin and an immediate addition of pancreatin 9



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(4% w/w protein basis) while maintaining the solution at 37 °C to initiate duodenal digestion.

197

Small amounts of samples were taken at 30, 60, 90, 120 and 180 min during the pepsin–

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pancreatin digestion, and the digestion was ultimately terminated by the addition of an equal

199

volume of 30% TCA solution. After chilling to 4 °C, the digests were centrifuged at 10,000 g for

200

10 min. The resultant precipitate was dissolved in 1 mL of 1 M NaOH and the protein

201

concentration was measured using the biuret method. In vitro protein digestibility of the gel

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samples was calculated using the following formula, where '( and ') represent, respectively,

203

total and TCA-precipitated protein concentrations.

204

Digestibility (%) =

'( − ') × 100 '(

205

Furthermore, Tricine–SDS–PAGE was carried out to analyze the peptides of the

206

pepsin-pancreatin digests using 16% polyacrylamide for separating gel and 4% polyacrylamide

207

for stacking gel. Enzymes in digested MP samples were inactivated by boiling for 5 min then

208

cooling to room temperature. Additional sample preparations for electrophoresis and other

209

details were described by Schägger.30 Aliquots of 35 µL of each sample (2 mg/mL protein) were

210

loaded into each sample well.

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Statistical Analysis. Data obtained from three independent trials (n = 3) each employing a

212

new set of MP preparation were submitted to the analysis of variance using the general linear

213

model’s procedure of Statistix software 9.0 (Analytical Software, Tallahassee, FL). Significant

214

(P < 0.05) differences between means were identified by the least significance difference (LSD)

215

all–pairwise multiple comparisons.

216 217

RESULTS AND DISCUSSION 10


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Lipid Oxidation. Muscular fat plays an important role in meat product flavor and human

219

nutrition. However, unsaturated fatty acids in lipids are susceptible to oxidative stressors during

220

meat processing, and hydroxyl radical (●OH) has been recognized as a primary initiator of lipid

221

oxidation in muscle foods.31 Even for extremely lean meat products where visible fat tissues are

222

trimmed, membrane-derived phospholipids can still generate various secondary products via

223

lipid peroxidation, including both volatile (hexanal, pentanal, etc.) and non-volatile

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(4-hydroxy-2-nonenal, MDA, etc.) components.32 The TBARS method is broadly adapted to the

225

assessment of oxidative status of muscle foods although it is relatively non-specific and does not

226

measure volatile compounds. As reported previously,20 MP extracted from lean porcine muscle

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tissue under non-denaturing conditions would always contain residual phospholipids (0.49%, dry

228

basis). This is also true for fish surimi, a fat-depleted protein concentrate used to make

229

crustacean seafood analogs, such as crab meat.33 These residual lipids could be oxidized to

230

generate TBA-reactive secondary oxidation products, such as MDA.

231

As presented in Figure 1, the concentration of TBARS in control (NonOx) MP was about

232

0.70 mg/kg protein, which was produced from meat storage and the protein isolation process.

233

The TBARS content increased drastically (P < 0.05) to 3.07 mg/kg when the MP samples were

234

exposed to the

235

●

236

increase over that of the non-oxidized control (P > 0.05). This inhibitory effect was not GA

237

dose-dependent. The result was in agreement with many previous findings that plant extracts rich

238

in phenolic compounds were capable of curtailing lipid oxidation, for example, in cooked beef

239

treated with oleoresin rosemary, grape seed, and pine bark extract,34 and in refrigerated and

240

frozen precooked pork patties containing licorice extract or rosemary extract.35

●

OH-generating system. The addition of GA significantly inhibited the

OH-induced lipid oxidation as the TBARS values of GA-added samples showed no apparent

11



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241

Modification of Amino Acid Residue Side-chain Groups. Carbonyls. The formation of

242

carbonyls (aldehydes and ketones) is one of the primary consequences of protein oxidation and

243

has been widely applied to the assessment of protein oxidative modification in muscle foods.1–3

244

As shown in Figure 1, the carbonyl content of non-oxidized MP was 1.21 nmol/mg protein

245

which was close to that reported previously for freshly prepared MP.16 However, exposures of

246

MP to ●OH resulted in a remarkable rise in the carbonyl content (P < 0.05), showing a net

247

increase of 2.02 nmol/mg over the control MP sample. The presence of GA significantly

248

inhibited the carbonyl formation, especially at high dosage levels, lowering the carbonyl content

249

by as much as 55.4% when compared with oxidized MP. It is understood that protein-bound

250

carbonyl groups can be generated via several pathways, including direct oxidation of amino acid

251

residues, fragmentation of the peptide backbone, and adduction of carbonyl compounds

252

generated from lipid oxidation, such as HNE and MDA, via lysine, histidine, and cysteine

253

residues.1,36 Because in the presence of ●OH, GA is oxidized into a quinone derivative, it may

254

contribute to the total protein carbonyl content while inhibiting other carbonylation processes as

255

a metal ion chelator and radical chain reaction breaker. The apparent correlation between the

256

TBARS content and that of protein carbonyls would suggest that in the MP system a significant

257

amount of the latter was derived from the secondary products of lipid oxidation. The effects of

258

polyphenols on protein carbonyl formation have been investigated by other researchers; however,

259

contradictory results were reported,10,11 which may be attributed to different polyphenols and

260

oxidation conditions implemented.

261

Total Sulfhydryls. MP is rich in SH groups that can be readily converted to disulfide bond

262

(S−S) upon oxidative stress. In the present study, the total SH content of non-oxidized MP was

263

68.3 nmol/mg protein (Figure 2). Approximately 13.5% (P < 0.05) was lost when MP samples

12


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264

were exposed to ●OH. The addition of 6 µmol/g GA effected no protection, and at 30 and 150

265

µmol/g, GA in fact slightly promoted the SH loss. The binding of oxidized GA (quinone) with

266

thiol groups in MP is believed to be responsible for the further SH loss, which was also reported

267

in mixed MP and artificially generated quinone solutions.37 This viewpoint was confirmed in

268

other studies.14,37,38

269

Free Amines. The ε–NH2 groups of lysine residues in MP are readily accessible by radicals

270

and are converted to carbonyls, which may subsequently react with available NH2 groups.22 As

271

presented in Figure 2, the free amine content of oxidized MP declined by 12.3% from

272

non-oxidized MP (P < 0.05). Modification of amino groups by oxidants and Schiff’s base

273

adduction of oxidation-generated carbonyls and ε–NH2 were the primary causes for loss of free

274

amines.22 The addition of GA did not prevent the free amine loss; on the contrary, samples

275

treated with GA displayed an additional decline of free amine content, and the amount in the MP

276

sample treated with 150 µmol/g GA was 17.2% less than that of oxidized MP without GA (P

Dual Role (Anti- and Pro-oxidant) of Gallic Acid in ... - ACS Publications

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