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Mar 22, 2016 - ABSTRACT: The dose-dependent effects of gallic acid (GA; at 0, 6, 30, and 150 μmol/g protein) on chemical changes and gelling properti...
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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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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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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United States

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

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(Submitted to: Journal of Agricultural and Food Chemistry)

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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

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

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

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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

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

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corresponding

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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

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

35

antioxidants; gallic acid.

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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

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

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

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

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

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

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

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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

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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

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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

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

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

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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

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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

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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%,

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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

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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,

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

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

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

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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

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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

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

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

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

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was used as the control.

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

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

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

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

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

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

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

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

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

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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

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containing 0.6 M NaCl (pH 6.25).

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

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

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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

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

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

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

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

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

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

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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

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

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

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used for sulfhydryl content calculation.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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regression of a plot of  ⁄ versus  .

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Differential Scanning Calorimetry. The conformational stability of MP samples was

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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

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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)

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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

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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–

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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

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

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

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

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

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

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

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

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

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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

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

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Journal of Agricultural and Food Chemistry

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

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

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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,

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

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

glass

vials,

loosely

covered

with

plastic

caps,

then

heated

in

a

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

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

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

 × 100 

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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

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

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

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

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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.

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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

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

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

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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,

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total and TCA-precipitated protein concentrations.

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Digestibility (%) =

'( − ') × 100 '(

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

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

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

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

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

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loaded into each sample well.

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

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

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

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

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all–pairwise multiple comparisons.

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RESULTS AND DISCUSSION 10

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

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

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

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

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

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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

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

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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

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

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

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

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

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

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

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exposed to the

235



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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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oxidation conditions implemented.

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

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

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

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

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

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

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

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other studies.14,37,38

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

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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

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

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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

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

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