Binding of Aroma Compounds with Myofibrillar Proteins Modified by

Article pubs.acs.org/JAFC

Binding of Aroma Compounds with Myofibrillar Proteins Modified by a Hydroxyl-Radical-Induced Oxidative System Feibai Zhou,† Mouming Zhao,† Guowan Su,† and Weizheng Sun*,† †

College of Light Industry and Food Science, South China University of Technology, Guangzhou 510640, China ABSTRACT: The objective of this study was to investigate the influence of oxidation-induced structural modifications of myofibrillar protein on its binding ability with aroma compounds such as 2-methyl-butanal, methional, 2-pentanone, 2heptanone, and nonanal. A method using solid-phase microextraction (SPME) combined with gas chromatography/mass spectrometry (GC/MS) was used to determine the corresponding binding ability. The binding with aroma compounds was found to be strongly affected by the oxidation levels of proteins, probably due to the varying modifications in protein structure and surface. Incubation with oxidants around or below 1 mM mainly caused the refolding of protein structure and accelerated the protein aggregation, which reduced the affinity of the aroma compounds, thus decreasing the binding ability. Nevertheless, treatment with oxidants over 2.5 mM would cause protein reaggregation and partial degradation, and thus, the subsequent modification of protein surface properties. The aggregated protein with wrinkled surfaces favored the hydrophobic interactions with aroma compounds, forming the protein-aroma compound complex, thus enhancing the resultant binding ability as evidenced by fluorescence quenching and SPME-GC/MS analysis. KEYWORDS: myofibrillar protein, oxidative modification, aroma compounds, fluorescence quenching, SPME

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on the influence of protein structure.11,12 It has been reported that the reaction of radicals with proteins and peptides in the presence of oxygen gives rise to alterations in both the backbone and amino acid side chains, resulting in cleavage of peptide bonds, modification of amino acid side chains, and formation of covalent intermolecular cross-linked protein derivatives.13−15 Moreover, in our previous studies, we also found that oxidation could alter the secondary and tertiary structure of myofibrillar proteins, which could lead to changes in physicochemical state of proteins and thus their functional properties.16−18 In consideration of the fact that myofibrillar proteins in meat and meat products would unavoidably undergo attack from ROS during meat maturation, storage, and industrial processing,19−21 which can strongly influence the structure of proteins, it is of interest to study the influence of the oxidation modifications of myofibrillar proteins on their binding of aroma compounds in order to establish their possible contribution to flavor release in meat products. Moreover, to the best of our knowledge, no report about the influence of structural changes caused by oxidation other than frozen on the binding ability of myofibrillar proteins with aroma compounds has been found to date.10 In this study, myofibrillar protein, the most predominant muscle protein component, was exposed to a free-radicalgenerating system most commonly involved in meat processing. The accompanied protein structural changes (carbonyl content, surface hydrophobicity, particle size, and microstructure, etc.) were assessed for better investigating the influence of oxidatively modified protein structure on its binding ability

INTRODUCTION Acceptability of foods mainly depends on their sensory attributes and, in particular, their flavor. The quality of meat and meat products, a major source of protein of high biological importance, is highly influenced by their flavor perception.1 With the increasing demand for meat and meat products, understanding the mechanisms influencing flavor binding or release behavior from the meat matrix is very important not only for flavor modulation but also to improve the sensory properties of meat products.2,3 So far as concerned, flavor perception in meat products depends on the concentration and odor threshold of volatile compounds and on their interactions with other food components, especially with protein.4 It is well-documented that the main influence of proteins on flavor perception is caused by the interaction of the flavor component with the protein.3−5 A better comprehension of the factors affecting this interaction is important for flavor binding, and several authors have even attempted to investigate it through the study of model solution.6−8 As has been reported, the critical factors mainly include the nature of protein and aroma compound, ionic strength of the medium, concentration of other food components, temperature, and pH.3 Damodaran and Kinsella9 investigated the binding of alkanones to fish actomyosin, showing that the binding affinity increased along with the chain length of the carbonyl. Recent studies have also been carried out to investigate the ability of protein homogenates and peptides from skeletal muscle to interact with typical flavor compounds.3,6,10 These previous works demonstrated that the binding ability was largely dependent on protein structure and conformation. With the increasing understanding that food proteins are sources and targets for reactive oxygen species (ROS), numerous studies have now dealt with the occurrence of protein oxidation in muscle foods and have tried to shed light © XXXX American Chemical Society

Received: May 28, 2014 Revised: August 12, 2014 Accepted: August 31, 2014

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dx.doi.org/10.1021/jf502540p | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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according to the following formula (eq 1) and expressed as an index of hydrophobicity.

toward aroma compounds (2-methyl-butanal, methional, 2pentanone, 2-heptanone, and nonanal) using solid phase microextraction combined with gas chromatography−mass spectrometry (SPME-GC/MS) analysis. In addition, the interactions between oxidized protein and aroma compounds were further investigated using fluorescence quenching studies to better understand the underlying binding mechanism.

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BPB bound (μg) = 100 μg × (A control − A sample)/A control (1) Mean Particle Size of Oxidized Proteins. The mean particle size and size distribution of oxidized proteins were measured by a Mastersizer 2000 (Malvern Instruments Co. Ltd., Worcestershire, U.K.) at 25 °C, as described by Sun et al.18 The refractive index was taken as 1.57, and the absorption index was 0.001. The particle sizes measured are reported as the surface-weighted mean diameter d3,2 (d3,2= ∑ nidi3/∑ nidi2) and volume-weighted mean diameter d4,3 (d4,3= ∑ nidi4/∑ nidi3), where ni is the number of particles with diameter di. SDS−PAGE Analysis. SDS−PAGE was performed according to the method of Flores.26 Briefly, myofibrillar protein suspensions (5 mg/ mL in 20 mM phosphate buffer at pH 6.0) were mixed with sample buffer with and without 5% β-mercaptoethanol (β ME) at a ratio of 1:1. The ones with β ME were incubated for 5 min at 100 °C before centrifugation (10000g, 10 min). Then, 14 μL of samples were loaded onto the polyacrylamide gel made of 12% running gel and 5% stacking gel and subjected to electrophoresis using a Mini-PROTEAN 3 Cell apparatus (Bio-Rad Laboratories, Hercules, CA). Microstructure. The surface morphology of oxidized proteins were examined using field emission scanning electron microscopy (FE-SEM, model S4800, Hitachi, Japan) at an accelerator voltage of 10 kV. Before imaging, freeze-dried protein samples were mounted on a holder with double-sided adhesive tape and sputter-coated with gold (JEOL JFC-1200 fine coater, Japan). Protein−Aroma Binding Measurements. Preparation of Aroma Compounds Solution. A stock solution containing each aroma compound was prepared in methanol. The aroma compounds were then simultaneously added to protein suspension (control or oxidized, 8 mg/mL in 20 mM phosphate buffer containing 0.5 M NaCl at pH 6.0), resulting in a final concentration of 1 ppm for 2-pentanone, 2-heptanone, and nonanal and 2 ppm for 2-methyl-butanal and 5 ppm for methional. Both the control and the oxidized protein samples at different levels were prepared in duplicate. The selection of the five aroma compounds was based on their presence in the headspace and the contribution to the flavor of typical Cantonese sausage as well as other dry-cured meat products.2,27 Solid-Phase Microextraction (SPME) and Gas Chromatography− Mass Spectrometry (GC/MS) Analysis. Eight milliliters of the mixture was placed in a 20 mL headspace vial and sealed with a PTFE-faced silicone septum (Supelco, Bellefonte, PA). The control vial contained exactly the same buffer just without protein. Control and protein vials were stored overnight (12 h) at 30 °C to allow equilibration.9 The quantity of aroma compounds in the headspace of protein and control vial was determined using SPME and GC/MS analysis using conditions as described by Gianelli et al.6 with slight modifications. A 75 μm carboxen/poly(dimethylsiloxane) (CAR/PDMS) fiber (Supelco, Bellefonte, PA) was exposed to the headspace for sampling the aroma compounds. After 30 min of adsorption, the aroma compounds were desorbed by inserting the fiber into the GC injection port of a gas chromatograph set at 220 °C for 5 min in splitless mode. The split valve was opened for 1 min after injection. The fiber was heated for 5 min in the injection port at 220 °C to avoid analyte carryover between the samples. Analysis of the aromas was performed using Trace GC/MS system equipped with an Ultra GC, a Trisplus automated sampler, and a TR-Wax column (30 m × 0.32 mm × 0.25 μm, J&W Scientific, Folsom, CA). Helium was used as the carrier gas with a linear velocity of 20.4 cm s−1. The fiber was placed in the injector, and the GC oven temperature was started at 38 °C and held for 6 min; next, the temperature was increased to 105 °C at a rate of 6 °C min−1, then raised to 220 °C at the rate of 15 °C min−1, and held for 5 min. The detector temperature was set at 240 °C. The mass spectrometer was operated in electron impact (EI) mode. The ionization energy, detector voltage, scan range, and scan rate applied for the analysis were 70 eV, 350 V, m/z 35−350, and 3.00 scans/s, respectively. Chromatograms and mass spectra were evaluated using Xcalibur version 2.0 (Thermo Finnigan, San Jose, CA). The results are

MATERIALS AND METHODS

Materials. Longissimus muscle from three pork carcasses (48 h post-mortem) was purchased from a local commercial abattoir (Zhongshan, China), and the pigs were slaughtered about 6 months of age following standard industrial procedures. Fat was trimmed away and muscle was cut into cubes, minced, vacuum packaged (ca. 100 g), and frozen at −80 °C until use (used within 1 week). The aroma compounds 2-methylbutanal and 3-(methylthio) propanal (methional) were purchased from Sigma−Aldrich (Steinheim, Germany). 2Pentanone, 2-heptanone, and nonanal were obtained from Aladdin (Shanghai, China). Methanol used was of the highest commercial grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All other chemicals were of analytical reagent grade. Preparation of Myofibrillar Proteins. Raw muscle was thawed at 4 °C and then used for the myofibrillar protein preparation according to the method of Park, Xiong, Alderton, and Ooizumi22 with slight modifications. The pH of myofibrillar protein suspension at 0.1 M NaCl in the last wash was adjusted to 6.25 before centrifugation. The pellet was finally suspended in 20 mM sodium phosphate buffer (pH 6.0) containing 0.5 M NaCl, and the protein concentration was determined by the Biuret method using BSA as standard. Protein Oxidation. Myofibrillar protein suspension (28 mg/mL containing 0.5 mg/mL NaN3) was incubated at 37 °C for 3 h in 20 mM sodium phosphate buffer (pH 6.0) containing 0.5 M NaCl with a hydroxyl radical-generating system.22,23 The hydroxyl radicals were produced by a 100 μM FeCl3/100 μM ascorbic acid solution with H2O2 at various concentrations (0−25 mM). The same myofibrillar protein treated without incubation or H2O2 was chosen as the control. To prevent further oxidation, butylated hydroxyl toluene (BHT) (1 mM final concentration) was then added. Evaluation of Oxidative Changes. Carbonyls. The content of carbonyls was determined by the reaction with 2,4 dinitrophenylhydrazine (DNPH) using a Varioskan Flash (Thermo Scientific 3001, Thermo Fisher Scientific Oy, Finland), as described by Oliver et al.24 with some modifications. Briefly, 200 μL of 5 mg/mL protein suspension in 20 mM sodium phosphate buffer (pH 6.0) was mixed with 800 μL of 2 M HCl (control) or 10 mM DNPH in 2 M HCl and incubated at room temperature (25 °C) for 1 h. The DNPH-reacted samples after 10% trichloroacetic acid (TCA) precipitation were recovered by centrifugation at 8000g for 10 min and then washed three times with 1 mL of ethanol:ethyl acetate (1:1, v/v) to eliminate free DNPH. The final protein pellets were dissolved in 1.2 mL of 6 M guanidine HCl with 20 mM sodium phosphate buffer (pH 6.0) and were then centrifuged at 8000g for 10 min. Protein concentration in the supernatant was calculated at 280 nm in the HCl control using BSA in 6 M guanidine as standard. The carbonyl concentration was calculated using an absorption of 21.0 mM−1cm−1 at 370 nm against the HCl control for protein hydrazones. Protein Surface Hydrophobicity. The hydrophobicity was determined using the hydrophobic chromophore bromophenol blue (BPB) method reported by Chelh et al.25 with slight modifications. The 0.5 mL protein suspension (5 mg/mL in 20 mM sodium phosphate buffer at pH 6.0) was thoroughly mixed with 100 μL of 1 mg/mL BPB (in distilled water). The resulting samples were continually shaken at room temperature (25 °C) for 10 min and then centrifuged at 4000g for 15 min (4 °C). The absorbance of supernatant was measured at 595 nm against a phosphate buffer blank. The sample with the same treatment in the absence of protein was used as the control. The amount of bound BPB was calculated B



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expressed as a percentage of the free aroma compound found in the headspace without protein in the solution. Fluorescence Spectroscopy Measurements. Protein intrinsic fluorescence was measured at a constant protein concentration (0.8 mg/mL) with various methional concentrations (0.05−0.5 ppm) in 20 mM phosphate buffer (pH 6.0) using a Varioskan Flash (Thermo Scientific 3001, Thermo Fisher Scientific Oy, Finland). Emission spectra were recorded from 300 to 400 nm at an excitation wavelength of 283 nm. Both the excitation and emission slit widths were set at 5 nm. The fluorescence spectra of the phosphate buffer were subtracted from the respective spectra of the samples. Fluorescence quenching is described according to the Stern−Volmer equation (eq 2):28 F0/F = 1 + kqτ0[Q ] = 1 + KSV[Q ]

(2)

In this equation F0 and F are the fluorescence intensities in the absence and presence of a quencher, respectively, [Q] is the quencher concentration (mol/L), KSV is the Stern−Volmer quenching constant, kq is the bimolecular quenching rate constant, and τ0 is the lifetime of fluorescence in the absence of a quencher (∼10−8 s). Hence, eq 1 was applied to determine KSV by linear regression of a plot of F0/F versus [Q]. Statistical Analysis. Statistical calculation was investigated using the statistical package SPSS 11.5 (SPSS Inc., Chicago, IL) for one-way ANOVA. Student−Newman−Keuls test was used for comparison of mean values among determinations using a level of significance of 5%. Triplicate preparations of myofibrillar proteins were carried out to confirm the accordance. Data were expressed as means ± standard deviations (SD) of triplicate determinations unless specifically mentioned.

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RESULTS AND DISCUSSION Oxidative Modifications of Myofibrillar Proteins. Carbonyls. The carbonyl contents of the control and oxidized myofibrillar proteins are presented in Figure 1A. In this work, carbonyl content of the control protein was 1.66 nmol/mg protein, similar to those reported by Traore et al.29 on raw longissimus thoracis muscle and Morzel et al.23 on skeletal muscle myofibrillar proteins from pig longissimus dorsi. Compared to the control, carbonyl content of the incubated protein sample (0 mM) exhibited no significant difference (p < 0.05). Nevertheless, in the presence of oxidant, protein carbonyl content linearly increased (R2 = 0.998) with the increasing H2O2 concentrations, and a maximum value of 14.03 nmol/mg protein was observed at 25 mM H2O2. This indicated that the formation carbonyl was weakly affected by the thermal process alone but was markedly exacerbated by the presence of H2O2, in agreement with previous studies.19,22 In addition, considering the increase in carbonyl content during storage of meat for 10 days could be reached at approximately 2.5−10 mM oxidizing agent in our experiment,19 the oxidative system used could be compared to real meat products to some extent. Protein Surface Hydrophobicity. In view of its capacity to monitor subtle changes in chemical and physical states of protein, surface hydrophobicity could be a suitable parameter to estimate protein denaturation.30 Figure 1B shows the surface hydrophobicity of the control and oxidized myofibrillar proteins. Compared to that of the control (44.74 μg bound BPB), the hydrophobicity of incubated protein sample (0 mM H2O2) was significantly increased (p < 0.05). This indicated that the thermal treatments could markedly increase the protein surface hydrophobicity, which could be related to the unfolding of actomyosin fibers, which begins at 35 °C.31 For oxidized proteins, as can be seen, relatively low addition of H2O2 (0.5−1 mM) decreased the hydrophobicity level, while the further (H2O2 ≥ 2.5 mM) significantly (p < 0.05) enhanced it. The

Figure 1. Physicochemical changes (A: carbonyl content; B: protein surface hydrophobicity) of the myofibrillar proteins upon oxidation by 100 μM FeCl3/100 μM Ascorbic acid/0−25 mM H2O2. For the letters a−f, means (n = 3) without a common letter differ significantly (p < 0.05).

hydrophobicity for protein at 25 mM H2O2 was 68.61 μg, indicating an obvious increase by about 53% when compared with that of control (44.74 μg). Similar results have also been obtained stating that heating at temperatures close to the physiological temperature under slight oxidative stress might lead to protein refolding,32,33 which could further lead to the decrease in hydrophobicity. However, with the further increase in H2O2 concentration (>2.5 mM), the hydrophobic amino acids, which are buried in protein inside, were exposed and thus enhanced protein surface hydrophobicity.30,34 Moreover, the cleavage of certain peptide under oxidative attack might also contribute to the enhancement of surface hydrophobicity.13 Mean Particle Size and Distribution. The mean particle size (d3,2 and d4,3) and size distributions for the oxidized proteins were used to assess the aggregation behavior of protein during oxidation, as shown in Figure 2. It can be clearly seen that, compared to the control, the incubated protein sample (0 mM H2O2) exhibited a better size distribution, although no significant difference (p > 0.05) was observed in the mean particle size values (d3,2 and d4,3). Upon the presence of H2O2, it is noted that, compared to the control, the proteins showed markedly higher particle size values (d3,2 and d4,3) and the particle size distribution was monomodal and narrow. MoreC



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SDS−PAGE. The aggregation behavior of oxidized proteins was further studied by the SDS−PAGE analysis (Figure 3). As shown in Figure 3, oxidative modification significantly altered the electrophoretic pattern of myofibrillar protein. In the absence of β ME, the exposure of myofibrillar protein to •OH caused a gradual disappearance of several subunits in a H2O2 dose-dependent manner (Figure 3A), among which changes in myosin were most remarkable, similar to those previous reports.15,35 Herein, both myosin heavy chain (MHC) and heavy meromyosin−myosin heavy chain (HMM-MHC) began to reduce at 0.5 mM of H2O2 and almost completely vanished since H2O2 reached 1 mM. A similar, albeit less conspicuous, actin loss was also observed with increasing addition of H2O2 despite remaining faint even at high H2O2 concentrations. Simultaneously, upon oxidation, high molecular weight polymers, which barely entered the stacking gel, became accentuated (Figure 3A). These results indicated the occurrence of cross-linking under oxidation, which was consistent with the changes in particle size distributions as aforementioned (Figure 2), and myosin and actin were mainly involved. In addition, changes in troponin T (TnT) and tropomyosin (Tm) were also noticeable for their band intensity both declined upon oxidation and almost disappeared when the oxidant reached 1 mM. Nevertheless, they reappeared with further oxidation. The results implied that TnT and Tm were also involved in the formation of aggregates within the H2O2, ranging from 0 to1 mM, well explaining the corresponding significant (p < 0.05) decrease in protein surface hydrophobicity (Figure 1B). Nevertheless, further oxidation caused the reappearance of TnT and Tm, indicating that they were gradually no longer involved in the aggregates, which might be related with the degradation of protein backbone under •OH attack.36 In the presence of β ME, MHC, actin, TnT, and Tm were mostly recovered (Figure 3B), suggesting that these polymers were formed largely by disulfide bonds (S−S) or mainly through this mechanism. Nevertheless, samples treated with more than 2.5 mM of H2O2 exhibited rather diffused bands at the top of the resolving gel, and the band intensity increased along with H2O2 concentration. This suggested the presence of other covalent polymers containing no S−S bonds, such as Tyr−Tyr and active carbonyl−NH2 interaction, as

Figure 2. Particle size distributions of the myofibrillar proteins upon oxidation. Inset: Mean particle diameter (d3,2 and d4,3) of the control and oxidized proteins.

over, with the increasing addition of oxidants, an evident shift of the size distribution peak to the right was observed (Figure 2). These results should be attributed to the fact that the H2O2induced protein oxidation might promote the intermolecular aggregation behaviors,15 thus resulting in the change on the size distribution and the increase in particle size values (d3,2 and d4,3) of proteins. On the basis of the result of protein surface hydrophobicity (Figure 1B), we can thus conclude that the increase in surface hydrophobicity might favor the association between proteins mainly due to hydrophobic interactions and thereby the increase of intermolecular aggregation. In addition, it is interesting to note that, within the H2O2 concentration range from 0.5 to 2.5 mM, no marked changes on the values of d4,3 and d3,2 for the oxidized proteins were observed. However, with further increase in H2O2 concentrations (2.5−25 mM), the d4,3 and d3,2 values for proteins began to dramatically increase. These results should probably be related to the surface modification of myofibrillar protein under the H2O2-induced oxidative stresses and were in accordance with the sudden increase in protein surface hydrophobicity when the H2O2 added reached 2.5 mM (Figure 1B).

Figure 3. Representative SDS−PAGE patterns of the control and myofibrillar proteins oxidized by 100 μM FeCl3/100 μM ascorbic acid/0−25 mM H2O2 (A) without and (B) with 5% β ME. MHC, myosin heavy chain; HMM-MHC, heavy meromyosin−myosin heavy chain; TnT, troponin T; Tm, tropomyosin; MLC-1, myosin light chain-1; MLC-2, myosin light chain-2. D



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Figure 4. Scanning electron microscope micrographs at (A) ×1000 and (B) ×5000 magnification of the control and oxidized (100 μM FeCl3/100 μM ascorbic acid/0−25 mM H2O2) myofibrillar proteins. Scale bars indicate (A) 10 μm and (B) 2 or 1 μm.

previously reported.15,23 These covalent polymers, previously linked together by S−S (therefore too large to enter the stacking gel),37 were present in those oxidized protein samples. In addition, new but hazy bands with molecular weight similar to MCL-1 and MCL-2 generated in samples treated with more than 2.5 mM of H2O2, suggesting the fragmentation or degradation of protein backbone under •OH attack, as has been previously reported by Berlett et al.,36 which could contribute to the increase in protein surface hydrophobicity (Figure 1B). Microstructure. The changes in the physicochemical properties of oxidized myofibrillar proteins were further investigated by the measurements of their morphologic properties. Microstructures of oxidized proteins under different H2O2 levels, in comparison with the control, are presented in Figure 4. For the control, protein appeared in an intactly dense and firm pattern with the heterogeneous surface (Figure 4, panels A and B). Compared to the control, the incubated protein sample (0 mM H2O2) showed a more flat surface with few visible pores (Figure 4A), which might be due to the unfolding and slight shrinkage of myofibrillar protein upon incubation.38 Additionally, evenly distributed granular aggregates, which has been reported by Astruc et al.,38 were also observed when further magnified (Figure 4B). For proteins with oxidants, at low H2O2 concentrations (0.5 and 1 mM), the surface microstructures of samples were not appreciably different despite the increasing roughness of overlapped surface (Figure 4A). Moreover, the

increase in granular size of the aggregates (Figure 4B) well indicated the accelerated state of aggregation, in agreement with the results of surface hydrophobicity and mean particle size (Figures 1B and 2). When H2O2 concentrations were increased to higher levels (2.5−25 mM), it was noteworthy that an obvious morphological change was observed and this morphological transition occurred between 1 and 2.5 mM of oxidants. This trend was in accordance with changes in the data of protein surface hydrophobicity, mean particle size, and results revealed by SDS−PAGE (Figures 1B, 2, and 3). It was more likely that samples treated with 2.5 mM of oxidants, which underwent steric modifications, rendered the layered surface with discontinuous aggregates (Figure 4). The enlarged surface area thus resulted in the sudden increase in protein surface hydrophobicity (Figure 1B), which was also consistent with the results revealed by SDS−PAGE (Figure 3). When being further oxidized (10−25 mM H2O2), myofibrillar protein showed a surface with more wrinkles at a larger size (Figure 4). This suggested that higher levels of oxidation would further modify the protein surface properties and plausibly simultaneously involved protein reaggregation and degradation, as indicated by SDS−PAGE analysis (Figure 3). Binding of Aroma Compounds with Oxidized Myofibrillar Proteins. On the basis of the characterization of oxidized proteins, we attempted to investigate the influence of structural changes of proteins on their binding ability with aroma compounds. Thus, the free percentages of each aroma E



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leading to the formation of protein aggregates and overlapped/ reduced surface, as confirmed by SDS−PAGE and microstructure analysis (Figures 3 and 4). These changes (structural hindrance) impaired the affinity of the aroma compounds for the protein matrix by reducing the number of binding sites for the aroma compound2 and thus decreased the binding ability of protein with all the selected aroma compounds (Figure 5). However, for protein treated with oxidants more than 2.5 mM, the binding ability of oxidized proteins toward the aroma compounds (especially for methional) increased (Figure 5). This could be explained by the remarkable increase in the protein surface hydrophobicity and modified protein surface properties (wrinkled surfaces) within the oxidant range (Figures 1B and 4), which might contribute to the interaction between protein and these compounds mainly driven by hydrophobic forces, thus enhancing the corresponding binding ability (Figure 5). Interactions between Oxidized Proteins and Methional. Fluorescence spectroscopy is commonly used to study the binding between protein and small molecules, and fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample.40−42 A variety of mechanisms can cause quenching, such as excited state reaction, molecular rearrangement, energy transfer, ground state complex formation, and collision quenching.40,43 Since myofibrillar proteins have tryptophan (Trp) residues that possess intrinsic fluorescence, the interaction mechanisms between myofibrillar proteins and aroma compounds could be further studied by the fluorescence quenching method. In the present work, we have demonstrated that oxidation-induced structural changes could markedly influence the protein binding ability with aroma compounds (Figure 5), especially for methional. Thus, methional was first selected as a model aroma compound, and the interaction between oxidized myofibrillar proteins and methional in the aqueous phase was subsequently investigated using fluorescence quenching studies (Figure 6). Figure 6A displays the example of fluorescence emission spectra of oxidized myofibrillar protein (25 mM H2O2) with various concentrations of methional. The fluorescence spectra of control and other oxidized protein (0−10 mM H2O2) samples in the presence of methional were also measured (data not shown). Evidently, the oxidized protein alone exhibited a strong fluorescence emission at 330 nm following an excitation at 283 nm. With the increasing of methional, a gradual attenuation of the fluorescence intensity of oxidized protein was observed (Figure 6A). Under the experimental conditions, methional revealed no fluorescence emission in the range of 300−400 nm, which did not affect protein intrinsic fluorescence. Thus, it could be concluded that there was interactions between oxidized myofibrillar protein (25 mM H2O2) and methional, in agreement with the results of protein binding ability with aroma compounds (Figure 5). To further discern the influence of protein oxidation on fluorescence quenching mechanism, Stern−Volmer eq 2 was used for data analysis, and the corresponding results fitted from Stern−Volmer plots (Figure 6B) are summarized in Table 1. As shown in Table 1, the Stern−Volmer quenching constant KSV varied among the oxidized protein samples. Compared to the control, the KSV value of the incubated protein sample (0 mM H2O2) significantly (p < 0.05) increased, in agreement with its enhanced binding ability with methional (Figure 5), which might be related to the increased surface hydrophobicity (Figure 1B). For oxidized proteins, with the low addition of

compound in the headspace in the presence of myofibrillar protein at different oxidizing levels were determined, as shown in Figure 5. One hundred percent of free aroma compound

Figure 5. Effect of protein oxidation on the binding of aroma compounds. Results are expressed as percentage of free aroma compounds found in the headspace without protein in solution. Bars with different letters are significantly different (p < 0.05) among oxidation levels.

corresponds to the headspace concentration present in the control vial that does not contain protein.10 As can be seen in Figure 5, the presence of the control protein produced a marked reduction of the percentages of free nonanal and a slight decrease of 2-methyl-butanal and 2-heptanone, suggesting that the control protein was able to bind those aroma compounds. In contrast, the increase in 2-pentanone implied its releasing behavior. These results were similar to that reported by Pérez-Juan et al.2,10 Compared to the control, the incubated protein sample (0 mM H2O2) exhibited significantly lower percentages of 2-methyl-butanal, nonanal, and methional (p < 0.05), suggesting that thermal treatment could enhance the binding ability of protein toward them, which could be mainly attributed to the fact that the unfolding of actomyosin fibers after heating (37 °C) increased the sites available for hydrophobic binding.10,31 For oxidized proteins, oxidation further affected the protein binding ability in a dose-dependent way. When the H2O2 added ranged from 0 to 1 mM, the percentages of all aroma compounds increased, indicating the decreased binding ability of protein. However, it was very interesting to note that, with the further increase in H2O2 concentration (>1 mM H2O2), an inverse effect on the percentages of aroma compounds was observed. As can be seen with increasing protein oxidation level, the percentages of all the selected aroma compounds decreased, implying the increased binding ability of protein toward them. Specifically, the percentages of free methional dramatically decreased with further oxidation (>1 mM H2O2) and even could not be detected when the highest concentration of H2O2 was added (25 mM). As has been reported, the concentration of free aroma compounds in the gas phase largely depends on their interactions with protein, and changes in the protein conformation affect the interaction between aroma compounds and proteins due to the modification of the available protein binding sites.3,39 As aformentioned, treatment with 0.5−1 mM of oxidants enhanced protein−protein interactions, mainly F



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Table 1. Stern−Volmer Quenching Constants for the Binding of Methional by Myofibrillar Proteins at Different Oxidation Levels H2O2

Ksva (× 104 M−1)

kq (× 1012 M−1 s−1)

R2

control 0 mM 0.5 mM 1 mM 2.5 mM 10 mM 25 mM

± ± ± ± ± ± ±

± ± ± ± ± ± ±

0.992 0.972 0.983 0.989 0.975 0.989 0.988

6.02 7.65 5.73 0.28 7.45 9.20 12.29

c

0.19 0.53d 0.30c 0.01b 0.45d 0.34e 0.56f

6.02 7.65 5.73 0.28 7.45 9.20 12.29

c

0.19 0.53d 0.30c 0.01b 0.45d 0.34e 0.56f

a

The mean value of three independent experiments with standard deviation (SD). bMeans in the same column with different superscript letters are significantly different (p < 0.05). cMeans in the same column with different superscript letters are significantly different (p < 0.05). dMeans in the same column with different superscript letters are significantly different (p < 0.05). eMeans in the same column with different superscript letters are significantly different (p < 0.05). f Means in the same column with different superscript letters are significantly different (p < 0.05).

compounds by food proteins could lead to the formation protein−aroma compound complex.42 On the basis of the above analysis, we can conclude that the oxidative system applied could either increase or decrease the binding ability of aroma compounds with myofibrillar proteins, depending on the structural changes, especially the surface modifications of protein under oxidative stress. Specifically, treatment with low oxidant concentration (0.5−1 mM H2O2) mainly decreased protein surface hydrophobicity due to the formation of aggregates and the subsequent refolding of protein surface structure (Figures 1B, 3, and 4). This led to the reduction of the affinity of the aroma compounds for the protein matrix and thus the decreased binding ability with all aroma compounds selected (Figure 5). However, treatment with oxidant over 2.5 mM further aggregated the protein and at the same time remarkably modified protein surface properties (Figures 3 and 4), which dramatically enhanced protein surface hydrophobicity (Figure 1B). This resulted in the increased binding ability of protein with aroma compounds (especially for methional) (Figure 5) mainly driven by hydrophobic interaction, forming the protein−aroma compound complex, as evidenced by fluorescence quenching (Figure 6). In addition, considering the real meat system, the link between protein structure and flavor binding is not straightforward. However, in view of all these present results, it could be speculated that there is a clear correlation between protein structure modified by hydroxyl radical-induced oxidative system and its aroma binding ability. In summary, this study clearly showed that the protein binding ability with aroma compounds was correlated with its structural changes, which was found to be strongly influenced by the hydroxyl radical-induced oxidative modifications. Incubation with low oxidant concentration (0.5−1 mM H2O2) mainly accelerated the aggregation and thus decreased protein surface hydrophobicity, reducing the binding ability with aroma compounds, while the presence of higher oxidant concentrations (>2.5 mM H2O2) would cause the protein reaggregation and partial degradation, and thus the subsequent modification of protein surface properties. The aggregated protein with wrinkled surfaces largely favored the binding with aroma compounds (especially for methional) mainly driven by hydrophobic interaction, forming the protein−aroma com-

Figure 6. (A) Example of fluorescence emission spectra of oxidized myofibrillar protein (25 mM H2O2) with various amounts of methional in 20 mM phosphate buffer B containing 0.5 M NaCl at pH 6.0: (a) 0.8 mg/mL oxidized protein alone; (b−j) 0.8 mg/mL oxidized protein with 0.05, 0.1, 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, and 0.50 ppm methional, respectively. (B) Stern−Volmer plots describing fluorescence quenching of the control and oxidized proteins at different levels in the presence of methional.

oxidants (0−1 mM H2O2), the values of KSV dramatically decreased. However, upon the further increase of oxidant concentration (2.5−25 mM H2O2), an obvious increase in the KSV values was observed when compared to that of the control. This result perfectly matched the general trends seen in the data of surface hydrophobicity (Figure 1B) and protein binding ability with aroma compounds (Figure 5), further implying the interesting influence of protein oxidation on the protein binding reaction with aroma compounds. Furthermore, all the values of kq calculated (Table 1) were much higher than the maximum value for diffusion-controlled quenching in water (∼1010 M−1 s−1), signifying the possible quenching mechanism for control and oxidized protein fluorescence by methional is a static type.28,40 These results indicated that the methional may bind with hydrophobic sites of protein, forming the protein− aroma compound complex. This is in agreement with previous studies, which have reported that hydrophobic binding of flavor G



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pound complex. These findings provided interesting correlation between protein structure and its aroma binding ability and are critical for the food industry to manufacture meat-flavored foods with myofibrillar protein that underwent oxidative stress.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +86 20 22236089. Funding

This work was supported by the National Natural Science Foundation of China (Grant 31201387), the Natural Science Foundation of Guangdong Province (Grant S2012040007533), and the Doctoral Fund of Ministry of Education of China (Grant 20120172120017). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The assistance provided by Dr. Zhi-Li Wan is gratefully acknowledged and appreciated. REFERENCES

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Binding of Aroma Compounds with Myofibrillar Proteins Modified by

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