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Protein carbonyls in meat systems,A review

Review

Protein carbonyls in meat systems:A review

Mario Estévez ?

Animal Production and Food Science Department,Food Technology,University of Extremadura,10003,Cáceres,Spain

a b s t r a c t

a r t i c l e i n f o Article history:

Received 1March 2011

Received in revised form 20April 2011Accepted 25April 2011Keywords:DNPH

α-Aminoadipic semialdehyde γ-Glutamic semialdehyde Metal-catalyzed oxidation Meat quality

Protein oxidation

Protein oxidation (P-OX)is an innovative topic of increasing interest among meat researchers.Carbonylation is generally recognized as one of the most remarkable chemical modi ?cations in oxidized proteins.In fact,the quanti ?cation of protein carbonyls by the dinitrophenylhydrazine (DNPH)method is the most common procedure for assessing P-OX in meat systems.Numerous studies have investigated the occurrence of protein carbonylation right after slaughter and during subsequent processing and cold storage of meat.However,the signi ?cance of protein carbonylation in meat systems is still poorly understood.Beyond their role as markers of protein oxidation,speci ?c protein carbonyls such as α-aminoadipic and γ-glutamic semialdehydes (AAS and GGS,respectively)are active compounds that may be implicated in several chemical reactions with relevant consequences on meat quality.The formation of protein carbonyls from particular amino acid side chains contribute to impair the conformation of myo ?brillar proteins leading to denaturation and loss of functionality.Recent studies also highlight the potential impact of speci ?c protein carbonyls in particular meat quality traits such as water-holding capacity (WHC),texture,?avor and its nutritional value.As a truly emerging topic,the results from current studies provide grounds from the development of further investigations.The present paper reviews the current knowledge on the mechanisms and consequences of protein carbonylation in meat systems and aims to encourage meat researchers to accomplish further investigations on this fascinating research topic.

Contents 1.Introduction ..........................

....................................2602.Protein oxidation:An emerging topic ..................................................2603.

Chemical mechanisms and factors....................................................2613.1.Protein oxidation:General mechanisms..............................................2613.2.Protein carbonylation as expression of protein oxidation......................................2633.3.Carbonylation of meat proteins:Speci ?c mechanisms ..

.....................................2633.3.1.Role of MCO systems ..................................................2633.3.2.Role of myoglobin ...................................................2653.3.3.Role of oxidizing lipids .................................................2663.3.4.Other factors .................

.....................................2663.4.Reactivity of protein carbonyls ..................................................2664.

Assessment of protein carbonylation ..................................................2684.1.Total carbonyl content:The DNPH method ............................................2684.2.Analysis of speci ?c protein carbonyls ...............................................2685.

Protein carbonylation in meat and meat products ............................................2685.1.Protein carbonylation during meat aging and chill storage .....................................2685.2.Protein carbonylation during frozen storage of meat........................................2695.3.Protein carbonylation during meat processing...........................................2706.

Impact of protein carbonylation on meat quality .............................................2706.1.Protein conformation and functionality .........

.....................................

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Abbreviations:AAA,aminoadipic acid;AAS,α-aminoadipic semialdehyde;DNPH,dinitrophenylhydrazine;GGS,γ-glutamic semialdehyde;P-OX,protein oxidation;Mb,myoglobin;MetMb,Metmyoglobin;MCO,metal-catalyzed oxidation;MP,myo ?brillar proteins;ROS,reactive oxygen species;WHC,water-holding capacity.?Tel.:+34927257122;fax:+34927257110.E-mail address:mariovet@unex.es

10.1016/j.meatsci.2011.04.025

Contents lists available at ScienceDirect

Meat Science

j o u r n a l h o me p a g e :w w w.e l sev i e r.c o m /l oc a t e /me a t s c i

6.2.Nutritional value (272)

6.3.Sensory traits (273)

6.3.1.Texture (273)

6.3.2.Flavor (273)

7.Antioxidant strategies against protein carbonylation (273)

7.1.Nutritional strategies (274)

7.2.Technological strategies (274)

8.Final remarks (276)

Acknowledgments (276)

References (276)

1.Introduction

The occurrence of protein oxidation(P-OX)in biological systems has been known and studied for around50years owing to the connection between the oxidative damage to proteins and the development of age-related diseases(Berlett&Stadtman,1997;Shacter,2000;Stadtman, 1990;Stadtman&Oliver,1991).Only20years ago,in contrast,the fact that food proteins may be sources and targets for reactive oxygen species(ROS)was mostly ignored.The discovery that muscle proteins were susceptible to oxidative reactions leading to potential deleterious effects on meat quality(Decker,Xiong,Calvert,Crum,&Blanchard, 1993;Mercier,Gatellier,&Renerre,1995)greatly promoted the interest for this issue.The oxidation of food proteins is currently one of the most innovative research topics within the Food Science?eld.

P-OX is a complex phenomenon as the pathways and the nature of the products depend on the targets and how the oxidative reactions commence(Davies,2005).Numerous ROS are able to initiate the oxidative damage to proteins and transition metals as well as oxidizing lipids are known to be in?uential(Stadtman&Levine,2003).The chemical modi?cations caused to speci?c amino acid side chains and/or to the peptide backbone can lead to changes in the physical properties of the proteins,including fragmentation,aggregation,loss of solubility and functionality and decreased susceptibility to proteolysis(Xiong,2000). In meat systems,P-OX has been assessed through several of its multiple chemical manifestations including loss of sulfhydryl groups(Freder-iksen,Lund,Andersen,&Skibsted,2008;Martinaud et al.,1997),loss of tryptophan?uorescence(Estévez,Kylli,Puolanne,Kivikari,&Heinonen, 2008a;Ganh?o,Morcuende,&Estévez,2010a;Sun,Zhao,Yang,Zhao,& Cui,2011a),gain of carbonyl derivatives(Decker et al.,1993;Estévez, Ventanas,&Cava,2005;Ganh?o,Morcuende,&Estévez,2010b;Mercier, Gatellier,Viau,Remignon,&Renerre,1998)and formation of intra-and intermolecular cross-links(Ooizumi&Xiong,2006;Xiong,Park,& Ooizumi,2009).Among the aforementioned changes,the formation of carbonyl compounds has been highlighted as one of the most salient modi?cations in oxidized proteins(Levine et al.,1990;Stadtman& Levine,2003;Xiong,2000).In fact,the quanti?cation of the total amount of protein carbonyls by using the dinitrophenylhydrazine(DNPH) technique(Oliver,Ahn,Moerman,Goldstein,&Stadtman,1987)is probably the most frequent method for assessing P-OX in meat and biological systems(Estévez,Morcuende,&Ventanas,2008b;Nystr?m, 2005;Requena,Levine,&Stadtman,2003;Tornv?ll,2010).It remains contradictory that protein carbonyls are commonly employed as indicators of P-OX while the actual signi?cance of protein carbonylation in food systems is inde?nite.The impact of P-OX on meat quality is still the subject of multiple studies but it is generally accepted that the activity of muscle proteases and the functionality of myo?brillar proteins(MP)are affected by oxidative reactions(Carlin,Huff-Lone-rgan,Rowe,&Lonergan,2006;Xiong,2000).The digestibility and water-holding capacity(WHC)of muscle proteins as well as the post-mortem tenderization of meat are believed to be affected by oxidative reactions (Bertram et al.,2007;Huff-Lonergan,Zhang,&Lonergan,2010;Liu, Xiong,&Chen,2010).The precise mechanisms by which protein carbonylation could in?uence these undesirable P-OX-induced changes are not fully clear.The identi?cation of the routes and mechanisms involved in the formation of speci?c protein carbonyls is essential to establish the potential implication of such compounds on particular quality traits.Recently,speci?c carbonyls,namely,α-amino adipic and γ-glutamic semialdehydes(AAS and GGS,respectively)were identi?ed in oxidized MP by using a derivatization procedure described by Akagawa et al.(2006)followed by liquid chromatography–electrospray ionization–mass spectrometry(LC–ESI–MS)analysis(Estévez,Ollilai-nen,&Heinonen,2009).This accurate methodology enabled the accomplishment of subsequent studies devoted to investigate the role of transition metals and phenolic compounds on the carbonylation of MP(Estévez&Heinonen,2010),the potential role of the semialdehydes in Strecker-type reactions with non-oxidized amino acids(Estévez et al., 2011)and the plausible implication of protein carbonylation in the loss of WHC occurred during freezing storage of meat(Estévez et al.,2011).

Xiong(2000)and more recently Lund,Heinonen,Baron,and Estévez(2011)reported general overviews on the occurrence of P-OX in muscle foods.The analytical methods employed for the assessment of P-OX in biological and food systems have also been reviewed (Estévez et al.,2008b;Hawkins,Morgan,&Davies,2009;Requena et al.,2003;Tornv?ll,2010).The present paper focuses on protein carbonylation as an expression of the oxidative damage to meat proteins.This review covers the current knowledge on this topic and aims to elucidate future challenges.

2.Protein oxidation:An emerging topic

As a major component of muscle tissue,proteins play a decisive role in meat products regarding sensory,nutritional and technological aspects(Lawrie,1998).Muscle proteins have been subjects of multiple research studies focused on the modi?cations undergone during post-mortem changes,processing and storage of meat and meat products. These modi?cations mostly comprise denaturation(=loss of their native tertiary structure)and hydrolytic degradation(=proteolysis) by endogenous and/or exogenous enzymes.These biochemical phe-nomena have been profusely studied in relation to their impact on meat quality(Kazemi,Ngadi,&Gariépy,2011;Kemp,Sensky,Bardsley, Buttery,&Parr,2010;Toldrá,1998;Van Laack,Liu,Smith,&Loveday, 2000).In contrast,the fact that food proteins may be susceptible to oxidative reactions has been ignored for decades.Taking into consideration the great heights of sophistication reached in other food research?elds(e.g.‘lipid oxidation’),the lack of basic knowledge on P-OX in food systems is considerably surprising.According to the SciVerse Scopus database,‘Food Science and Technology’SCI journals have published574articles devoted to‘protein oxidation’in total while the ‘lipid oxidation’topic has been covered in4054articles.Several may be the reasons for this uneven situation including i)the high complexity of the chemistry behind the oxidation of food proteins,ii)the lack of speci?c methodologies for assessing P-OX in food systems and iii)the belief that other biochemical phenomena such as lipid oxidation or microbial spoilage explained all deleterious changes occurred in food systems.Interestingly,half of the studies devoted to‘protein oxidation’

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have been accomplished in the last5years which re?ects the increasing interest of food researchers in this emerging topic.

The?rst approach to P-OX carried out by meat scientists took place in mid nineties by using knowledge and techniques from medical research(Decker et al.,1993;Mercier et al.,1995).The discovery that meat proteins were susceptible to oxidative reactions and that oxidized proteins may have an impact on meat quality boosted the interest among meat researchers(Xiong,2000).Numerous subsequent studies shed light on unknown aspects on the oxidative modi?cations of muscle proteins during post-mortem changes,handling,processing and storage of meat(Estévez et al.,2005;Fuentes,Ventanas,Morcuende,Estévez,& Ventanas,2010;Lund,Lametsch,Hvii,Jensen,&Skibsted,2007a;Rowe, Maddock,Lonergan,&Huff-Lonergan,2004;Salminen,Estévez,Kivikari, &Heinonen,2006;Ventanas,Ventanas,Tovar,García,&Estévez,2007; Xia,Kong,Liu,&Liu,2009).In some cases,the lack of speci?c knowledge and scienti?c grounds prevented from meaningful discussions about the impact of P-OX in meat products and plain descriptive studies were accomplished instead.Some authors,for instance,quanti?ed the gain of protein carbonyls during meat processing although the technological meaning of such measurement remained unknown(Cava,Ladero, González,Carrasco,&Ramírez,2009;Ventanas,Estévez,Tejeda,&Ruiz, 2006).Some other authors described the ef?cacy of certain antioxidant strategies against meat protein carbonylation while the actual bene?t of such protection in the meat product was not fully recognized(Estévez& Cava,2006;Mercier et al.,1998).In order to progress towards the understanding of the signi?cance of P-OX in meat systems,subsequent works accomplished more challenging studies devoted to the chemistry behind the oxidation of muscle proteins(Gatellier,Mercier,Rock,& Renerre,2000;Batifoulier,Mercier,Gatellier,&Renerre,2002;Estévez& Heinonen,2010;Estévez,Kylli,Puolanne,Kivikari,&Heinonen,2008c; Estévez et al.,2009;Ooizumi&Xiong,2006;Park&Xiong,2007; Lametsch,Lonergan&Huff-Lonergan,2008;Liu&Xiong,2000). Nevertheless,numerous and relevant issues regarding the impact of P-OX in meat systems require further investigations on basic meat chemistry.As long as the chemical nature of particular protein oxidation products is inde?nite,the potential effect of oxidized meat proteins on meat quality or human's health will remain unknown.The current lack of knowledge may explain that most review articles and academic books still ignore that meat proteins and the products of their hydrolysis (peptides and amino acids)are actively implicated in numerous and complex oxidative reactions.It is worth noting that the global knowledge on Food Chemistry is clearly incomplete without the full comprehension of the biochemical reactions in which oxidizing proteins may be implicated.It is plausible to consider that the onset of P-OX in complex food systems such as meat products,may not only have an impact on the protein fraction but also in other food components affected by such reactions(i.e.lipids).In this sense,lipid oxidation and other biochemical reactions with great impact on food quality such as the Maillard reaction are known to interact in complex food systems and share common chemical mechanisms and intermediate compounds (Zamora&Hidalgo,2005).As a matter of fact,the understanding of the chemical mechanisms,routes and products formed as a result of the oxidative degradation of food proteins would enable a full comprehen-sion of the potential interactions between P-OX and other biochemical changes and the consequences of such interactions on food quality.The development of innovative methodologies for the analysis of speci?c protein oxidation products and the advanced knowledge gained during the last years may serve as a solid ground to endure working on this fascinating topic.

3.Chemical mechanisms and factors

3.1.Protein oxidation:General mechanisms

Numerous ROS such as the superoxide(O2??),the hydroperoxyl (HO2?)and hydroxyl(HO?)radicals and other nonradical species such as the hydrogen peroxide(H2O2)and hydroperoxides(ROOH)have been recognized as potential initiators of P-OX(Butter?eld&Stadtman, 1997).Natural components of the muscle tissue such as unsaturated lipids,heme pigments,transition metals and oxidative enzymes are potential precursors or catalysts for the formation of ROS and hence, play a relevant role in the initiation of muscle P-OX(Xiong,2000).The peptide backbone and the functional groups located in the side chain of amino acid residues are common targets for ROS.As a direct consequence of the abstraction of a hydrogen atom from a susceptible target(PH),a carbon-centered protein radical(P?)is formed(Reac-tion1)(Stadtman&Levine,2003).The initial P?is consecutively converted into a peroxyl radical(POO?)in the presence of oxygen,and to an alkyl peroxide(POOH)by abstraction of a hydrogen atom from another susceptible molecule(Reactions2and3).Further reactions with ROS such as the HO2?radical or with reduced forms of transition metals(M n+)such as Fe2+or Cu+lead to the formation of an alcoxyl radical(PO?)(Reactions4and5)and its hydroxyl derivative(POH) (Reactions6and7)as follows:

PHtHO?→P?tH2Oe1TP?tO2→POO?e2TPOO?tPH→POOHtP?e3TPOOHtHO?2→PO?tO2tH2Oe4T

POOHtM nt→PO?tHO?tMent1Tte5TPO?tHO?2→POHtO2e6T

PO?tHttM nt→POHtMent1Tt:e7TThe speci?c routes and chemical nature of the?nal oxidation products depends on the target,the oxidizing system and the intensity of the oxidation conditions(Davies,2005).The oxidative modi?cation of the amino acid side chains,the conversion of one amino acid into a different one,the fragmentation of the peptide backbone and the formation of intra-and inter-molecular cross-links are common consequences of ROS-mediated protein oxidation (Stadtman&Levine,2000).Besides the intrinsic susceptibility of particular amino acid residues to undergo oxidative reactions,their position within the protein structure largely affects their exposure to oxidation promoters and hence,their oxidative degradation.Certain amino acids such as cysteines and methionine would be?rstly oxidized at mild oxidation conditions(low concentration of ROS) owing to the high susceptibility of sulfur centers.Tryptophan residues are also promptly oxidized in the presence of transition metals with this phenomenon being regarded as an early protein oxidation manifestation(Estévez et al.,2008a,c;Viljanen,Kivikari,&Heinonen, 2004).Interestingly,the early and preferential oxidation of certain amino acids such as methionine would involve the so-called ‘sacri?cial protection’,by which certain amino acids with antioxidant potential and insigni?cant role in the protein functionality would scavenge ROS and hence,protect other susceptible amino acids against oxidation(Levine,Berlett,Moskovitz,Mosoni,&Stadtman, 1999).At intense oxidation conditions,such as those existing during

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severe metal-catalyzed oxidation(MCO)of proteins,large amounts of protein carbonyls are generated.ROS-mediated oxidation of proteins leads to the conversion of histidine to oxohistidine and imidazolone derivatives,tryptophan residues to kynurenine or N-formylkynur-enine,and leucine and valine to hydroxy derivatives(Stadtman& Levine,2000).Sulfur-containing amino acids such as cysteine and methionine are highly susceptible to oxidation in the presence of oxidizing lipids and to yield varied sulfur-containing compounds such as sulfone,sulfoxide and disulphide derivatives(Stadtman&Levine, 2000).Cross-linking is usually attributed to the formation of cystine (disulphide bonds)and dityrosines from two cysteine and two tyrosine residues,respectively(Lund et al.,2011).The formation of carbonyl compounds principally derives from the oxidation of threonine,proline,arginine and lysine residues(Stadtman&Levine, 2003).Previous book chapters and review articles have reported in detail the large variety of oxidation-induced changes in proteins from food and biological systems(Butter?eld&Stadtman,1997;Davies, 2005;Stadtman&Berlett,1989;Stadtman&Levine,2003;Lund et al., 2011;Xiong,2000).The present paper will only cover in detail the formation of carbonyl derivatives(Section3.2)with particular stress on mechanisms and factors speci?cally ascribed to meat proteins (Section3.3).

3.2.Protein carbonylation as expression of protein oxidation

Carbonylation is an irreversible and non-enzymatic modi?cation of proteins that involves the formation of carbonyl moieties induced by oxidative stress and other mechanisms(Berlett&Stadtman,1997). Carbonyls(aldehydes and ketones)can be formed in proteins through four different pathways,namely,i)direct oxidation of the side chains from lysine,threonine,arginine and proline(Requena,Chao,Levine,& Stadtman,2001),ii)non-enzymatic glycation in the presence of reducing sugars(Akagawa,Sasaki,Kurota,&Suyama,2005);iii) oxidative cleavage of the peptide backbone via theα-amidation pathway or via oxidation of glutamyl side chains(Berlett&Stadtman, 1997;Garrison,1987)and iv)covalent binding to non-protein carbonyl compounds such as4-hydroxy-2-nonenal(HNE)or malondialdehyde (MDA)(Feeney,Blankenhorn,&Dixon,1975)(Fig.1).Among the four pathways,the direct oxidation of susceptible amino acid side chains has been highlighted as the main route for protein carbonylation and the most potent and major source of direct oxidative attack to proteins (Shacter,2000;Stadtman,1990;Stadtman&Levine,2000).In addition, this is,to our knowledge,the only mechanism that has been proved to yield carbonyls from meat proteins(Estévez et al.,2009;Estévez& Heinonen,2010).Park,Xiong,and Alderton(2006a)reported a negligible impact of peptide scission on the production of carbonyls during in vitro oxidation of MP.Whereas the other three mechanisms may be applicable to complex food systems,the relative contribution of such pathways to the carbonylation of meat proteins remains unknown.

The formation of carbonyl derivatives from lysine,threonine, arginine and proline side chains is usually attributed to MCO systems (Stadtman&Levine,2003).According to this mechanism,reduced forms of transition metals would reduce H2O2to form a reactive intermediate(hydroxyl radical;?OH)through the Fenton reaction (Reaction8)in the immediate proximity of a susceptible amino acid side chain.

M nttH2O2→Ment1TttHO?tHO?e8TThe presence of metal binding sites in the proteins explains that amino acid residues situated at such locations are uniquely sensitive to MCO by a site-speci?c mechanism(Stadtman&Levine,2003).In this sense,certain authors consider that MCO is limited to the metal binding sites of proteins at mild oxidation conditions while virtually all amino acid residues would be affected at high concentrations of H2O2and metal ions such as Fe2+.The oxidized forms of such ions such as Fe3+could generate HO2?radicals from H2O2through a Fenton-like reaction(Reaction9)(De Laat&Gallard,1999):

Fe3ttH2O2→Fe2ttHO?2tHt:e9TThere are enough scienti?c evidences to support that both reduced and oxidized forms of iron(Fe2+and Fe3+,respectively)are able to promote the in vitro formation of protein carbonyls through ROS-mediated reactions(Akagawa et al.,2006;Estévez et al.,2009).In fact, the two oxidation states of certain metal ions such as iron(Fe2+/Fe3+) and copper(Cu+/Cu2+)would coexist in most in vitro and biological systems where they act as electron donors(the reduced forms)or acceptors(the oxidized forms).This redox cycle impart important catalytic properties including reduction of molecular oxygen to form superoxide anion radical(Reaction10),which undergoes successive reactions to form hydrogen peroxide(Reaction11)and hydroxyl radical(Haber–Weiss reaction,12),and the cleavage of lipid hydroperoxides(LOOH)to form peroxyl and alkoxyl radicals(Re-actions13and14)(Kanner,Hazan,&Doll,1988).

M nttO2→Ment1TttO??2e10T2O??2t2Ht→H2O2tO2e11TO??2tH2O2→O2tHO?tHO?e12TM nttLOOH→Ment1TttLO?tHO?e13T

Ment1TttLOOH→M nttLOO?tHt:e14TAs a consequence of MCO,threonine is converted intoα-amino-3-keto butyric acid,lysine intoα-amino adipic semialdehyde(AAS),and arginine and proline intoγ-glutamic semialdehyde(GGS).The two latter(AAS and GGS)were originally proposed as biomarkers of oxidative damage to proteins by Daneshvar,Frandsen,Autrup,and Dragsted(1997).Later on,both semialdehydes were highlighted as the main carbonyl products from the MCO of proteins with both semialdehydes accounting between23and60%of the total carbonyl compounds in oxidized plasma and liver proteins(Akagawa et al., 2006;Requena et al.,2001).According to the formation pathway, thoroughly described?rst by Stadtman and Oliver(1991)and afterwards by Akagawa et al.(2006),the side chains of the susceptible amino acids are oxidatively deaminated in the presence of transition metals such as iron and copper(Fig.2).The reactive species would attack the amino group from the amino acid side chain by abstracting a hydrogen atom from the neighboring carbon,leading to the formation of a carbon-centered protein radical.In a further step, oxidized forms of the metal ions would accept the lone electron of the carbon radical to form an imino group which is spontaneously hydrolyzed to yield the corresponding aldehyde moiety(Akagawa et al.,2006).

3.3.Carbonylation of meat proteins:Speci?c mechanisms

3.3.1.Role of MCO systems

Meat proteins are susceptible to oxidative reactions leading to the formation of carbonyl compounds.The carbonylation of meat proteins can be induced in vitro by using several ROS-generating systems including MCO,myoglobin-mediated oxidation and lipid-oxidizing systems(Decker et al.,1993;Estévez et al.,2009;Estévez&Heinonen, 2010;Park et al.,2006a;Park,Xiong,Alderton,&Oozumi,2006b)

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(Table 1).In agreement with reports from medical research,the combination of transition metals with H 2O 2has been proved to have an effective pro-oxidant effect on meat proteins (Decker et al.,1993;Martinaud et al.,1997;Xiong &Decker,1995).Decker et al.(1993)found that a combination of metal ions (Fe 3+/Cu 2+)with ascorbic acid was effective at inducing the formation of carbonyls from in vitro oxidized MP.The results indicate that addition of H 2O 2is not required as transition metals could generate reactive species from oxygen and/or existing H 2O 2.The ascorbic acid creates a ‘redox-cycle ’by reducing the oxidized form of the metal ion to the reduced counterpart,which,in turn,sustains the formation of ROS from oxygen and/or H 2O 2(Kanner et al.,1988).The same authors found that Fe 3+was more effective than Cu 2+at promoting protein carbonylation.Xiong and Decker (1995)reported that the signi ?cant increase of carbonyls in muscle proteins incubated with Fe 2+/ascorbate occurred concomi-tantly with a loss of ε-NH 3groups,which emphasizes the oxidative deamination of amino acid side chains as a main route for the formation of carbonyl moieties in meat proteins.Uchida,Kato,and Kawakishi (1992)also reported a decrease of basic amino acids during in vitro oxidation of collagen.According to the results from that study,Cu 2+/H 2O 2exerted a more intense pro-oxidant effect than Fe 2+/H 2O 2.The effectiveness of ferric and ferrous iron in combination with H 2O 2for the formation of carbonyls from meat proteins was also con ?rmed by Martinaud et al.(1997),Mercier,Gatellier,and Renerre (2004)and Park et al.(2006a,b).It is worth to mention that the methodology employed by the above mentioned authors (DNPH-method;Section 3.5.1)provides an estimation of the total amount of protein carbonyls regardless of the formation pathway (see Section 3.4.).Taking into consideration that an inde ?nite proportion of DNPH-derivatized carbonyls may not derive from metal-catalyzed oxidative reactions,the inference on the oxidation mechanisms by using such methodology is of limited value.Twelve years after the report by Daneshvar et al.(1997),major protein carbonyls,AAS and GGS,were found to be present in food proteins oxidized in vitro by

MCO systems (Estévez et al.,2009).In a recent study,AAS and GGS were found to account up to 70%of the total protein carbonyls yielded in a moderately oxidized meat product (Utrera et al.,in press ),which is in accordance with reports from medical research.By analyzing such speci ?c carbonyls,Estévez and Heinonen (2010)found that Cu 2+was more effective at promoting the formation of AAS and GGS from MP than Fe 3+.Since iron has been reported to be more effective than copper at catalyzing the formation of hydroxyl radicals (Rowley &Halliwell,1983),the results obtained by Uchida et al.(1992)and Estévez and Heinonen (2010),among others,should respond to other mechanisms.Hawkins and Davies (1997)found similar results and ascribed to a site-binding mechanism the higher and more speci ?c pro-oxidant effect of copper on collagen compared to that exerted by iron.Unlike iron,copper is able to bound to particular binding sites in proteins such as collagen,leading to an enhanced and “caged ”pro-oxidant action towards amino acid side chains in such locations.Knott,Baoutina,Davies,and Dean (2002),Thanonkaew,Benjakul,Visessanguan,and Decker (2006)and Letelier,Sánchez-Jofré,Peredo-Silva,Cortés-Troncoso,and Aracena-Parks (2010)also referred to this mechanism for explaining the severe pro-oxidant effect of copper ions towards different animal proteins.In contrast to other authors,Letelier et al.(2010)claimed that the site-binding mechanism of copper on macromolecules may be non-speci ?c.Interestingly,copper is co-factor of enzymes such as the lysyl oxidase,aimed to catalyze the formation of carbonyls from lysine residues in collagen and ellastine (Feeney et al.,1975).

3.3.2.Role of myoglobin

Besides transition metals,other natural components of muscle such as myoglobin (Mb),have been proved to promote P-OX and in particular,protein carbonylation.In fact,Estévez and Heinonen (2010)found that H 2O 2-activated Mb promoted the formation of AAS and GGS from MP to a greater extent than Cu 2+/H 2O 2and Fe 3+/H 2O 2.Consistently,Park et al.(2006a,b)reported that metmyoglobin

Table 1

Summary of studies in which carbonylation a of muscle proteins was induced in vitro by several oxidizing systems.Model system Oxidation initiators Oxidative conditions Additional analyses Reference

SSP (6mg/mL)25μM FeCl 3+(0–25mM)A 0.12M KCl,pH 6,23°C,6h Protein solubility,WHC,Gel Strength

Decker et al.(1993)SSP (6mg/mL)25μM CuCl 2+(0–25mM)A 0.12M KCl,pH 6,23°C,6h Protein solubility,WHC,Gel Strength

Decker et al.(1993)MPI (5mg/mL) 2.5mM FeSO 4+2.5mM H 2O 250mM KCl,pH 7.4,37°C,5h Protein thiol oxidation,SDS-PAGE

Martinaud et al.(1997)MPI (5mg/mL)

0.1mM FeCl 3+2.5mM H 2O 2

50mM KCl,pH 7.4,37°C,5h Protein thiol oxidation,SDS-PAGE Martinaud et al.(1997)Turkey muscle extract (0.1g/mL)

0.11mM FeSO 4+2.19mM NADPH +2.63mM ADP

pH 7.4,20°C,5h TBA-RS,ESR Gatellier et al.(2000)Turkey microsomal fraction (1mg/mL)

0.1mM FeCl 3+0.5mM A pH 7.4,37°C,5h TBA-RS,Glucose-6-phosphatase Mercier,Gatellier,Vincent,and Renerre (2001)Turkey microsomal fraction (2mg/mL)

50μM MetMb +50mM H 2O 2pH 7,37°C,24h TBA-RS,ESR Batifoulier et al.(2002)Pork homogenate (0.1g/mL) 2.5mM FeSO 4+1mM H 2O 2

pH 7,37°C,5h

TBA-RS

Mercier et al.(2004)MPI (40mg/mL)(0.01and 0.1mM)FeCl 3+(0.00–10mM)H 2O 20.6NaCl,pH 6,4°C,24h ATPase activity,TBA-RS,DSC Park et al.(2006a)MPI (30mg/mL)10μM FeCl 3+0.1mM AA+(0.05–5.0mM)H 2O 20.6NaCl,pH 6,4°C,24h ATPase activity,TBA-RS,DSC Park et al.(2006b)MPI (30mg/mL)(0.05–5.0mM)LA +3750u.lipoxidase/mL 0.6NaCl,pH 6,4°C,24h ATPase activity,TBA-RS,DSC Park et al.(2006b)MPI (30mg/mL)(0.05–0.5mM)metmyoglobin

0.6NaCl,pH 6,4°C,24h

ATPase activity,TBA-RS,DSC Park et al.(2006b)MPI (20mg/mL)10μM FeCl 3+0.1mM AA+1mM H 2O 20.6NaCl,pH 6,37°C,14days –

Estévez et al.(2009)b MPI (25mg/mL)10μM FeCl 3+0.1mM AA+1mM H 2O 20.1M NaCl,pH 6.2,4°C,6h Protein solubility,Hydration Liu et al.(2009)

MPI (20mg/mL)10μM FeCl 3+1mM H 2O 2

0.6NaCl,pH 6,37°C,20days Phenolic compounds Estévez and Heinonen (2010)b

MPI (20mg/mL)10μM Copper acetate +1mM H 2O 20.6NaCl,pH 6,37°C,20days Phenolic compounds Estévez and Heinonen (2010)b

MPI (20mg/mL)10μM MetMb +1mM H 2O 2

0.6NaCl,pH 6,37°C,20days

Phenolic compounds

Estévez and Heinonen (2010)b

Porcine loin disks

10μM FeCl 3+0.1mM AA+(1–20mM)H 2O 2

0.1M NaCl,pH 6.2,4°C,40min TBA-RS,WHC,Hydration

Liu et al.(2010)

a Assessed as total protein carbonyls by the DNPH-method.

Assessed as total protein carbonyls by the DNPH-method and speci ?c protein carbonyls (AAS,GGS)by LC –ESI –MS SSP:salt soluble proteins;MPI:myo ?brillar protein isolate;A:Ascorbate;AA:Ascorbic acid;LA:linoleic acid;MetMb:metmyoglobin;TBA-RS:Thiobarbituric acid-reactive substances;WHC:water-holding-capacity;DSC:Differential scanning calorimetry;ESR:Electron spin resonance;SDS-PAGE:Sodium dodecyl sulfate polyacrylamide gel electrophoresis.

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(MetMb)promoted the formation of carbonyls from MP to a greater extent than a metal-catalyzed oxidizing system(Fe3+/ascorbic acid/ H2O2).In a further study,Park and Xiong(2007)con?rmed that a MetMb oxidizing system(MetMb/H2O2)was more effective at degrading individual amino acids such as lysine,than an iron-catalyzed oxidizing system.In the presence of H2O2,Mb form hypervalent species such as ferrylmyoglobin(MbFe(IV)=O)which have been found to initiate lipid and protein oxidation(Baron& Andersen,2002).Hydrogen peroxide has also been shown to be capable of causing the release of iron from heme molecule and the non-heme iron would eventually catalyze oxidative reactions(Rhee, Ziprin,&Ordó?ez,1987).Moreover,the oxidation of oxymyoglobin to metmyoglobin results in the formation of superoxide radical that dismutates to hydrogen peroxide,following a similar catalytic redox cycle previously described for transition metals(Reactions10–12). Using proteomics,Promeyrat et al.(2011)recently reported that myoglobin is a good predictive marker of carbonyl formation in meat systems which highlight its role as an ef?cient promoter of protein carbonylation.Whereas the relative contribution of each iron form to protein oxidation in meat and meat products might remain inde?nite, heme and non-heme iron,as well as copper are potential contributors to the formation of protein carbonyls in meat systems.

3.3.3.Role of oxidizing lipids

Lipid-derived reactive oxygen species,such as peroxyl radicals (ROO?)are also potential initiators of protein carbonylation.According to Park et al.(2006b),the incubation of MP with linoleic acid and lipooxidase leads to several biochemical changes including the formation of carbonyl compounds.The same authors observed a coupling of lipid and protein oxidation processes during in vitro oxidation of MP by metal-catalyzed and lipid-oxidizing systems. Whereas protein carbonylation can take place in the absence of lipids (Stadtman&Oliver,1991),the concomitant occurrence of lipid and protein oxidation in meat systems suggests a plausible interaction between both phenomena(Estévez et al.,2008a;Estévez et al.,2008c; Mercier et al.,1995;Mercier et al.,1998;Park et al.,2006a,b).These interactions may involve the reciprocal transfer of reactive and non-reactive species between lipids and proteins.According to the rate constants reported by Davies(2005),the?OH radical would react faster and preferentially with certain proteins such as albumin(8×1010dm3 mol?1s?1)or collagen(4×1011dm3mol?1s?1)than with unsatu-rated lipids such as linoleic acid(9×109dm3mol?1s?1).As afore-mentioned(Section3.1.),the early degradation of readily oxidizable sulfur-containing groups in MP could be regarded as an endogenous protection mechanism against ROS(Saigas,Tanabe,&Nishimura,2003). Both,proteins and lipids,may be bene?ted from such antioxidant defense.In fact,Estévez et al.(2008a)observed a protecting role of MP against lipid oxidation in oil-in-water emulsions.When the oxidative stress exceeds the antioxidant capacity of proteins,both lipids and proteins would undergo oxidative damage manifested as free-radical chain reactions(Gardner,1979).Once the oxidative reactions com-mence,the measurable changes indicate that lipid oxidation would progress faster than the oxidative degradation of MP(Estévez et al., 2008a,b;Parkington et al.,2000;Viljanen et al.,2004;Vuorela et al., 2005).Like this,radicals and hydroperoxides formed from unsaturated lipids would attack susceptible amino acid side chains to yield carbonyl moieties in accordance to the mechanisms described by Park et al. (2006b).

3.3.

4.Other factors

Besides the presence of transition metals,myoglobin and oxidizing lipids,the oxidation of proteins and amino acids is affected by numerous environmental factors including pH,temperature,water activity and the presence of other promoters and/or inhibitors such as phenolic compounds(Estévez&Heinonen,2010;Park et al.,2006a,b; Park&Xiong,2007;Viljanen et al.,2004;Xiong,2000).Light and irradiation are also able to initiate protein oxidation(Garrison,1987; Rowe et al.,2004)but the knowledge the impact of such physical factors on the oxidation of meat proteins is rather limited. Furthermore,proteins display an intrinsic susceptibility to undergo oxidative reactions and yield carbonyl compounds(Estévez et al., 2009;Requena et al.,2001).Like this,MP and other animal proteins such as bovine serum albumin(BSA)and whey proteins are more susceptible than soy proteins to carbonylation induced by Fe3+/H2O2 (Estévez et al.,2009).The tertiary structure of the proteins,their size, their amino acid composition and sequence,and the distribution of amino acids on the protein structure could in?uence the susceptibility of proteins to undergo carbonylation processes(Estévez et al.,2008a; Estévez et al.,2009;Viljanen et al.,2004;Xiong,2000).

3.4.Reactivity of protein carbonyls

In general,carbonyl moieties from biomolecules are known to participate in multiple reactions with great biological signi?cance (Feeney et al.,1975).Until recently,the lack of knowledge on the exact chemical nature of carbonyls formed from food proteins, hindered any chance to investigate the involvement of such‘unknown’compounds in further reactions.Even before AAS and GGS were con?rmed to be present in muscle proteins,Xiong(2000)hypothe-sized about the involvement of protein-bound carbonyls in reactions with free amines for the formation of cross-links between poly-peptides.In line with this postulation,Stadtman and Levine(2000) and Akagawa et al.(2006)reported the likely implication of protein carbonyl residues in condensation reactions with amino groups from neighboring amino acid side chains to form cross-links via Schiff base formation.The detection of speci?c protein carbonyls,namely AAS and GGS(Daneshvar et al.,1997)and the con?rmation of their presence in oxidized food proteins(Estévez et al.,2009)enables the accomplishment of studies speci?cally devoted to shed light on the reactivity of such compounds in food systems.Results obtained from initial studies on the formation of AAS and GGS in meat model systems lent weight to the hypothesis proposed by Xiong(2000).Estévez et al. (2009)and Estévez and Heinonen(2010)reported that the initial increase of AAS and GGS as a result of the in vitro MCO of MP was followed by a signi?cant decrease of both compounds.The authors hypothesized whether the apparent net loss of protein carbonyls at prolonged oxidation rates may be caused by the implication of such compounds as reactants in advanced reactions.

The aldehyde moiety from speci?c protein carbonyls can be involved in several reactions including,i)a further oxidative degradation by which the aldehyde moiety is oxidized into a carboxylic acid(Sell,Strauch,Shen,&Monnier,2008),ii)the reaction with an aldehyde moiety from another protein-bound carbonyl residue to form an aldol condensation product(Dolz&Heidemann, 1989;Eyre,1984),iii)the reaction with an-amino group from a neighboring protein-bound amino acid(mainly lysine)to form a covalent bond via Schiff base formation(Dolz&Heidemann,1989; Eyre,1984),iv)and the reaction with anα-amino group from a free amino acid to form a Strecker aldehyde via Strecker-type degradation –oxidative deamination and decarboxylation of the amino acid in the presence of a carbonyl compound–(Estévez et al.,2011)(Fig.3).

Under intense oxidative conditions,the ongoing oxidation of AAS would lead to the formation of a stable end-product:α-aminoadipic acid(AAA).This compound has been known for decades to be present in acid hydrosylates of certain proteins such as bovine skin collagen and ellastine(Bailey,Ranta,Nicholls,Partridge,&Elsden,1977).In subsequent studies,AAA was identi?ed as an oxidation product of lysine via formation of AAS in cell cultures and model systems(Sell et al.,2008).It is worth noting that the formation of AAA from AAS requires the presence of oxygen and an oxidizing agent.In biological systems,several ROS and transition metals could be involved in such oxidative degradation.The resulting molecule has only one carbon

266M.Estévez/Meat Science89(2011)259–279

more than glutamic acid and can be easily detected by HPLC subsequently to derivatization with 9-?uorenylmethylchloroformate (FMOC)or analyzed as tri ?uoroacetyl methyl esters by GC-MS (Sell et al.,2008).Whereas the investigations of AAA as a protein oxidation product are scarce even in medical research,Sell et al.(2008)recently highlighted that AAA may be a more reliable marker of P-OX than its carbonyl precursor.As expected,recent experiments have con ?rmed the formation of AAA in MP oxidized in vitro with a hydroxyl radical generating system (Fe 3+/H 2O 2/ascorbate)(Unpublished results).

As aforementioned,the carbonyl moiety of an AAS residue reacts promptly with protein-bound lysine as well as with other AAS residues to form Schiff bases and aldol condensation structures,respectively (Dolz &Heidemann,1989).These reactions are known to take place in vivo to form,under physiological conditions,cross-links between peptides chains within collagen and ellastine proteins (Eyre,1984).The carbonyl-amine condensation between oxidized amino acids and amino acid residues also occur as a result of diverse age-related disorders which involve increased protein oxidation,intense AAS formation and uncontrolled protein cross-linking (Eyre,1984;Sell et al.,2008).Upon base hydrolysis,the cross-links are can be detected as lysinonorleucine (AAS-Lysine)and allysine aldol (AAS-AAS)(Feeney et al.,1975).These bifunctional cross-links can undergo successive condensation reactions with additional lysine and AAS residues to form complex cross-links structures such as desmosine.It is worth noting that these condensation reactions may take place in the absence of oxygen but require a favorable stereochemical distance between reacting groups.Several meat researchers have proposed the formation of carbonyl-amine condensations in MP (Estévez et al.,in press-b;Park et al.,2006a,b;Xiong,2000;Xiong et al.,2009).

A recent study investigated the potential implication of protein carbonyls,AAS and GGS on the formation of Strecker aldehydes from particular amino acids (Estévez et al.,2011).The formation of Strecker aldehydes is usually ascribed to the oxidative deamination and decarboxylation of the free amino acid in the presence of α-dicarbonyl compounds formed in the Maillard reaction and/or from lipid oxidation such as alkadienals and ketodienes (Zamora &Hidalgo,2005).The aforementioned study con ?rmed that protein carbonyls,AAS and GGS,promoted the in vitro degradation of leucine and isoleucine to yield the corresponding Strecker aldehydes,3-methylbutanal and 2-methylbutanal.These volatile compounds are common volatile components and aroma contributors in foods such as meat products (Estévez,Morcuende,Ventanas,&Cava,2003).The study carried out

222

H 2-amino adipic semialdehyde

-amino adipic

acid

-amino adipic semialdehyde -amino adipic semialdehyde

Aldol condensation product

-amino adipic semialdehyde

2Azomethine

-amino adipic semialdehyde

Amino acid

R NH 2

O H 2

2Diamino-carboxilic acid

Strecker aldehyde

O i)

iii)

Fig.3.Fate of a speci ?c protein carbonyl (AAS)by implication in further reactions.i)Oxidation of the carbonyl moiety (Sell et al.,2008).ii)Formation of an aldol condensation product by reaction with a neighboring AAS (Dolz &Heidemann,1989;Eyre,1984).iii)Formation of an azomethine (Schiff-base structure)by reaction with an amino group of a neighboring amino acid (Dolz &Heidemann,1989;Eyre,1984).iv)Implication in the formation of a Strecker aldehyde by a Strecker-type degradation of a free amino acid (Estévez et al.,2011).

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by Estévez et al.(2011)highlights that protein degradation products–free amino acids and free oxidized amino acids–would be sources of Strecker aldehydes in the absence of reducing sugars and oxidizing lipids.Unlike the previously described carbonyl-amine reactions,a previous proteolysis is required as the aldehyde moiety of the protein carbonyl would react,in this case,with theα-amino group from a free amino acid.

Beyond their role as P-OX markers,AAS and GGS may be active compounds in food systems and their interactions with other food components could lead to relevant consequences on meat quality as reported in Section6of the present review.

4.Assessment of protein carbonylation

4.1.Total carbonyl content:The DNPH method

The DNPH method is a routine procedure that enables the quanti?cation of the total amount of carbonyls from a protein sample. The results are widely used as a general index of P-OX in meat and meat products(reviewed by Estévez et al.,2008b;Lund&Baron,2010;Lund et al.,2011).The method is based on the reaction between the DNPH with protein carbonyl compounds to form a2,4-dinitrophenyl(DNP) hydrazone product which displays a maximum absorbance peak at around370nm.The procedure involves a simultaneous determination of carbonyl derivatives and protein content of the sample(Oliver et al., 1987).The concentration of DNP hydrazones is calculated by measuring reacted DNPH spectrophotometrically on the basis of an absorption of 22,000M?1cm?1at370nm.Concentration of protein is determined in a control sample(without added DNPH)at280nm using BSA as standard.Results are usually expressed as nmols DNP hydrazones per mg of protein.The original method(Oliver et al.,1987)was developed for analyzing oxidative stress in biological samples and has been subsequently employed with minor modi?cations by food scientists. These modi?cations include treating the samples with a hydrochloric acid–acetone solution in order to remove potentially interfering chromophore substances(e.g.hemoglobin,myoglobin,and retinoids) and using high ionic strength buffers to facilitate the suspension of particular food proteins such as MP(reviewed by Estévez et al.,2008b). The spectrophotometric assay has been extensively reviewed and details can be found elsewhere(Estévez et al.,2009;Levine et al.,1990; Reznick&Packer,1994;Shacter,2000).Whereas the simplicity and convenience of this assay make of it a widespread method for assessing P-OX,meat researchers often complain for the dif?culty of obtaining consistent and reliable results.According to Cao and Cutler(1995),the DNPH method is unsuitable for measuring the actual level of protein carbonyls in animal tissues because it is plagued with artifacts.Highly credited medical researchers who promoted the DNPH method (Requena et al.,2001),accepted the criticisms from Cao and Cutler (1995).Beyond delicate procedural issues,the method can be criticized for the limited value of the information provided.Whereas the total carbonyl content is employed as indicator of the extent of P-OX in a meat sample,this oxidation index do not re?ect consistently the extent of P-OX as multiple oxidative modi?cations in proteins do not lead to the generation of carbonyl compounds(see Section3.1).In addition, carbonyl moieties can be present in proteins as a result of mechanisms that do not involve the oxidation of amino acid residues(Requena et al., 2003).Like this,the DNPH method would underestimate the overall oxidative damage to proteins and would overestimate the total amount of protein carbonyls by accounting absorbance from artifacts,exceeding derivatization agent,and lipid-derived carbonyls(Armenteros,Heino-nen,Ollilainen,Toldrá,&Estévez,2009;Cao&Cutler,1995;Estévez et al.,2008b).In addition,the method lacks speci?city as the information regarding the speci?c nature of the oxidation products is missing.As a result,an accurate deduction of oxidation mechanisms and reaction pathways by using the DNPH method is not feasible(Xiong&Decker, 1995).The DNPH could be useful to accomplish descriptive studies devoted to the occurrence of P-OX in meat systems as long as complementary techniques(loss of sulfhydryl groups,formation of cross-links,tryptophan oxidation etc.)are employed to have a reliable overall picture on the extent of the oxidative damage.The DNPH method has enabled numerous studies on the occurrence of protein carbonylation during processing and storage of meat and meat products.Section5of the present review covers that speci?c topic.

4.2.Analysis of speci?c protein carbonyls

Soon after AAS and GGS were highlighted as reliable biomarkers of oxidative damage to proteins in relation to age-related diseases (Daneshvar et al.,1997),several methodologies were developed for the accurate detection and quanti?cation of both semialdehydes from biological samples.AAS and GGS are sensible to acid hydrolysis and therefore,a derivatization procedure is required for stabilization. Protein carbonyls are,in addition,highly reactive(Requena et al., 2001;Xiong,2000)and hence,the derivatization procedures usually involve modi?cation of the carbonyl moiety by reduction or amination.Climent,Tsai,and Levine(1989)proposed a derivatization procedure with?uoresceinamine(FINH2)prior to the analysis of the derivatized semialdehydes by HPLC–MS.Requena et al.(2001) developed a method based on gas chromatography(GC)-MS for the detection of AAS and GGS in the form of their corresponding hydroxyl derivatives,hydroxyaminovaleric acid(HAVA)and hydroxyaminoca-proic acid(HACA),after their reduction with sodium borohydride (NaBHO4).Both procedures have been criticized as FINH2-and hydroxyl derivatives of AAS and GGS are partially degraded during hydrolysis with HCl(Akagawa et al.,2006).Recently,Akagawa et al. (2006)proposed an alternative procedure for the detection of AAS and GGS that involves the reductive amination of both semialdehydes in the presence of sodium cyanoborohydride(NaCNBH3)and p-aminobenzoic acid(ABA)(Fig.2).This procedure provides some advantages as ABA-derivatized semialdehydes show great stability against acidic hydrolysis and cold storage(Akagawa et al.,2005). Following this derivatization procedure,Estévez et al.(2009)detected AAS and GGS in oxidized MP by using HPLC–ESI–MS.Taking into account that the original derivatization procedure was originally conceived for plasma proteins,in a recent paper,Utrera et al.(in press)accomplished an adaptation for the preparation of meat samples in terms of the required initial protein concentration,ABA concentration and hydrolysis time.These authors also described a simple and fast HPLC program for the separation of ABA and the derivatized forms of the semialdehydes prior to the quanti?cation using a?uorescent detector.The elucidation of the exact chemical nature of the protein carbonyls enables the understanding of the mechanisms leading to their generation(Section3.3),the potential implication of the speci?c compounds in further reactions(Section3.4)and their potential signi?cance in biological and food systems(Section6).This technique should be employed whenever a chemical insight of protein oxidation mechanisms is thoroughly analyzed.

5.Protein carbonylation in meat and meat products

5.1.Protein carbonylation during meat aging and chill storage

The formation of ROS is an inevitable consequence of aerobic metabolism and hence,oxidative reactions affecting to muscle lipids and proteins take place in vivo(Holloszy&Coyle,1984).The total protein carbonyl content is estimated to be≈1–2nmol/mg protein in a variety of human and animal tissues(Requena et al.,2001),which represent modi?cation of about10%of the total cellular protein (Starke-Reed&Oliver,1989).After slaughter,the in vivo antioxidant mechanisms partially collapse while the biochemical changes oc-curred during the conversion of muscle to meat,favor oxidation (Morrissey,Sheehy,Galvin,Kerry,&Buckley,1998).The pH decline

268M.Estévez/Meat Science89(2011)259–279

from 7.0to 5.5and subsequent storage at 0°C has been shown to enhance oxidation of beef MP as measured by the DNPH-method (Srinivasan,Xiong,&Decker,1996).High concentrations of H +are thought to favor the redox cycle of myoglobin and hence,its pro-oxidant potential (Caughley &Watkins,1985).Other low pH-driven effects such as aggregation,denaturation and decreased solubility of muscle proteins could affect their susceptibility to oxidation.Accord-ing to a recent study,the induction of pre-slaughter stress on broilers leads to muscles with low ?nal pH which are in turn,more susceptible to protein carbonylation,protein denaturation and decreased solu-bility (Wang,Pan,&Peng,2009).In the same line,Zhang,Xiao,Lee,and Ahn (2011)reported that breast muscles from broilers subjected to in vivo oxidative stress through dietary means,displayed lower ?nal pH,increased susceptibility to protein carbonylation and more intense drip loss than muscles from non-stressed birds.In contrast,Chan,Omana,and Betti (2011)found no effect of pH at 24h on the extent of protein carbonylation in turkey breast muscle.The authors assumed the in ?uential impact of pH on P-OX and ascribed their results to the better protective system of turkey muscles against oxidative stress than that from broiler muscles.

Besides the pH decline,other post-mortem biochemical changes such as alteration of cellular compartmentalization,release of free-catalytic iron and oxidizing enzymes and the propagation of lipid oxidative reactions likely promote the formation of protein carbonyls in post-rigor meat.The occurrence of early postmortem P-OX was ?rstly described in beef muscles by Martinaud et al.(1997).According to these authors,10days chill storage of beef longissimus lumborum and diaphragma pedialis increased the total carbonyl content in these muscles from 3.1to 5.1nmol/mg protein and from 4.8to 6.9nmol/mg protein,respectively.Subsequent studies con ?rmed the occurrence of protein carbonylation during aging/chill storage of beef (Lindahl,Lagerstedt,Ertbjerg,Sampels,&Lundstr?m,2010;Rowe et al.,2004)pork (Herring,Jonnalongadda,Narayanan,&Coleman,2010;Lund et al.,2007a;Ventanas et al.,2006),poultry (Rababah et al.,2004;Zhang et al.,2011)turkey (Chan et al.,2011;Mercier et al.,1998;Santé-Lhoutellier,Théron,Cepeda,Grajales,&Gatellier,2008a ),lamb (Petron et al.,2007;Santé-Lhoutellier,Engel,Aubry,&Gatellier,2008b ),rhea (Filgueras et al.,2010;Terevinto,Ramos,Castroman,Cabrera,&Saadoun,2010)and ostrich meat (Leygonie,Britz,&Hoffman,2011).The large variability of the data reported by different authors for the amount of protein carbonyls in similar meat samples analyzed using the same technique (DNPH-method),prevents from inferring general patterns.For instance,the amount of protein

carbonyls reported by Martinaud et al.(1997)in beef muscles at the beginning of the chilling assay were already larger than those found by Lund,Hviid,and Skibsted (2007b)in beef patties chilled for six days in a high-oxygen atmosphere (3.1–4.8nmol/mg protein vs .1.9nmol/mg protein).Similarly,the carbonyl content found by Mercier et al.(1998)in turkey muscles chilled for 9days in atmospheric air was larger than that reported by Lund et al.(2007a)in porcine muscles chilled for a longer time (14days)in a high-oxygen atmosphere (3.1–3.4nmol/mg proteins vs. 1.1nmol/mg protein).In the same line,the total amount of protein carbonyls found by Wang et al.(2009)in broiler muscle pectoralis major at slaughter were 17-fold times higher than those recently reported by Zhang et al.(2011)in breast muscle (9.7nmol/mg protein vs.0.55nmol/mg protein).As another example,the extent of carbonyl-ation displayed by fresh pork loin (Lund et al.,2007a )remained similar to that reported by Ventanas et al.(2006)in dry-cured loins subjected to salting and a subsequent 3-months ripening process.The lack of reliability between studies highlights the dif ?culty of applying consistently the DNPH method in different laboratories and questions the real signi ?cance of the results provided by the spectrophotometric method when compared between studies.Individual works,however,show that the extent of protein carbonylation is highly dependent on the origin of the meat,type of muscle,specie and the storage conditions (Filgueras et al.,2010;Gatellier et al.,2000;Lund et al.,2007a,b;Santé-Lhoutellier et al.,2008a,b ).Beef has been found to be more susceptible to protein carbonylation than pork.The authors ascribed the differences to the noticeably larger amounts of iron and myoglobin in the cattle muscles (Lund et al.,2007a,b ).Similar results were found by Mercier et al.(1998)at comparing beef and turkey muscles.The authors ascribed the differences to the noticeably larger amounts of iron and myoglobin in the cattle muscles (Lund et al.,2007a,b ).The differences found between oxidative and glycolytic muscles for the susceptibility to protein carbonylation could also be ascribed to similar compositional factors while the amount of residual glycogen has also been proposed to be in ?uential (Filgueras et al.,2010).

5.2.Protein carbonylation during frozen storage of meat

Recent studies have demonstrated that meat proteins also undergo carbonylation during frozen storage of pork (Estévez et al.,2011;Xia et al.,2009),beef (Popova,Marinova,Vasileva,Gorinov,&Lidji,2009),poultry (Rababah et al.,2010;Soyer,?zalp,Dalm ?s,&Bilgin,

Table 2

Summary of studies in which carbonylation a of meat proteins were found to be affected by processing technologies.Technology Meat product Studied effects

Additional analyses Reference

Irradiation

Beef meat

Oxidation system

Protein thiol oxidation Martinaud et al.(1997)Chicken breast Antioxidant effect of plant extracts TBA-RS

Rababah et al.(2004)Beef sausage

Antioxidant effect of carrot juice TBA-RS,sensory evaluation Badr &Mahmoud (2011)Cooking

Pork patties Antioxidant effect of plant phenolics Hexanal Vuorela et al.(2005)Pork patties Antioxidant effect of grain meals Hexanal

Salminen et al.(2006)Pork patties Antioxidant effect of fruit extracts Tryptophan depletion

Ganhao et al.(2010a)b Pork patties Antioxidant effect of fruit extracts Instrumental color,texture

Ganhao et al.(2010b)Beef meat

Composition of feeds Aromatic amino acids,free thiol Gatellier et al.(2010)Dry-curing

Dry-cured loin Feeding regime TBA-RS,hexanal

Ventanas et al.(2006)Dry-cured ham

Feeding regime TBA-RS,sensory evaluation Ventanas et al.(2007)Sausage fermentation

Cantonese sausage Processing Protein solubility,thiol groups Sun et al.(2011b)Cantonese sausage

Processing

Proteolysis,digestibility Sun et al.(2011a)High-hydrostatic pressure

Sliced dry-cured ham Pressure and holding time TBA-RS,instrumental color Cava et al.(2009)Sliced dry-cured ham

Vacuum-packaging

Volatiles,sensory evaluation

Fuentes et al.(2010)b

a Assessed as total protein carbonyls by the DNPH-method.

Assessed as speci ?c protein carbonyls (AAS,GGS)by LC –ESI –MS.

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M.Estévez /Meat Science 89(2011)259–279

2010),turkey(Chan et al.,2011),and rhea meat(Filgueras et al., in press).Soyer et al.(2010)reported signi?cant increases of the total amount of protein carbonyls after6months of frozen storage at?18°C(from1.78to2.88nmol/mg protein).Xia et al.(2009) reported a slight but signi?cant increase of total protein carbonyls in porcine longissimus dorsi subjected to5freeze/thaw cycles(from1.09 to1.16nmol/mg protein).By analyzing speci?c protein carbonyls, Estévez et al.(2011)found a signi?cant increase of AAS and GGS during the?rst2months of frozen storage at?18°C while a signi?cant decrease was detected by the end of the freeze storage (4months).According to the authors,those results suggest that AAS and GGS might be involved in further reactions,which is in good agreement with the hypothesis proposed in Section3.4.of the present review.The carbonylation of meat proteins during frozen storage seems to be linked to the occurrence of lipid oxidative reactions and is affected by the type of muscle(Estévez et al.,2011;Filgueras et al.,in press;Soyer et al.,2010),freezing temperature(Soyer et al.,2010), the packaging conditions and previous operation such as pre-mincing (Estévez et al.,2011).

5.3.Protein carbonylation during meat processing

Numerous processing technologies have been reported to affect protein carbonylation of meat including irradiation(Badr&Mahmoud, 2011;Martinaud et al.,1997;Rababah et al.,2004,2010),cooking (Ganh?o et al.,2010a,b;Gatellier,Kondjoyan,Portanguen,&Santé-Lhoutellier,2010;Salminen et al.,2006;Santé-Lhoutellier,Astruc, Marinova,Grève,&Gatellier,2008c;Vuorela et al.,2005),ripening (Ventanas et al.,2006;Ventanas et al.,2007),sausage fermentation (Sun et al.,2011a;Sun,Cui,Zhao,Zhao,&Yang,2011b)and high-hydrostatic pressure(Cava et al.,2009;Fuentes et al.,2010)(Table2).

γ-Irradiation induces the formation of large amounts of ROS from molecular oxygen which are,as previously explained,initiators of protein carbonylation(Garrison,1987).The impact of irradiation on protein carbonylation has been described in raw meat(Martinaud et al.,1997)poultry(Rababah et al.,2004)and fresh sausages(Badr& Mahmoud,2011).The application of cooking technologies has been consistently found to promote the carbonyl gain in meat products and moreover,increase the susceptibility of meat proteins to undergo further carbonylation during the subsequent chill storage(Ganh?o et al.,2010a,b;Salminen et al.,2006;Santé-Lhoutellier et al.,2008c; Vuorela et al.,2005)The physico-chemical changes induced by high temperatures during cooking such as the disruption of cellular compartmentalization,the release of free catalytic iron and the formation and cleavage of hydroperoxides,also favors the onset of oxidative reactions during the following chill storage leading to intense protein carbonylation(Ganh?o et al.,2010a,b;Salminen et al., 2006).

The hydrolytic degradation of proteins during the manufacture of dry-cured fermented meats has been profusely studied and is known to play a relevant role in numerous technological and sensory aspects of the?nal products(Toldrá,1998).Results from recent studies highlight that besides proteolysis,meat proteins also undergo intense oxidative reactions,although the impact of such reactions is not fully clear.Studies carried out by Ventanas et al.(2006,2007)revealed the occurrence of protein carbonylation in dry-cured loins and hams which was found to be,once again,linked to lipid oxidative reactions. The hams,which are subjected to longer and more severe drying conditions,were found to have considerably larger amounts of protein carbonyls than the loins(≈9nmol/mg protein vs.≈1.3nmol/mg protein).The manufacture of dry-cured meats in-volves a lengthy process(up to36months for Iberian dry-cured hams)and several operations such as salting,post-salting,drying and cellar(Ventanas et al.,2007).Little is known about the impact of each step on the onset and intensity of the oxidative reactions affecting to meat proteins.Salting,which is a common operation for the manufacture of numerous meat products,may have an impact on protein carbonylation.The addition of sodium chloride has an impact on the ionic strength of the environment which in turn,affects the degree of assembly of MP(Wick,1999)their exposure to pro-oxidants and hence,their susceptibility to carbonylation(Montero,Giménez, Pérez-Mateos,&Gómez-Guillén,2005).In addition,several authors have proposed that NaCl could enhance the activity of Fe3+or that Cl?derived from NaCl would improve the solubility of such ion,hence, stimulating their pro-oxidant effects(Kanner,Salan,Harel,& Shegalovich,1991;Osinchak,Hultin,Zajicek,Kelleher,&Huang, 1992).It is worth to recall that Fe3+plays a relevant role as promoter of carbonylation in meat proteins(see Sections 3.2.and 3.3). Nevertheless,the very few studies devoted to study the impact of NaCl on the formation of carbonyls from muscle proteins do not supply de?nitive results(Montero et al.,2005;Shimizu,Kiriake, Ohtubo,&Sakai,2009;Srinivasan et al.,1996).Sun et al.(2011a,b) studied the carbonylation of different muscle proteins during processing of a fermented Cantonese sausage.According to these authors,sarcoplasmic and MP undergo a gradual and signi?cant carbonyls gain(from1.04to4.68nmol/mg protein and from1.32to 7.00nmol/mg protein,respectively)as a result of the manufacture process which involves fermentation and oven-drying of the sausages. The protein carbonylation was found to be related to the onset of lipid oxidation and other biochemical changes such as increased hydro-phobicity and secondary structural changes in proteins.

The extent of protein carbonylation has also been investigated in comminuted cooked meat products such as cooked sausages,cooked ham and liver paté(Estévez et al.,2005;Estévez&Cava,2004,2006; Estévez,Ventanas,&Cava,2007;Sun,Zhang,Zhou,Xu,&Peng,2010). Compared to the carbonyl level generally reported in raw meat samples(1–3nmol/mg protein),cooked meat products have been found to contain larger amounts of carbonyls(≈5nmol/mg protein) which suggest that manufacturing operations(cutting,mincing, cooking etc…)promoted the formation of protein carbonyls. Subsequent refrigerated storage leads to further protein carbonyla-tion with this process being closely related to the occurrence of other biochemical changes such as the increase of non-heme iron and the extent of lipid oxidation(Estévez&Cava,2004).

Besides irradiation,other emerging technologies such as hydro-static pressure have been recently proved to promote protein carbonylation in cured meats(Cava et al.,2009;Fuentes et al., 2010).The application of600MPa causes a signi?cant increase on the amount of speci?c protein carbonyls,AAS and GGS,as a likely result of physico-chemical changes induced by hydrostatic pressure including tissue disruption and increase of free catalytic iron(Fuentes et al., 2010).

6.Impact of protein carbonylation on meat quality

As a major component of muscle tissue,proteins play a decisive role in muscle foods regarding technological,nutritional and sensory aspects(Lawrie,1998).The modi?cation of the native structure and/ or integrity of muscle proteins as a result of denaturation and/or proteolysis phenomena is known to affect meat quality including texture traits(Kemp et al.,2010),color(Kazemi et al.,2011),aroma (Toldrá,1998),?avor(Toldrá,1998),water-holding capacity(Van Laack et al.,2000)and biological functionality(Ahhmed&Muguruma, 2010).P-OX causes multiple physico-chemical changes in proteins including amino acid destruction,decreases in protein solubility due to protein polymerization,loss of enzyme activity and impaired protein digestibility(reviewed by Xiong,2000).Considering these severe modi?cations,it looks reasonable to hypothesize that oxidation-induced changes in meat proteins could also affect meat quality.The understanding of the signi?cance of P-OX in meat systems has been a challenge since the?rst studies on P-OX were conducted (Decker et al.,1993;Martinaud et al.,1997;Mercier et al.,1995).The

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onset of P-OX in muscle-based systems was initially linked to loss of protein functionality including alterations in solubility,viscosity,gelation,emulsi ?cation,and water-holding capacities (Decker et al.,1993;Liu &Xiong,1996;Srinivasan et al.,1996;Wan,Xiong,&Decker,1993).In a recent review,Lund et al.(2011)outlined the conse-

quences of P-OX in muscle foods,and reported deleterious effects on texture traits and nutritional value.

The link between P-OX and the alleged P-OX-driven deterioration is usually made on the basis of signi ?cant correlations between protein carbonyl measurements (DNPH-method)and the assessed quality trait (Chan et al.,2011;Estévez et al.,2005;Estévez &Cava,2004;Ganh?o et al.,2010b;Morzel,Gatellier,Sayd,Renerre,&Laville,2006;Rowe et al.,2004;Sun et al.,2011a;Ventanas et al.,2006,2007;Zakrys,O'Sullivan,Allen,&Kerry,2009).However,the scienti ?c arguments provided for supporting the potential causality connection rarely include precise mechanisms explaining the implication of protein carbonyls in the deleterious effects.In some cases,when a likely mechanism is proposed,the deterioration is ascribed to another P-OX manifestation or to general P-OX-induced changes such as loss of functionality.Like this,carbonyls are then regarded as mere indicators of P-OX with no apparent connection with the alleged deleterious https://www.wendangku.net/doc/fe7122648.html,forting exceptions,however,can be found in the literature (Morzel et al.,2006).As stated by Xiong (2000),“the in ?uence of oxidation on the functionality of muscle proteins and its implications for meat quality cannot be de ?ned without due consider-ation of,and reference to,the speci ?c physico –chemical reactions taking place within and between the individual protein molecules ”.The recent advances on the understanding of the mechanisms involved in the formation of protein carbonyls from meat proteins may facilitate the clari ?cation of the potential implication of protein carbonylation on meat quality (see Sections 3.3and 3.4.).Previous papers reviewed the signi ?cance of P-OX on biological (Dalle-Donne et al.,2006;Davies,2005;Nystr?m,2005;Shacter,2000;Stadtman,1990;Stadtman &Oliver,1991)and food systems (Lund et al.,2011;Lund &Baron,2010;Xiong,2000).The present paper will only cover meat quality traits in which protein carbonyls may play a relevant role (Figs.4and 5

Fig.4.Plausible mechanisms by which protein carbonylation could affect technological and sensory traits of meat and meat products according to the existing literature.(In some cases,the relationship between protein carbonylation and the alleged alteration is based on correlations and the causality connection may not be

proved).

Fig.5.Plausible mechanisms by which protein carbonylation could affect nutritional value of meat and meat products according to the existing literature.(In some cases,the relationship between protein carbonylation and the alleged alteration is based on correlations and the causality connection may not be proved).

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6.1.Protein conformation and functionality

MP(actin and myosin)are the most abundant muscle proteins and play a main role in the muscle contractile function as components of the myo?brils.This organelle make up between82and87%of the muscle cell volume and around85%of water is mostly physically entrapped inside this protein structure(Huff-Lonergan&Lonergan, 2005;Puolanne&Halonen,2010).The functional properties of MP in meat systems,including their WHC,largely determines the quality of fresh meat and the success of numerous technological processes applied during the manufacture of meat products.

The speci?c amino acid composition and peptide sequence (primary structure)of MP largely in?uence their native(secondary and tertiary)structure which in turn determines their functionality. To this regard,the characteristics and distribution of the amino acid side chains play a relevant role.While surrounded by a water environment such as in muscle foods,the polar(hydrophyllic) residues are exposed to the water phase while the non-polar (hydrophobic)groups are generally occluded in the molecule.The chemical interaction between the polar groups from MP and water molecules is essential for their water-functionality including their gelling,emulsifying and WHC(Huff-Lonergan&Lonergan,2005; Puolanne&Halonen,2010).As polar residues,the side chains of basic amino acids such as lysine or arginine,would be orientated outwards with respect to the bulk water(Puolanne&Halonen,2010).These polar amino acid side chains are also more exposed and accessible to potent oxidation promoters such as metal ions and hence,more susceptible to oxidative reactions.As a result of oxidative stress in the presence of metal ions,protein carbonyls would be generated from basic amino acids such as lysine and arginine through the oxidative deamination pathway described in Section3.3.The loss of protonable amino groups as a result of protein carbonylation would lead to an alteration of the distribution of the electrical charges and the overall electrical arrangement of MP as seen in bovine serum albumin and other animal proteins(Davies&Delsignore,1987;Stadtman,1993). At intense oxidative conditions,a major protein carbonyl,AAS,would undergo additional oxidative degradation to yield a carboxylic moiety (Sell et al.,2008;Section 3.4.).This further modi?cation would worsen the impaired electric arrangement of meat proteins including their isoelectric point.The modi?cation of the isoelectric point of proteins as a result of oxidative stress has been described by Stadtman (1993)amongst others,and mainly attributed to the oxidative modi?cation of basic amino acids.As a direct consequence of these chemical modi?cations,a shift of the balance between protein intramolecular interactions and protein–water interactions would take place,causing a loss of protein solubility,favoring protein–protein interactions and eventually protein denaturation.The P-OX-induced alteration of the tertiary structure is also known to cause protein unfolding,increased surface hydrophobicity,formation of aggregates and then,irreversible denaturation(Xiong,2000).Consis-tently,oxidized proteins are known to be more prone to thermal denaturation and less soluble than non-oxidized proteins,and protein carbonyls have been proposed to play a relevant role in such biochemical and structural changes(Chan et al.,2011;Liu&Xiong, 2000;Park et al.,2006a,b;Parkington et al.,2000;Rowe et al.,2004; Santé-Lhoutellier,Aubry,&Gatellier,2007;Sun et al.,2011a;Wang et al.,2009).Therefore,the carbonylation of proteins may play a relevant role in the physico-chemical modi?cations in oxidized proteins which have been described as leading causes for the alteration in gelation, emulsi?cation,viscosity and hydration of muscle foods(Bertram et al., 2007;Xiong,2000;Xiong&Decker,1995).These modi?cations may be directly derived from the oxidative damage to proteins and/or by inducing several conformational changes leading to denaturation (Xiong,2000).

Protein carbonyls may also contribute to increase the loss of protein functionality through the formation of cross-links according to the reactions pathways described in Section 3.4.Intra-and intermolecular cross-linking in oxidized proteins is considered a major cause to the structural changes leading to loss of functionality (Xiong,2000;Liu&Xiong,2000;Liu,Xiong,&Chen,2009;Liu et al., 2010).Cross-links contribute to the formation and stabilization of protein aggregates which causes the shrinkage of myo?brils and a mechanic constriction against swelling(Liu et al.,2009).Moreover, many of the functional properties of MP are dependent on the association among individual proteins and hence,oxidative modi?-cations leading to polymerization and massive aggregation may cause signi?cant deleterious effects in muscle foods(Xiong,2000).While cross-linking between polypeptide chains can occur through a variety of mechanisms(Xiong,2000),most studies on muscle foods recognize disulphide bonding and to a minor extent,dityrosine formation as main routes for protein cross-links in muscle foods(Lund et al.,2011). The condensation of protein carbonyls with amino groups from neighboring amino acids has been recurrently proposed as an additional mechanism for cross-linking in meat systems by Liu and Xiong(2000),Xiong(2000),Park et al.(2006a,b),Liu et al.(2010), among many others.However,the exact nature of such reaction remained unde?ned.The recent identi?cation of speci?c protein carbonyls in oxidized MP enables the proposal of speci?c pathways for the formation of cross-links.As described in Section3.4,the pathways would comprise not only cross-links via carbonyl-amine condensa-tion but also the reaction between of two neighboring AAS to form an aldol condensation product.The occurrence of such cross-links in meat systems and their potential negative effect on meat functionality is supported by recent?ndings by Estévez et al.(2011).According to this study,keeping pork under freeze storage leads to an initial gain of AAS and GGS,followed by a net loss of both semialdehydes which timely coincide with a dramatic loss of water-holding capacity of the meat.The implication of AAS and GGS in the formation of cross-links in accordance to the proposed mechanisms would explain the decrease of these semialdehydes by the end of the freeze storage and would reinforce their liability in the loss of WHC of porcine muscles.The negative effect of P-OX on WHC of meat might affect speci?c technological processes such as brine irrigation,meat marinating and subsequent cooking(Liu et al.,2009;2010)and particular eating quality traits such as juiciness(Huff-Lonergan& Lonergan,2005)(Fig.4).

Finally,it is worth mentioning that some authors support that meat processing conditions that favor mild P-OX and specially, protein carbonylation,may have a positive effect on the gelation and emulsifying abilities of MP(Srinivasan et al.,1996;Xiong,2000). In the same line,controlled enzymatic-induced oxidation of MP from chicken has also been successful for improving the texture charac-teristics of surimi-based products(Lantto,Puolanne,Kalkkinen, Buchert,&Autio,2005).Xiong(2000)hypothesized that slow and mild-to-moderate oxidation of MP could lead to an improvement of the stability and rheological properties of gels through the formation of cross-links via carbonyl-amine condensations.In contrast,exten-sive P-OX would result in decreased functionality due to random and excessive aggregation phenomena.

6.2.Nutritional value

The potential negative effect of food P-OX on nutritional aspects and consumer's health has been recognized as a major issue that requires further investigation(Lund et al.,2011;Xiong,2000).Early studies on dairy proteins investigated the oxidative modi?cations of essential amino acids and the bioavailability of the oxidized de-rivatives(Cuq,Besancon,Chartier,&Cheftel,1978).Meat consump-tion guarantees an adequate delivery of amino acids which are whether not found in plant-derived foods or have poor bioavailability. Oxidation of meat proteins causes to a net loss of particular amino acids and a considerable modi?cation of the amino acid pro?le(Park&

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Xiong,2007).Since the formation of protein carbonyls involves the irreversible oxidative modi?cation of essential amino acids such as lysine,arginine and threonine,protein carbonylation is a clear expression of the detrimental impact of P-OX on the nutritional value of food proteins.However,it is not de?nite to which extent the formation of carbonyls in meat proteins would cause a signi?cant deleterious effect on dietary meat products.

In addition to the loss of essential amino acids,the impact of P-OX on protein digestibility would contribute to impair the nutritional value of oxidized proteins.According to Grune,Jung,Merker,and Davies(2004)low levels of oxidation induced subtle changes in protein structure which may favor their recognition by proteases leading to increased susceptibility to proteolysis.At higher oxidation levels,the formation of protein aggregates and the oxidative degradation of speci?c amino acid side chains would alter recognition sites both chemically and physically leading to a decreased proteolytic susceptibility.Whereas both phenomena have been observed in meat model systems(Santé-Lhoutellier et al.,2007;Santé-Lhoutellier et al., 2008b),some recent studies con?rmed that intense oxidative modi?cation of MP leads to a decrease susceptibility to undergo proteolytic degradation,which has an effect on protein digestibility (Santé-Lhoutellier et al.,2008c).The decreased susceptibility of oxidized MP to papain was ascribed to the carbonylation of arginine and lysine as this enzyme hydrolyses at bonds involving such amino acids(Morzel et al.,2006).In this sense,the activity of pancreatic enzymes with a similar hydrolytic pattern like trypsin could also be affected by carbonylation of MP.Findings by Santé-Lhoutellier et al. (2007)support that hypothesis as negative and signi?cant correla-tions were found between protein carbonylation and the proteolytic activity of trypsin/α-chymotrypsin.According to these authors, protein carbonyls could also affect the digestibility of MP by contributing to form cross-links and aggregates via carbonyl-amine condensation(Fig.5).

In addition to the de?cient amino acid assimilation caused by protein aggregation,a reduced protein digestion rate would have a negative impact on human health.According to several studies,non hydrolyzed proteins are fermented by colonic?ora into phenol and p-cresol,which are mutagenic products,increasing in that way the risk of colon cancer(Evenepoel et al.,1998).The actual impact of the intake of oxidized proteins on human's health remains unknown.

6.3.Sensory traits

6.3.1.Texture

Several texture traits such as juiciness,tenderness and hardness have been recurrently studied in fresh meat and diverse meat products in relation to the potential impact of P-OX(Lund et al., 2011).Initial hypotheses based on the susceptibility of oxidized meat proteins to proteolysis,implicated P-OX with tenderization of beef during maturation(Martinaud et al.,1997).Rowe et al.(2004)found signi?cant correlations between total protein carbonyls and the instrumental texture(Warner-Bratzler shear force)of beef muscles. Numerous subsequent studies,including some recent reports(Zakrys et al.,2009)have con?rmed the link between P-OX,as assessed by the DNPH method,and decreased tenderness in beef muscles.Neverthe-less,the causality connection between protein carbonyls and meat tenderness is not fully clear.P-OX has been reported to in?uence meat tenderness by i)decreasing proteolytic degradation of meat proteins during aging and by ii)inducing protein cross-linking via disulphide-bonding.The?rst mechanism may comprise the collaborative in?uence of two P-OX-driven effects:the inactivation of proteolytic enzymes involved in meat tenderization and the oxidative alteration of the MP which would lead to decreased proteolytic susceptibility (Carlin et al.,2006;Huff-Lonergan et al.,2010;Rowe et al.,2004). Becauseμ-calpain and m-calpain enzymes contain both histidine and SH-containing cysteine residues at their active sites,the oxidative degradation of such groups could likely lead to the enzyme inactivation(Lametsch,Lonergan,&Huff-Lonergan,2008).According to the second mechanism protein cross-linking would strengthen the myo?bril structure,hence,causing the toughening of the muscle tissue(Lund et al.,2007a,b).The contribution of protein carbonyls to massive cross-linking during meat aging,could explain the correla-tions found between protein carbonylation and decreased tenderness but this extent has not been con?rmed(Fig.4).

The timely coincidence between P-OX and texture changes has also been reported in various meat products such as liver patés(Estévez& Cava,2004),frankfurters(Estévez et al.,2005)and emulsi?ed cooked patties(Ganh?o et al.,2010b).These authors found signi?cant correlations between the instrumental hardness of the meat products and the extent of P-OX as measured by the DNPH-method.Similar results have been reported by Fuentes et al.(2010)while studying the effect of high-hydrostatic pressure on the onset of P-OX in dry-cured meats and the impact on particular texture traits such as tenderness and juiciness.The implication of protein carbonylation in the alleged effects was attributed to the formation of cross-linking.

6.3.2.Flavor

A recent study(Estévez et al.,2011)highlighted the potential implication of speci?c protein carbonyls,AAS and GGS,on the formation of Strecker aldehydes from leucine and isoleucine accord-ing to the reaction mechanism described in Section3.4.The Strecker degradation of amino acids is one of the main reactions leading to the ?nal aroma compounds in the Maillard reaction.It involves the oxidative deamination and decarboxylation of free amino acids in the presence ofα-dicarbonyl compounds formed in the Maillard reaction. Certain carbonyls derived from lipid peroxidation such as alkadienals and ketodienes have been demonstrated to promote the oxidative degradation of amino acids and yield the corresponding Strecker aldehydes via Strecker-type reactions(Zamora&Hidalgo,2005). According to the study carried out by Estévez et al.(2011),the carbonyl moieties of AAS and GGS could react with the amino group of free amino acids,namely leucine and isoleucine,to form Schiff base structures and eventually trigger the formation of the corresponding Strecker aldehydes.AAS was found to be more reactive than GGS and the ability of the semialdehydes to promote the formation of the Strecker aldehydes is inhibited by blocking their carbonyl moiety and signi?cantly affected by the pH of the media.Like this,protein degradation products–free amino acids and free oxidized amino acids–would be sources of Strecker aldehydes in meat systems, hence,having an effect on their?avor(Fig.4).Whereas the actual implication of AAS and GGS in the Strecker-type degradation of free amino acids in food systems remains unknown,it is plausible that protein semialdehydes would play a relevant role as sources of these odorants in particular ripened meats such as dry-cured products. Armenteros et al.(2009)reported signi?cantly higher amounts of AAS and GGS in dry-cured meats compared to other muscle foods.Strecker aldehydes are common volatile components of dry-cured meats and active contributors to their?avor.The simultaneous occurrence of high rates of proteolysis,protein oxidative reactions and the formation of Strecker aldehydes during ripening of meat products (Toldrá,1998)suggests that protein semialdehydes may be implicat-ed in the formation of Strecker aldehydes by reacting with neighboring free amino acids.

7.Antioxidant strategies against protein carbonylation

The recognition of the implication of protein carbonyls in severe deleterious effects in meat systems,justi?es the development of antioxidant strategies against protein carbonylation.In fact,the discovery that antioxidant compounds improved the rheological and gelling abilities of bovine cardiac MP led to the plausible deduction that oxidative reactions may be responsible for the loss of protein

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functionality(Wan et al.,1993).The belief that lipid and protein oxidation were governed by similar chemical mechanisms explains that most strategies aimed to control P-OX in meat and meat products were those reported to be effective against lipid oxidation.Results from early studies displayed the complexity of the chemistry behind protein oxidation as certain antioxidant strategies with proved effectiveness against lipid oxidation were not as effective against P-OX(Gatellier et al.,2000;Haak,Raes,&De Smet,2009;Mercier et al., 2004)or even displayed pro-oxidant effects(Estévez&Cava,2006; Srinivasan et al.,1996).It is then understood that the development of reliable antioxidant strategies against P-OX required the fully comprehension of the speci?c mechanisms involved in the oxidative degradation of food proteins.As far as protein carbonylation is concerned,some recent advances have contributed to shed light on the complex mechanisms implicated in the interaction between MP and natural antioxidants from plant kingdom(phenolic compounds). According to the existing literature,meat proteins can be protected against carbonylation following two main strategies:enhancing the oxidative stability of the animal tissues through dietary means or adding substances with antioxidant activity directly to the meat system.

7.1.Nutritional strategies

It is generally accepted that the oxidative stability of meat and meat products can be improved by managing animals'feeds(Decker, Faustman,&Lopez-Bote,2000).The fatty acid composition of muscle tissues of monogastric meat animals can be modi?ed by dietary means.In addition,animal tissues and in particular skeletal muscles can be enriched in natural antioxidants–tocopherols–by supplementing such substances in the diet.Hence,the development of dietary strategies aimed to enhance the oxidative stability of animal tissues generally involves reducing the ratio PUFA/FA in animal tissues and supplement animal feeds withα-tocopherol(≈200mg tocopherol/kg feed).As far as protein carbonylation is concerned,the supplementation withα-tocopherol and carotenoids has been proved to be effective while no concluding effects have been ascribed to the modi?cation of the fatty acid composition.Several authors have reported the antioxidant protection of dietary tocopherol and vitamin C against the carbonylation of muscle proteins during aging/chill storage of beef(Rowe et al.,2004),turkey(Mercier et al.,1998)and pork(Ventanas et al.,2006).On the other hand,Gatellier et al.(2000) reported that the nature of the dietary fat given to turkeys had a higher impact on lipid oxidation than in protein carbonylation.In the same line,Lund et al.(2007a,pork)and Gatellier et al.(2010) reported that the level of unsaturation of dietary fat was unrelated to the extent of protein carbonylation in pork and beef samples, respectively.Ventanas et al.(2006,2007)reported that the protective role of supplemented tocopherols would remain during long-term processing of meats.The amount of protein carbonyls in dry-cured loins and hams pigs fed on tocopherol-supplemented feeds was signi?cantly lower than that found in products from pigs fed on conventional feeds.In this case,signi?cant positive correlations were found between protein carbonyls and the PUFA/tocopherol ratio.

Feeding animals on pasture and other natural resources such as acorns have also been described as successful strategies to reduce the extent of protein carbonylation in porcine muscles owing the high concentration of tocopherols in such feeds(Ventanas et al.,2006).In agreement,meat emulsions(frankfurters and liver patés)manufac-tured with tissues from Iberian pigs fed on pasture and acorns showed a higher stability against the formation of protein carbonyls than products produced from pigs fed on concentrates(Estévez et al.,2007; Estévez&Cava,2006).In the same line,Santé-Lhoutellier et al. (2008b)observed a lower level of protein carbonyls in lamb fed pasture compared to lamb fed concentrates.Additionally,a study on chicken fed with a high antioxidant diet containing apple and broccoli revealed a lower level of protein carbonyl content in the muscle protein soluble fraction compared to chickens fed a low antioxidant diet(Young,Steffensen,Nielsen,Jensen,&Stagsted,2002).

7.2.Technological strategies

According to the existing literature,protein carbonylation can be inhibited in meat and meat products by reducing the exposure to molecular oxygen through packaging strategies packaging or by formulating meat products with substances with potential antioxi-dant effect.

Lund et al.(2007a,b)reported that the susceptibility of beef patties to protein carbonylation increases in high-oxygen atmosphere(80% O2/20%N2vs.100%N2)while pork seems to be unaffected by packing conditions.Recently,some other authors reported a promoting effect of high-oxygen packaging on protein carbonylation of beef muscles (Lindahl et al.,2010;Zakrys-Waliwander,O'Sullivan,Allen,O'Neill,& Kerry,2010)while others found no signi?cant impact of such modi?ed atmosphere packaging on the formation of protein carbonyls in ostrich meat(Leygonie et al.,2011).According to Filgueras et al. (2010),the employment of vacuum-packaging was appropriate for controlling ef?ciently protein carbonylation during chilled storage of rhea meat.Taking the results altogether,we can conclude that the effect of packaging conditions on meat protein carbonylation is variable and dependent on the specie.Existing literature suggests that storage of meat in high-oxygen atmosphere holds a potential risk for elevated oxidation but the onset of the oxidative reactions leading to protein carbonylation requires the presence of pro-oxidants(transi-tion metals,oxidizing lipids etc…)or technologies(irradiation, cooking…)capable of activating the molecular oxygen and yield ROS.Therefore,the observations in real meat systems are in good agreement with the chemical mechanisms described in detail in Section3.

Among substances with proved antioxidant potential,plant phenolics are compounds of increasing interest for their natural origin and additional bioactive effects(Frankel&Meyer,2000).The ability of phenolic compounds to act as antioxidants depends on intrinsic factors such as its own chemical structure and extrinsic ones such as the composition and characteristics of the substrate,stage and intensity of the oxidative reactions and localization of the phenolic compound(Frankel&Meyer,2000).Plant phenolics have been shown to be effective at reducing lipid oxidation and rancidity in a large variety of muscle foods(Estévez et al.,2007;Haak et al.,2009; Salminen et al.,2006;Vuorela et al.,2005).The antioxidant effect of plant phenolics against lipid oxidation is expected to reduce P-OX to some extent by minimizing the formation of secondary lipid oxidation products and thereby blocking their pro-oxidant effects on proteins. However,certain compounds that are able to prevent lipid oxidation may not be effective at inhibiting protein carbonylation(Haak et al., 2009).The intrinsic mechanisms of the protein oxidation damage, nature of the target,location of the site of attack together with the type of attacking species have an impact on the development of P-OX and hence,on the ability of the compound to prevent the oxidative damage(Dean,Hunt,Grant,Yamamoto,&Niki,1991).In addition,the effect of plant phenolics against P-OX is governed by speci?c interaction mechanisms between the phenolic compounds and the proteins.These interactions are dependent on the amount and chemical state of the phenolic compound and the size,conformity and overall charge of the protein(Kroll&Rawel,2001).Until recently, the lack of knowledge on the complex chemistry behind the protection of meat proteins by phenolic compounds prevented from designing reliable successful antioxidant strategies by using such compounds.Some phenolic-rich plant and fruit extracts have been shown to inhibit protein carbonylation in chicken meat,cooked patties, frankfurters,and liver patés(Estévez et al.,2005;Ganh?o et al.,2010b; Rababah et al.,2004;Rodríguez-Carpena et al.,2011;Vuorela et al.,

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2005),but the antioxidant effect was in general lower than that displayed against lipid oxidation.Lund et al.(2007b)found a clear antioxidant effect of a rosemary extract against lipid oxidation in beef patties,while the impact of such extract on protein carbonylation was negligible.Similar results were obtained by Haak et al.(2009)while evaluating the effect of rosemary and green tea extracts in porcine patties.In accordance,Sun et al.(2010)reported a signi?cant antioxidant effect of apple polyphenols on lipids from sliced cooked hams while the same dose had no effect against the formation of protein carbonyls.Estévez and Cava(2006)reported a dose-dependent pro-oxidant effect of a rosemary essential oil in proteins from cooked sausages.In model systems,some polyphenols andα-tocopherol have been reported to be ef?cient at protecting MP against oxidation,while others promoted protein carbonylation(Estévez et al.,2008c;Estévez& Heinonen,2010;Srinivasan et al.,1996).

A recent study proposed particular chemical mechanisms through which phenolic compounds could exert both antioxidant and pro-oxidant actions on MP(Estévez&Heinonen,2010).According to this study,phenolic compounds(i.e.gallic acid,catechin,cyanidin-3-glucoside and rutin)would inhibit the formation of speci?c protein carbonyls,AAS and GGS,from MP through their ability to act as metal chelators and inactivate the pro-oxidant effect of non-heme iron and as scavengers of radical species including lipid-derived ones(Fig.6).The protective effect of phenolic-rich fruit extracts against the formation of AAS and GGS in cooked patties,has recently been ascribed to these antioxidant mechanisms(Ganh?o et al.,2010a).Based on original ?ndings by Akagawa and Suyama(2001),Estévez and Heinonen(2010) also proposed a likely mechanism by which phenolic compounds could promote the formation of AAS and GGS from MP.In the presence of transition metals such as iron or copper,phenolics such as chlorogenic acid would undergo an auto-oxidation process leading to the formation of the corresponding quinones which display amine-oxidase activities. According to this proposal,quinone forms of plant phenolics could catalyze the oxidative deamination of susceptible amino acids to form the corresponding semialdehydes(Fig.6).

8.Final remarks

The present review collects the most relevant knowledge on the mechanisms and consequences of protein carbonylation in meat systems.Numerous challenges are still waiting to be accomplished all the way from the initiation of P-OX in meat systems to the management of P-OX in the meat industry to produce muscle foods with enhanced quality characteristics.The in?uence of in vivo factors, such as the rearing system and the feeding regime,on the occurrence of P-OX in muscle foods has been slightly studied.AAS and GGS are considered reliable indicators of oxidative stress in vivo and disease. Pre-slaughter exposure to stress by several means is known to affect the oxidative stability of animal tissues upon slaughter.May these speci?c protein carbonyls serve as indicators of animal welfare and/or early markers of the meat quality?The impact of numerous intrinsic (i.e.metabolic pro?le of muscle,composition,and structure)and extrinsic factors(i.e.exposure to light or irradiation)on the onset of P-OX in meat and meat products is still poorly understood.The fate of particular amino acids during handling,processing and storage of meat products requires further elucidation.The identi?cation of the oxidation pathways and products would clarify the potential role of P-OX,and in particular protein carbonylation,on the quality of diverse meat products.For instance,the identi?cation of the precise mechanisms by which P-OX could in?uence the loss of WHC in frozen meat is of high importance due to the enormous economical impact of such phenomenon on the global meat industry.Further investigations should also cover the potential impact of P-OX in consumer's health and the in?uence of oxidative reactions on the nutritional value and bioactive effects of proteins and peptides.

The ful?llment of these objectives requires a determined dedica-tion to the study of basic P-OX chemistry through the application of innovative and highly precise methodologies.The accomplishment of research studies on P-OX is paramount for having,eventually,a complete picture of the map of biochemical reactions affecting to major food components.As a truly emerging topic,ful?lling partial objectives unavoidably leads to a new challenge that would provide, in turn,grounds for further studies.The present review aimed to display the enormous potential of this highly unexplored?eld and may serve as encouragement to those who look for novel research lines.Protein carbonylation and hence,P-OX will be a leading topic during the upcoming years.

Acknowledgments

Mario Estévez is very much thankful to his teachers and mentors for their enduring encouragement and support:Prof.J.Ventanas (University of Extremadura,Spain),Prof.E.Puolanne and Prof.M. Heinonen(University of Helsinki,Finland).Mario Estévez thanks the Spanish Ministry of Science and Innovation for the contract through the“Ramón y Cajal(RYC-2009-03901)”program and the support through the project“Protein oxidation in frozen meat and dry-cured products:mechanisms,consequences and development of antioxidant strategies;AGL2010-15134”.Mario Estévez thanks the European Community for the economical support from the Marie Curie Reintegration Fellowship(PERG-GA-2009-248959-Pox-MEAT). References

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