Article pp.181-188 du Vol.27 n°2 (2007)

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FOCUS : JSMTV

Post mortem metabolism and meat qualities : how proteomics can bring new information

M. Morzel, C. Terlouw, E. Laville

1 – INTRODUCTION

Muscle post mortem metabolism may be defined as the whole set of bio- chemical processes occurring between animal death and the final processing of meat before consumption. The most documented post mortem events are glycolysis, leading to lactate accumulation and pH decline, lipid oxidation and modifications of proteins, namely denaturation, proteolysis and oxidation. Numerous factors influence the nature and/or extent of post mortem metabolism, depend- ing on the animal (genetics, rearing conditions and feeds, reactivity to slaughter stress etc.) or on the technology used: stunning method, chilling of carcases, packaging etc. (LEBRET et al., 2002, TERLOUW et al., 2005).

Studies on post mortem metabolism classically focus on one type of reaction, and are based on the quantification of one or a few effectors or products of that reaction. For example, glycolysis is studied by quantifying selected enzymes in the pathway, or by measuring a resulting characteristics (pH, lactate concentration…). Proteolysis is investigated by quantifying proteases and their inhibitors, or by observation of proteolytic profiles. However, all events interact with each other, and it is the result of the interaction that determines technolog- ical (e.g. water-holding capacity), sensory (flavour, tenderness, juiciness) or nutritional properties of meat. This justifies the use of global methods such as proteomics or metabolomics.

Proteins are key elements in post mortem metabolism, firstly because they constitute a target of chemical (proteolysis, oxidation) or structural (denaturation) modifications but also because they are the mediators of all biochemical reactions, may they be enzymes, inhibitors of regulatory proteins. Thus, in this communication, we will present the influence of the muscle cell protein content at the time of slaughter and the influence of protein post mortem modifications, on the quality of raw meat. We will provide illustrations based on recent studies performed in our research group, using proteomics.

Unité Qualité des Produits Animaux – INRA – Theix – 63122 Saint-Genès-Champanelle – France.

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2 – PROTEIN EXPRESSION AND MEAT QUALITIES

In the present article, “protein expression” is the muscle cell content in proteins at the time of slaughter. It depends both on the animal genetics (breed, mutations) and on modulation factors, acting throughout the whole animal lifespan or during shorter periods, in particular during the pre-slaughter steps which are well-known to be great disturbers of muscle physiology. Taking into account the level of expression of proteins and their biological function, hypoth- eses on their interactions can be formulated and mechanisms underlying meat quality development can be proposed. For that purpose, we have studied spe- cifically the proteome soluble fraction (sarcoplasmic fraction) since it contains the majority of mediators of biochemical reactions.

2.1 Genetic factors

Between-muscle differences are often the consequence of specificity in the function of a given muscle. For example, comparing proteomes of three sheep muscles (tensor fasciae latae – fast glycolytic, vastus medialis – oxidative, and semimembranosus – intermediate type), we have found that muscles were dif- ferentiated by proteins of the oxidative metabolism (HAMELIN et al., 2007) while expression of the glycolytic metabolism enzymes was relatively constant between muscles. Associated to the oxidative metabolism, we have evidenced proteins linked to the oxidative stress, i.e. involved in cell detoxification, in refolding of denatured proteins, in degradation of damaged proteins and in protein synthesis. Such proteins confirm that an oxidative metabolism generates toxic compounds, for example ROS (reactive oxygen species), leading to a high rate of protein turnover. As a consequence, oxidative type fibres have a particular protein composition which enables them to withstand oxidative stress. This specificity of red muscles might explain why they are less susceptible than white muscles to denaturation phenomena, independently from the fact that post mortem glycogenolysis is lower in red muscles.

The mutation leading to muscle hypertrophy in the “Belgian Texel” ovine breed involves the myostatin gene (CLOP et al., 2006). Animals carrying the mutation have a higher muscle mass and less fat, but alteration of meat sensory qualities has not been reported. Comparing muscle proteins in animals carrying the mutation or not (HAMELIN et al., 2006a), we have shown in the first group over-expression of enzymes of both oxidative and glycolytic energy metabolisms, which is concomitant with over-expression of several chaperone proteins. Finally, transferrin and alpha-1-antitrypsin are over-expressed and under- expressed, respectively, in all muscles of the mutation carriers, whether the muscles are affected by hypertrophy or not, as for example in vastus medialis.

These two proteins, which genes are highly expressed during foetal stages (ARNm) have probably a role in enhancing the proliferation signal during myo- genesis (HAMELIN et al., 2006b). In this study, myostatin – encoded by the mutation carrier gene – was not evidenced, but proteome analysis revealed proteins that may be involved in the regulation of myostatin, thereby contributing to muscularity increase.

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Another mutation affecting the gene encoding for the RyR calcium channel results in a great metabolic distrubance, leading to severely defective pork meat quality. Between susceptible and non-susceptible pigs, differences in protein expression are limited to modifications in enzymes of the energetic metabolisms. Susceptible pigs are characterised by higher levels of glycolytic enzymes (Sayd, personal communication).

In a pig population with a high genetic variability, the proteomic tool has also been applied in order to clarify the mechanisms by which defective PSE zones, resulting in so-called “destructured” meat, appear in cooked ham (SAYD et al., 2006). This meat quality defect may be observed firstly by discoloration of the inner muscles of fresh hams. After cooking, the affected zones are torn during mechanical slicing which leads to considerable economical losses. We have compared the proteome of two types of ham muscles, dark and light, selected within 12 families of an F2 population presenting a large genetic variability. Dark muscles have a more oxidative metabolism and contain more chaperone proteins. Such a protein composition would decrease the rate of pH decline and would limit protein denaturation. Conversely, light muscles have a more glycolytic metabolism and over-express transferrin, an iron transporter. This hypoxia- related protein suggests a deficiency in muscle oxygenation, due to a lesser vascularity or to the animal inability to reach an appropriate blood flow at the time of slaughter. Also in light muscles, over-expression of glutathione S-trans- ferase omega (GSTO) could contribute to a constant higher sarcoplasmic calcium concentration, and therefore to the production of oxidative compounds.

Associated to a predominantly glycolytic metabolism, over-expression of GSTO may accelerate ATP depletion, increase the rate of pH decline and the conse- quent protein denaturation, all those events leading to meat discoloration.

Those results explain in part why slaughter conditions have a large influence on prevalence of PSE zones in hams.

Through the examples presented above, it appears that a genetic mutation has often indirect consequences on differential expression of proteins. The dif- ference on protein content does not necessarily indicate the causative mutation, but it may be used as a marker of a phenotype which had better be retained or eliminated, through genetic selection or through use of appropriate breeding or slaughter conditions. In some cases, differences in proteome also suggest mechanisms by which meat qualities are developed.

Finally, proteomics may also contribute to understanding differences in meat quality between pigs of different genetic origins. The study used an experimental design set up within the European project Susporkqual: it aimed at determin- ing the relative contribution of the genetic type (sires: Duroc or Large White, dams: Large White x Landrace), of breeding conditions (indoors, outdoors) and of slaughter conditions (mixing of groups or not, lairage or not) on the techno- logical qualities of pig meat. Analysis of the sarcoplasmic protein fraction of longissimus lumborum from 24 animals evidenced that 10 proteins are quantita- tively influenced by the sire breed only. For example, a Ca²⁺ binding protein (CBP), involved in the regulation of intracellular Ca²⁺ concentrations, is negatively correlated with exudation in pigs from Large White sires and explains 50% of variability. The two measures are probably indirectly linked through the relationship between ultimate pH (itself correlated to exudation) and CBP. In pigs from Duroc sires, CBP spots intensity is higher but is not correlated to exudation. Proteomics also allows to identify proteins discriminating for the sire

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breed type. Thus, a principal component analysis using the 10 proteins allows to correctly classify 22 out of 24 pigs according to their breed origin. After exclusion of 8 of the least contributing proteins, 20 pigs are still properly classi- fied using only two proteins, namely heat shock protein 70 (HSP70) and pyru- vate kinase M2 (PK2). The two proteins are strongly influenced by the genetic origin, but only HSP70 is correlated to a meat quality indicator, cooking loss.

This study shows that the link between protein expression and meat qualities are dependent on the animal genetic type.

2.2 Conditions of the animal environment

2.2.1 Breeding

It is well-known that breeding conditions can modify the muscle contractile properties and the muscle concentration in some proteins such as myoglobin (BIDNER et al., 1986), antioxidant enzymes (GATELLIER et al., 2004) or enzymes of the energetic metabolisms (BEE et al., 2004). This has been especially investi- gated when comparing conventional vs outdoors breeding. Modifications in protein content result from different components of the breeding conditions, particularly feeding regimes (e.g. access to pasture), and exercise dependent on the space allocated to the animal.

As an example, using the Susporkqual experimental set up, it was evidenced that 18 protein spots were significantly influenced only by the breeding type (KWASIBORSKI et al., 2006). Compared to indoors pigs, longissimus lumbo- rum of outdoors pigs had higher levels of myoglobin and lower levels of glyc- erol-3-phosphate dehydrogenase (G3PDH, involved in NADH synthesis), indicating a more oxidative metabolism. The 2 proteins were negatively correlated to L* value. Compared to the genetic origin (cf above), breeding conditions affect a larger number of proteins, and more proteins are also necessary to correctly classify the animals into their breeding class: 18 proteins allow the correct classification of 22 pigs. The 10 most contributing proteins are required for a proper classification of 18 pigs.

2.2.2 Slaughter stress

Slaughter stress, with its emotional and physical components, induces a profound disturbance of muscle physiology, which can have consequences on meat quality. This has been largely documented in most meat-producing animals, and even more in pig, poultry and fish.

In pig and poultry, a consequence of intense muscle activity immediately before killing is a drop in water-holding capacity. This phenomenon is due among other factors to an increased muscle temperature and an acceleration of post mortem glycolysis, the resulting combination of low pH and high tempera- ture favouring protein denaturation (BENDALL et WISMER-PEDERSEN, 1962), for example of myosin. This was reported as the major cause of excessive exudation (OFFER, 1991).

Another example of a detrimental consequence of slaughter stress is unde- sirable muscle softening in several fish species (SIGHOLT et al., 1997 ; MORZEL et al., 2003). In order to investigate the underlying mechanisms, we have

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performed a comparative proteome analysis of muscles from two batches of rainbow trout. The first batch was slaughtered in conditions limiting muscle activity (fast netting followed by chemically induced anaesthesia and bleeding while fish were sedated). The second batch was subjected to lowering of the water level in the tank for 15 minutes, which generates intense physical activity in the group, before proceeding to the same netting, anaesthesia and bleeding protocol. Intense muscle activity for 15 minutes is sufficient to modify the relative proportion of several enzymes of the energy-producing pathways, but also of structural proteins, very early (15 minutes) post mortem. At 24 h post mor- tem, differences between the two batches are less pronounced, but desmin, a structural protein, remains less represented in trout subjected to high levels of muscle activity. Desmin has been proposed as a marker of fish freshness. Thus, physical activity induced by prolonged crowding would influence muscle texture by affecting the muscle cell cytoskeleton (MORZEL et al., 2006a).

3 – PROTEIN MODIFICATIONS AND MEAT QUALITIES

Post mortem, events of a physico-chemical or enzymatic nature lead to altera- tion of the muscle structures. This phenomenon is known as ageing in mammals and birds, where it contributes to improved meat sensory properties, including tenderness. During this period, muscle proteins are modified by three main types of reactions interacting with each other: denaturation, oxidation, proteolysis.

3.1 Denaturation

Denaturation can be defined as the spatial reorganisation of proteins without hydrolysis of the peptide bonds. It may result from muscle acidification, dessi- cation, exposure to high levels of salts or to low (< 0°C) or high temperatures (LAWRIE, 1998). Several experimental measurements are indicators of denaturation, the most commonly used being solubility (protein concentration in the muscle fraction soluble in a phosphate buffer) or the degree of protein hydro- phobicity.

As previously described, the combination of low pH and high temperature increases the level of protein denaturation, as for example in PSE (Pale Soft Exu- dative) meat in pig or poultry. In a recent study (LAVILLE et al., 2005) semimem- branosus muscles of pigs affected by PSE zones in deep ham areas were characterised. Denaturation of the sarcoplasmic proteins (reduced solubility) was evidenced and we have observed one-dimensional electrophoretic patterns of myofibrillar and sarcoplasmic proteins similar to those previously described in PSE meat, for example by JOO et al. (1999). The originality of the study, however, was provided by the use of 2D electrophoresis for characterising defective muscles. For example, we have observed the lesser representation of one form of HSP27 in destructured meat. Since this protein is involved in the stabilisation of actin myofilaments, it is plausible that a lower quantity of HSP27 enhances denaturation of the proteins constitutive of thin myofilaments. A mechanism leading to protein denaturation was later proposed, as described above (SAYD et al., 2006).

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3.2 Oxidation

After slaughter, alteration of the antioxidant protection systems (e.g. lower activity of antioxidant enzymes, degradation of molecules such as vitamin C) creates a cell environment favourable for protein oxidation. Oxidation is expressed as the formation of carbonyls groups on amino-acids with NH₂ groups (R-NH₂ converted into R-CHO), oxidation of SH groups and the conse- quent formation of disulfide bridges between two cysteins, oxidation of OH groups leading to butyrosines or hydroxylation of aromatic amino-acids (DAVIES, 1987; STADTMAN, 1990). Protein oxidation has received some interest in research on muscle as food, whether in meat (LIU et XIONG, 1996; MARTINAUD et al., 1997) or in fish flesh (KJÆRSGÅRD et JESSEN, 2004) because of its role in protein loss of solubility (DECKER at al., 1993) and in modulation of post mortem proteolysis. For instance, results obtained on isolated myofibrils, chemically oxi- dised by hydroxyl radicals OH° and subsequently hydrolysed by papain, show that oxidation induced the formation of disulfide bridges and bityrosines, gener- ated aggregates and decreased the proteolytic susceptibility of myofibrillar proteins (MORZEL et al., 2006b).

3.3 Proteolysis

Alteration of muscle integrity after resolution of rigor mortis results from the action of proteolytic enzymes on structural proteins. Thus, degradation of several myofibrillar and cytsokeletal proteins during meat ageing is widely accepted, for example degradation of titin (FRITZ et GREASER, 1991), nebulin (TAYLOR et al., 1995; HUFF-LONERGAN et al., 1995), desmin (KOOHMARAIE et al., 1991; TAKAHASHI, 1996) and troponin T (HO et al., 1994). In fish muscle, a specific marker of freshness is α-actinin (TSUCHIYA et al., 1992; PAPA et al., 1996). The proteolytic systems involved in meat ageing are the same ones as those ensuring protein degradation during cell turnover in the live animals (ASGHAR et BHATTI, 1987). Three main enzymatic systems are likely to intervene: calpains, cathepsins and proteasome. The role of proteases involved in apoptosis, caspases, has also been recently documented (SENTANDREU et al., 2002; HERRERA-MENDEZ et al., 2006).

Use of the proteomic tool has brought about new information on proteolytic events. Firstly, in most of our studies, we have observed protein fragments in muscles sampled very early post mortem, suggesting that such fragments exist in the live animal muscle (MORZEL et al., 2004, LAVILLE et al., 2005, SAYD et al., 2006). In the latter study, we recorded a higher abundance of fragments of cre- atine kinase and of enolases in pig muscle sampled very early after death and leading to a darker meat. We therefore propose a mechanism whereby the higher degree of proteolysis is linked to an oxidative metabolism, itself indicated by larger amounts of mitochondrial enzymes of the respiratory chain. The consequence on meat quality is not direct, but the degree of proteolysis is indica- tive of the peculiar metabolic conditions.

The second type of information we have obtained is the identification of unpublished post mortem proteolyis targets, at the protein or ultrastructural level. Such targets belong to for example to the sarcoplasmic compartment (α-crystallin, myokinase) or are constitutive of the Z-line (cypher proteins, myozenin), which certainly contributes to the weakening of this structure during meat ageing (MORZEL et al., 2004).

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4 – CONCLUSIONS

The studies presented above show that the muscle energy metabolism is involved in the meat quality specificities, particularly its texture attributes. The type of metabolism and the presence of « protective » proteins (HSP, chaper- ones…), in interaction with events preceding animal death, have an influence on meat qualities. This is well accepted to explain meat qualities in pig and poultry meat, and may also apply to bovine meat.

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