Potential Biomarker of Myofibrillar Protein Oxidation in Raw and

Article pubs.acs.org/JAFC

Potential Biomarker of Myofibrillar Protein Oxidation in Raw and Cooked Ham: 3‑Nitrotyrosine Formed by Nitrosation Xianchao Feng,†,‡ Chenyi Li,† Niamat Ullah,†,# Robert M. Hackman,§ Lin Chen,*,† and Guanghong Zhou‡ †

College of Food Science and Engineering, Northwest A&F University, No. 28 Xinong Road, Yangling, Shaanxi 712100, China National Center of Meat Quality and Safety Control, Key Laboratory of Meat Processing and Quality Control, Ministry of Education, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China § Department of Nutrition, University of CaliforniaDavis, One Shields Avenue, Davis, California 95616, United States # Department of Human Nutrition, The University Of Agriculture Peshawar, Khyber Pakhtunkhwa 25000, Pakistan ‡

S Supporting Information *

ABSTRACT: The stability of cured meat products is increased by the protection of its proteins from oxidation by sodium nitrite (NaNO2) during processing. This study investigated the effects of NaNO2 (0, 50, 100, 200, and 400 mg/kg) on the physiochemical and structural characteristics of myofibrillar protein (MP) in raw and cooked ham. The NaNO2 showed a dosedependent antioxidant effect, by inhibiting carbonyl formation, dityrosine formation, and denaturation of MP, and a nitrosative effect, through the formation of 3-Nitrotyrosine (3-NT). The 3-NT content within MP of raw ham had distinct negative correlations with sulfhydryl content and surface hydrophobicity. The 3-NT content within MP of cooked ham had significantly negative correlations with carbonyl, sulfhydryl content and turbidity and had significantly positive correlations with disulfide content. These results indicated that 3-NT may be a potential marker for protein oxidation in raw and cooked cured meat products. KEYWORDS: pork ham, sodium nitrite, protein nitrosation, 3-nitro-L-tyrosine, oxidative effect, nitrosative effect

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INTRODUCTION In cured meat products, sodium nitrite (NO2) contributes to the product’s attractive color and unique flavor and limits microbial growth and oxidation of lipids.1 One of the most noteworthy properties of NaNO2 is its strong antioxidant effect, which can effectively delay the development of oxidative rancidity.2 The antioxidant effect of NaNO2 is due to chelation of free radicals by nitric oxide and the formation of antioxidative nitroso and nitrosyl compounds.3 While this is the known antioxidative mechanism protecting against lipid oxidation,2 meat products can also turn rancid through oxidation of proteins by free radicals.4 It remains unknown if NaNO2 also acts as an antioxidant against protein oxidation. Few studies have reported research on the influence of NaNO2 on protein oxidation, while others looked for but failed to find a distinct effect.2,5,6 However, these studies used isolated MP as a simple meat model and a hydroxyl-radical-generating system to initiate protein oxidation.2,6 Investigation of interactions between NaNO2 and meat proteins can be helpful to understand the role of NaNO2 on cured muscle foods.6 However, meat products are more complex than isolated MP in actuality. Therefore, this model is insufficient for the study of protein modification in meat products, especially those that have been cooked. Therefore, the occurrence of these reactions in cured muscle foods requires further investigations. Hence, this study used ham, a popular pork product made around the world, to study the influence of curing compounds and processing conditions on the nitrosation and oxidation of proteins in the final processed meat product. © 2015 American Chemical Society

Food is processed through such techniques as mincing, salting, and/or cooking, in order to achieve a safe and palatable product. These treatments result in numerous chemical changes, such as the loss of sulfhydryls and free amines and the formation of carbonyls, disulfide bonds, dityrosines, and carbonyl-amine reactions, among other compounds.4,7,8 In addition to changes in the physical-chemical state of the meat proteins, oxidation and denaturation can also affect the structural properties of the meat proteins.9 Heat treatment is a common method of processing meatbased food products prior to consumption.10 Heat treatment initiates a cascade of both desirable and undesirable chemical reactions. For instance, heat treatment can trigger and increase the undesirable oxidation of meat proteins.8,10 The damages caused by heating occur not only at the whole food level, but also at the molecular level.8 Meat processing with the addition of NaNO2 can yield reactive species, such as nitric oxide and preoxynitrite, which might initiate nitrosation of proteins.2 The nitrosative stress on proteins can lead to the formation of specific compounds, such as 3-NT.6 In the processing of cured meat products, the oxidation of proteins simultaneously happens with nitrosation. However, most previous studies have focused on the antioxidative effect of NaNO2 in isolated MP, while few studies Received: Revised: Accepted: Published: 10957

August 26, 2015 November 17, 2015 November 21, 2015 November 21, 2015 DOI: 10.1021/acs.jafc.5b04107 J. Agric. Food Chem. 2015, 63, 10957−10964

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Journal of Agricultural and Food Chemistry

Table 1. Significance Values for the Main Effects of “Sodium Nitrite”, “Treatment”, and Their Interaction Term on the Protein Chemical Markers treatment (raw or cooked) sodium nitrite sodium nitrite × treatment

carbonyl

free amines

sulfhydryl

disulfide

dityrosine

hydrophobicity

turbidity

3-NT

0.000 0.000 0.003

0.000 0.010 0.000

0.119 0.189 0.000

0.051 0.127 0.000

0.000 0.000 0.000

0.000 0.000 0.000

0.000 0.000 0.000

0.000 0.000 0.000

have looked at the antioxidant effect in raw meat products.11 Vossen and De Smet reported that 3-NT was present in raw meat products, but whether other compounds and process conditions influence its occurrence post mortem and whether this is a suitable marker for protein oxidation in processed meat remain to be elucidated.2 There are few studies on the antioxidative effect of NaNO2 in cooked meat products, although these techniques are common and should comprise a particularly important line of investigation. Moreover, the connection between oxidation and nitrosation of proteins requires a fully comprehensive investigation. This investigation was designed to elucidate the influence of NaNO2 on protein oxidation and nitrosation in both raw and cooked meat products and to assess the use of 3-NT as a potential oxidative biomarker. The results of this study will help us to understand the influence of nitrosation on protein oxidation and the impact of NaNO2 on food quality. Furthermore, these results can lead to the optimization of the formula used to produce cured meat products.

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Carbonyl Analysis. The carbonyl levels in the MP samples were analyzed according to the method of Oliver et al.16 Carbonyl levels, in the form of hydrazones, were detected by 2,4-dinitrophenylhydrazine (DNPH). An absorption coefficient of 22 000 M−1 cm−1 was used at 370 nm. Free Amines. The free amine levels in the MP samples were measured as described by Liu et al.17 Absorbance was measured at 420 nm using 2,4,6-trinitrobenzenesulfonic acid (TNBS) as the indicator. Dityrosine. The dityrosine levels in the MP samples were measured using an F-4600 fluorescence spectrophotometer (Hitachi, Japan) with excitation set at 325 nm and emission at 420 nm.18,19 Dityrosine content was expressed as the difference of Absorbance Unit (A.U.) between samples and reagent blank. Total Sulfhydryl and Disulfide Bond Content. Sulfhydryl oxidation of MP was measured using 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB).20,21 Measurement of disodium 2-nitro-5-thiosulfobenzoate (NTSB) was used to determine disulfide bond content in MP.17 Electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE) was performed in a 4% stacking gel and 12.5% separating gel to observe the polymerization of protein samples induced by oxidation.22 The MP solution (2 mg/mL) was mixed with sample buffer (1:4 = v/v) containing 10% of βmercaptoethanol (βME) or βME free, and then boiled for 5 min. Each well of gel was loaded with 10 μL of samples. After electrophoresis, gels containing protein bands were stained with 0.1% coomassie brilliant blue. After clearing the free dye, the gels were photographed with a Gel Doc XRTM System (Bio-Rad Laboratories, Hercules, CA). Surface Hydrophobicity. Surface hydrophobicity of the MP was determined by hydrophobic chromophore bromphenol blue (BPB).19 Free BPB in the supernatant was measured at 595 nm. Control was prepared without MP. The percent of bound BPB (μg) by MP was calculated as an index of hydrophobicity:

MATERIALS AND METHODS

Materials. Fresh pork (from the gluteus medius muscle) was obtained from the Bensun Corporation (Yangling, Shaanxi, China). All chemicals were of reagent grade and were purchased from SigmaAldrich Co. (St. Louis, MO, U.S.A.) or Aladdin Industrial Corporation (Fengxian, Shanghai, China). Preparation of Ham. Ham was prepared according to previous methods.12,13 Briefly, ham was prepared as a mixture of 80% pork meat and 20% water. Fresh meat and half the volume of water (as ice) were added to a bowl cutter (BZBJ-5, Expro Machinery Engineering Co., Ltd., Hangzhou, China). Then, 2% salt, 0.03% ascorbic acid, and the other half volume of water were added before additional mixing in the bowl cutter for 5 min at low temperature (100 mg/kg) decreased the carbonyl content in both MPR and MPC, approximately 1.5-fold (p < 0.05) compared to untreated MP (Figure 1). The carbonyl content in MPC decreased similarly with increasing NaNO2 concentration, but did show a low level at 400 mg/kg NaNO2 (Figure 1). The negative correlation between carbonyls and NaNO2 indicated that sodium nitrite prevents carbonyl formation (Tables 2 and3). Since iron can catalyze the oxidative deamination of lysine, threonine, arginine, and proline residues to yield carbonyls, the chelation of iron by NaNO2 would be an important antioxidant property.2,26,27 Moreover, meat-based food products have various compounds which can react with the NaNO2 to form active antioxidants, such as nitrosylmyoglobin compounds of nitric oxide and intermediates of lipid oxidation.2,5,28 Vossen and Smet2 found a slight decrease in carbonyl levels in a model meat system, which is in accordance with the present result. Carbonyls can attack nucleophiles (such as ε-NH2 of lysine) and therefore contribute to the loss of total carbonyls and free amines (Figures 1 and 2).29,30 In cooked hams, heat treatment can promote the formation of carbonylNH2. This could explain why the MPC treated with NaNO2 had lower carbonyl levels compared to the MPR samples (Figure 1). However, studies using isolated MP found that NaNO2 showed pro-oxidative effect on the formation of carbonyls.2,6 This contrasts with our findings, but may be explained by the fact that reactive species, including reactive nitrogen species (RNS), in an in vitro hydroxyl-radicalgenerating system are rich enough to improve the formation of carbonyl compounds in the isolated MP.2,5 Free amines were also monitored to assess the physicochemical changes of MP. In the MPR samples, high NaNO2 concentrations (400 mg/kg) decreased the amine levels (p < 0.05) (Figure 2). This indicated that NaNO2 could also promote the formation of carbonylNH2. In MPC, the increased levels of free amines with increasing NaNO2 concentration could result in the cleavage of peptide bonds. However, the influence of NaNO2 on peptide bond cleavage and formation of carbonyl-NH2 during heat treatment is not clear. Dityrosine, an indicator of physicochemical changes in protein, has also been observed in model meat systems.31 Dityrosine is formed by the oxidation of tyrosine through the 10959

DOI: 10.1021/acs.jafc.5b04107 J. Agric. Food Chem. 2015, 63, 10957−10964

Article

Journal of Agricultural and Food Chemistry Table 2. Correlation Matrix between Parameters of Oxidative Modification and 3-NT Content of MPRa carbonyl free amine sulfhydryl disulfide bond dityrosine hydrophobicity turbidity 3-NT a

sodium nitrite

carbonyl

free amine

sulfhydryl

disulfide

dityrosine

hydrophobicity

turbidity

−0.628 −0.897** −0.758* 0.716 −0.549 −0.943** −0.607 0.874**

0.455 0.937** −0.973** 0.810* 0.756* 0.932** −0.698

0.623 −0.628 0.175 0.711 0.295 −0.638

−0.955* 0.781* 0.813* 0.859** −0.850*

−0.691 −0.769* −0.842* 0.703

0.779* 0.960** −0.731

0.813** −0.923**

−0.723

The relationships were tested using pearson correlation coefficients. Significance is noted as p < 0.05,* and p < 0.01,**.

Table 3. Correlation Matrix between Parameters of Oxidative Modification and 3-NT Content of MPCa carbonyl free amine sulfhydryl disulfide bond dityrosine hydrophobicity turbidity 3-NT a

sodium nitrite

carbonyl

free amine

sulfhydryl

disulfide

dityrosine

hydrophobicity

turbidity

−0.794* 0.250 −0.962** 0.969** −0.424 −0.375 −0.806* 0.998**

0.291 0.779* −0.884** 0.694 0.704 0.649 −0.756*

−0.156 0.006 0.449 0.624 −0.454 0.294

−0.957* 0.606 0.283 0.797* −0.965**

−0.548 −0.540 −0.708 0.957**

0.234 0.497 −0.406

−0.033 −0.324

−0.808*

The relationships were tested using pearson correlation coefficients. Significance is noted as p < 0.05, *; and p < 0.01, **.

Figure 2. Free amine levels in MPR and MPC treated with different NaNO2 doses (mg/kg). Asterisk denotes a significant difference between means (n = 5) of MPR and MPC treated with same NaNO2 dose (p < 0.05). Letters a−d denote significant differences between means (n = 5) of MPR or MPC treated with different NaNO2 doses (p < 0.05).

Figure 3. Dityrosine formation (fluorescence intensity) in MPR and MPC treated with different NaNO2 doses (mg/kg). Asterisk denotes a significant difference between means (n = 5) of MPR and MPC treated with the same NaNO2 dose (p < 0.05). Letters a, b, and c denote significant differences between means (n = 5) of MPR or MPC treated with different NaNO2 doses (p < 0.05).

formation of a tyrosyl phenoxyl radical. In this study, the dityrosine content of MPR treated with NaNO2 was 4-fold lower compared to that of MPR not treated with NaNO2 (Figure 3). This indicated that the addition of NaNO2 can decrease the formation of dityrosine in MPR. The mechanism of dityrosine formation in the presence of NaNO2 could be similar to that of carbonyl formation. In addition, under nitrosation stress induced by the presence of NaNO2, some tyrosine residues on the surface of the protein were modified into 3-NT which could also prevent the generation of dityrosine. But this interplay requires further study, as the formation of dityrosine in meat is rarely documented. Heat treatment strongly increased the formation of dityrosine in MPC, leading to an overall increase in dityrosine content in all MPC samples (Figure 3). Heat treatment could enhance the exposure of tyrosine residues inside the protein and thus increase the formation of dityrosine, as mentioned in previous studies.32,33 The promotion of dityrosine formation by heat treatment may partly cover up the inhibitory effects of NaNO2,

which were seen as a ten percent decrease in dityrosine at 50, 100, and 400 mg/kg NaNO2 (Figure 3). Sodium nitrite treatment also affected the levels of sulfhydryls in both the MPR and MPC samples. Treatment of MPR with NaNO2 resulted in lower sulfhydryl content than the MPR without NaNO2, in a dose-dependent manner (Figure 4). The dosedependent decrease in sulfhydryls in MPC was similar to that of MPR treated withNaNO2 (Figure 4). Sullivan and Sebranek28 found that decreasing sulfhydryls correlated with increasing input of NaNO2. Vossen and De Smet2 also observed a decrease in sulfhydryls in patties with 180 mg/kg NaNO2. The sulfhydryl of amino acid residue is highly susceptible to oxidation.4 Sodium nitrite is able to initiate oxidative reactions by abstracting an electron from a sensitive molecule in meat systems.5 Since the loss of sulfhydryls is mostly due to conversion to disulfide bonds,30,34 a concomitant increase in disulfide bond content in MPR and MPC treated with NaNO2 was observed, at approximately 1.4- to 4.6-fold higher compared to treatment without NaNO2 (Figure 4). The 10960


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Figure 4. Total sulfhydryl levels and disulfide bond content of MPR and MPC treated with different NaNO2 doses (mg/kg). Asterisk denotes a significant difference between means (n = 5) of MPR and MPC treated with the same NaNO2 dose (p < 0.05). Letters a−e denote significant differences between means (n = 5) of MPR or MPC treated with different NaNO2 doses (p < 0.05).

Figure 5. Representative SDS−PAGE patterns of MPR and MPC treated with different NaNO2 doses (mg/kg) (R, MFR; C, MPC; −, without βME; + , with βME).

significantly lighter MHC bands compared to the MPR samples with or without βME (Figure 5C,D). This indicated that heat treatment induced more polymerization of MPC (Figure 5C).36 Comparison of the MPR (more intense) and MPC (less intense) samples treated with βME indicated that a portion of unrecovered MHC in MPC was due to formation of other covalent bonds, such as Tyr-Tyr and active carbonyl-NH2 interactions.30 It is interesting that NaNO2 appeared to have both antioxidant and pro-oxidant effects on the meat protein.6 Sodium nitrite can react with myoglobin, resulting in nitrosylmyoglobin,37 which decomposes into nitric oxide, which can then eliminate activated oxygen species (ROS).5 In addition, compounds formed by reaction of nitric oxide with intermediates of lipid oxidation can also function as antioxidants.38 This may feed into the NaNO2-correlated protection against protein carbonylation (Tables 2 and 3). However, NaNO2 can initiate oxidative reactions of sensitive molecules5 and generate RNS that could stimulate oxidative reactions in meat products.39 Hence, the sulfhydryls had an NaNO2 dose-dependent decrease (Tables 2 and 3). We can conclude that the redox potential of the processed hams reached a balance in the antioxidant and pro-oxidant effects of NaNO2. While this balance should be further explored, the dual

formation of disulfide bonds cannot account for all the lost sulfhydryls, suggesting that some part of the sulfhydryls were changed to other oxidized species, perhaps sulfenic acid and sulfinic acid.28−30 In this study, heat treatment induced a notable loss of sulfhydryl content compared to the MPR sample in the absence of NaNO2 (Figure 4). Heat treatment has been reported to contribute to the formation of disulfide bonds in meat products.35 This could explain the significantly lower sulfhydryl levels and higher disulfide bond levels in MPC compared to those in MPR. The intra- or intermolecular covalent cross-linkings in MP were analyzed using electrophoretic mobility of the Myosin Heavy Chain (MHC). The intensity of the MHC bands in the MPR samples showed an NaNO2-dose dependent decrease (Figure 5 A). The MHC band intensities were higher when MPR-NaNO2 samples were treated with βME (Figure 5 A). This suggested that the disulfide bonds were the main factor contributing to the reduced mobility of the polymers (appearing near the top of the separating gel; Figure 5A,C). The intensity of the MHC bands in the MPC samples showed similar patterns (Figure 5B,C). These results were in accord with changes of the sulfhydryls and disulfide bonds in MPR and MPC (Figure 4). Heat treatment significantly decreased the intensity of the MHC bands. The MPC samples had 10961


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acids.40 Bax et al.8 found that heating promoted conformational changes or partial protein unfolding and protein oxidation that led to protein aggregation. In the present study, any amount of sodium nitrite decreased the surface hydrophobicity of MPC (Figure 6). This indicated that the NaNO2 had an antioxidant effect against protein denaturation, but it was only an approximate 3% decrease in surface hydrophobicity in the MPC treated with NaNO2 (Figure 6). 3-NT was found in proteins of raw and cooked ham in the absence of NaNO2. This level could be considered the basal level of 3-NT in muscle food. The 3-NT would likely be derived from the physiological nitrosative stress.6 RNS generated from NaNO2 can produce nitrosative stress and is able to initiate nitrosation of proteins.41 Under nitrosative stress, tyrosine might be modified though reactions between RNS (including peroxynitrite and nitrogen dioxide) and tyrosyl residues. 3-NT has been used as a marker for protein nitrosation,6 and its levels can be analyzed using HPLC or Western blot. In these results, a dose-dependent increase in 3NT can be seen when MPC or MPR was treated with NaNO2 compared to treatment without NaNO2 in present study (Figure 8 and Figure S2). The formation of 3-NT by the

effects of NaNO2 on protein modification further showed that NaNO2 is a useful food additive. Gel strength can be promoted by formation of bisulfide bonds in meat products.36 Physicochemical parameters within a foodstuff relate to changes in protein structure.36 Oxidative modification of amino acid residues on the surface of a protein can result in alteration of protein structure that can then expose the hydrophobic amino acids typically buried within the protein.40 The surface hydrophobicity of MPR showed an NaNO2-dose dependent decrease (p < 0.05) (Figure 6). The antioxidant effects of

Figure 6. Surface hydrophobicity of MPR and MPC treated with different NaNO2 doses (mg/kg). Asterisk denotes a significant difference between means (n = 5) of MPR and MPC treated with the same NaNO2 dose (p < 0.05). Letters a, b, and c denote significant differences between means (n = 5) of MPR or MPC treated with different NaNO2 doses (p < 0.05).

NaNO2 prevented the unfolding of the meat proteins during processing. Aggregates of proteins may rise in either a covalent or a noncovalent manner. Covalent aggregates form when a chemical bond, such as disulfide, distyrosine, and carbonyl-NH2 interactions, is formed between two or more monomers.30 Hydrophobic forces from exposed hydrophobic amino acid residue can also result in aggregation of MP.30 Turbidity is related to the presence of protein aggregates. The turbidity of MPR was lower with addition of NaNO2 (Figure 7). This may be explained by the fact that the aggregates caused by hydrophobic forces may cover up the covalent aggregates. Heat treatment had caused the denaturation of the protein, as seen as higher surface hydrophobicity compared to the raw ham (Figure 6). Heating could break the hydrogen bonds and van der Waals forces, thus exposing the interior hydrophobic amino

Figure 8. Effect of NaNO2 on protein nitrosation in MPR and MPC. Asterisk denotes a significant difference between means (n = 5) of MPR and MPC treated with the same NaNO2 dose (p < 0.05). Letters a−d denote significant differences between means (n = 5) of MPR or MPC treated with different NaNO2 doses (p < 0.05).

processing treatments showed a stark difference between the raw and cooked samples (Figure 8). Heat treatment decreases the stability of the nitro-amino acids residues. This may explain the significantly lower 3-NT content of MPC compared to that of MPR. In the present study, ascorbic acid was added to the hams to prevent the formation of the N-nitrosamines, which has been found to be carcinogenic.1 Villaverde et al.6 reported that nitric oxide could be produced from a reaction between ascorbic acid and NaNO2. Nitric oxide could further react with a superoxide radical to form peroxynitrite, which could initiate nitrosation of proteins and lead to the formation of 3-NT. There were contradictions between Villaverde et al.6 and Vossen and Smet,2 with no clear explanations for these contradictory results. Villaverde et al.6 reported that ascorbic acid can inhibit the nitrosation of MP dose-dependently. However, they also reported that the 3-NT content increased with increasing NaNO2 concentration at 250 mg/kg ascorbic acid.6 Other studies also found that nonheme muscle proteins can be nitrosated by NaNO2 to form 3-NT.42 Tyrosine nitration has been used as a marker for protein oxidation by RNS in human diseases.43 Herein, a correlation study established links between physicochemical and structural parameters and the measured 3-NT levels. The 3-NT content

Figure 7. Turbidity of MPR and MPC treated with different NaNO2 doses (mg/kg). Asterisk denotes a significant difference between means (n = 5) of MPR and MPC treated with the same NaNO2 dose (p < 0.05). Letters a−d denote significant differences between means (n = 5) of MPR or MPC treated with different NaNO2 doses (p < 0.05). 10962



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of MPR had significantly negative correlations with sulfhydryls and surface hydrophobicity (Table 2). More noticeably, the 3NT content of MPC had significantly negative correlations with carbonyls and sulfhydryls but a significantly positive correlation with disulfides (Table 3). It is challenging work to study the effect of nitrosation on protein structure and function. Protein oxidation is almost inevitable when protein nitrosation is induced in vitro.44 Sodium nitrite is able to yield RNS, such as peroxynitrite, which may be able to initiate both oxidation and nitration of protein.41 Tyrosyl residues in proteins might competitively react with RNS to form 3-NT, so the formation of carbonyls during protein oxidization could be partly inhibited. Protein residues like cysteine and methionine are susceptible to attack by nitrating agents.45 As a result, the formation of disulfide bonds could not be prevented. While nitration of tyrosine-containing peptides makes them more hydrophobic,46 the surface hydrophobicity of MP did not increase with increasing 3-NT (nitrosation degree). This may be due to masking of the effects of nitrosation on surface hydrophobicity by other physicochemical changes in the MP.45 Therefore, the effects of nitrosation of tyrosine residues on the structural properties of protein need to be studied in depth. As far as the authors know, this study is the first to provide evidence of potential relationships between 3-NT and other parameters of oxidative modification in meat products. These data also indicated that 3-NT can be a potential marker for protein oxidation in processed cured meat products.2

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REFERENCES

(1) Sindelar, J. J.; Milkowski, A. L. Sodium nitrite in processed meat and poultry meats: a review of curing and examining the risk/benefit of its use. AMSA White Paper Series 2011, 1−14. (2) Vossen, E.; De Smet, S. Protein Oxidation and Protein Nitration Influenced by Sodium Nitrite in Two Different Meat Model Systems. J. Agric. Food Chem. 2015, 63, 2550−2556. (3) Sebranek, J. G. Basic Curing Ingredients. Ingredients in Meat Products 2009, 1−23. (4) Lund, M. N.; Heinonen, M.; Baron, C. P.; Estevez, M. Protein oxidation in muscle foods: A review. Mol. Nutr. Food Res. 2011, 55, 83−95. (5) Skibsted, L. H. Nitric oxide and quality and safety of muscle based foods. Nitric Oxide 2011, 24, 176−183. (6) Villaverde, A.; Parra, V.; Estévez, M. Oxidative and nitrosative stress induced in myofibrillar proteins by a hydroxyl-radical-generating system: impact of nitrite and ascorbate. J. Agric. Food Chem. 2014, 62, 2158−2164. (7) Santé-Lhoutellier, V.; Astruc, T.; Marinova, P.; Greve, E.; Gatellier, P. Effect of meat cooking on physicochemical state and in vitro digestibility of myofibrillar proteins. J. Agric. Food Chem. 2008, 56, 1488−1494. (8) Bax, M.-L.; Aubry, L.; Ferreira, C.; Daudin, J.-D.; Gatellier, P.; Rémond, D.; Santé-Lhoutellier, V. r. Cooking temperature is a key determinant of in vitro meat protein digestion rate: investigation of underlying mechanisms. J. Agric. Food Chem. 2012, 60, 2569−2576. (9) Astruc, T.; Gatellier, P.; Labas, R.; Lhoutellier, V. S.; Marinova, P. Microstructural changes in m. rectus abdominis bovine muscle after heating. Meat Sci. 2010, 85, 743−751. (10) Deb-Choudhury, S.; Haines, S.; Harland, D.; Clerens, S.; Koten, C. V.; Dyer, J. M. Effect of cooking on meat proteins: mapping hydrothermal protein modification as a potential indicator of bioavailability. J. Agric. Food Chem. 2014, 62, 8187−8196. (11) Papagianni, M.; Sergelidis, D. Effects of the presence of the curing agent sodium nitrite, used in the production of fermented sausages, on bacteriocin production by Weissella paramesenteroides DX grown in meat simulation medium. Enzyme Microb. Technol. 2013, 53, 1−5. (12) Dineen, N.; Kerry, J.; Lynch, P.; Buckley, D.; Morrissey, P.; Arendt, E. Reduced nitrite levels and dietary α-tocopheryl acetate supplementation: effects on the colour and oxidative stability of cooked hams. Meat Sci. 2000, 55, 475−482. (13) Estrada, C. S. M. L.; Velázquez, L. D. C.; Genaro, S. D.; Guzmán, A. M. S. D. Comparison Of Dna Extraction Methods For Pathogenic Yersinia Enterocolitica Detection From Meat Food By Nested Pcr. Food Res. Int. 2007, 40, 637−642. (14) Sante-Lhoutellier, V.; Aubry, L.; Gatellier, P. Effect of oxidation on in vitro digestibility of skeletal muscle myofibrillar proteins. J. Agric. Food Chem. 2007, 55, 5343−5348. (15) Ouali, A.; Talmant, A. Calpains and calpastatin distribution in bovine, porcine and ovine skeletal muscles. Meat Sci. 1990, 28, 331− 348. (16) Oliver, C. N.; Ahn, B.-W.; Moerman, E. J.; Goldstein, S.; Stadtman, E. R. Age-related changes in oxidized proteins. J. Biol. Chem. 1987, 262, 5488−5491. (17) Liu, G.; Xiong, Y.; Butterfield, D. Chemical, Physical, and Gelforming Properties of Oxidized Myofibrils and Whey-and Soy-protein Isolates. J. Food Sci. 2000, 65, 811−818. (18) Davies, K.; Delsignore, M.; Lin, S. Protein damage and degradation by oxygen radicals. II. Modification of amino acids. J. Biol. Chem. 1987, 262, 9902−9907. (19) Cui, X.; Xiong, Y. L.; Kong, B.; Zhao, X.; Liu, N. Hydroxyl radical-stressed whey protein isolate: Chemical and structural properties. Food Bioprocess Technol. 2012, 5, 2454−2461. (20) Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82, 70−77. (21) Martinaud, A.; Mercier, Y.; Marinova, P.; Tassy, C.; Gatellier, P.; Renerre, M. Comparison of oxidative processes on myofibrillar

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04107. Figure S1, effect of NaNO2 on lipid oxidation; Figure S2, effect of NaNO2 on protein nitrosation; Figure S3, the emission spectra for dityrosine formation; Tables S1 and S2, analysis of variance between groups (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: 86029-87092486. E-mail: [email protected] (L.C.). Funding

This work was supported by the National Natural Science Fund for Young Scholars (Grant No: 29 31401515), Shaanxi Province Science and Technology Research and Development Project (Grant No: 2015NY022), Open Project of National Center of Meat Quality and Safety Control (Grant No: M2015K06), Doctoral Research Funding (Grant No: Z109021403), Shaanxi Province Research Funding for Recruiting Doctor (Grant No: Z111021507), Scientific and Technological Innovation Funding for General Project (Grant No: Z109021504) and Earmarked Fund for Modern Agroindustry Technology Research System, China (Grant No: nycytx-42-G5-01). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Wanqiang Wu for his skilled technical support. 10963


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Potential Biomarker of Myofibrillar Protein Oxidation in Raw and

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