Véronique Verrez-Bagnis

Trends in postmortem aging in fish: understanding of proteolysis and disorganization of the myofibrillar structure

Postmortem tenderization is caused by enzymatic degradation of key structural proteins in myofibrils as well as in extracellular matrix, and of proteins involved in intermyofibrillar linkages and linkages between myofibrils and the sarcolemma. The function of these proteins is to maintain the structural integrity of myofibrils. Current data indicate that calpains and cathepsins may be responsible for degradation of these proteins. Other phenomena occurring in cells postmortem (pH drop, sarcoplasmic Ca 2+ increase, osmotic pressure rise, oxidative processes) may act in synergy with proteases. Our understanding of the underlying mechanisms of muscle degradation should be improved for an accurate evaluation of the postmortem muscle changes and consequently of the fish quality.

In vitro proteolysis of myofibrillar and sarcoplasmic proteins of white muscle of sea bass (Dicentrarchus labrax L.): effects of cathepsins B, D and L

The purpose of this study was to obtain additional information regarding proteolysis mechanisms and disorganization of fish myofibrils resulting in a loss of flesh quality. The ability of cathepsins to degrade in vitro myofibrillar and sarcoplasmic proteins from fish muscle was investigated in order to explain their role in post mortem softening. This led to the identification of substrates of the enzymes. Cathepsins degraded myosin heavy chain and α-actinin. Tropomyosin and actin were only susceptible to the action of cathepsin L. Troponin T (assumed 32 kDa component) was resistant only to the action of cathepsin D. Desmin was degraded by cathepsins B and L. Slight changes of some other myofibrillar or cytosolic proteins were also observed (creatine kinase and other unidentified proteins). When compared with protein modifications observed in stored post mortem muscle, these results suggest that cathepsin D (if location is in the cytosol and if pH conditions for activity are met in post mortem muscle) could be involved in a post mortem myofibrillar degradation mechanism.

Post mortem release of fish white muscle α-actinin as a marker of disorganisation

alpha-Actinin release and its degradation from myofibrils Z-line were studied in post mortem white dorsal muscle from bass and sea trout stored at 4 degrees C and 10 degrees C. Using alpha-actinin specific antibodies, we show that this protein is rapidly released within the first 24 h for the two species, and reaches a plateau within 4 days. Proteolysis take place very rapidly in bass muscle yielding 80 and 40 kDa fragments from alpha-actinin as major bands of proteolysis. Sea trout muscle is more resistant, and muscle stored at 4 degrees C is not significantly alpha-actinin degraded even 10 days after death. In the case of sea trout muscle stored at 10 degrees C, an increasing quantity of 80 and 40 kDa fragment can be observed after the third day. These results show that release and proteolysis of alpha-actinin are time- and temperature-dependent processes that take place at the early stages of fish storage. Furthermore, we observed that proteolysis of alpha-actinin seems to be dependent on fish species. In both species studied, the early release of alpha-actinin comes before the degradation of released molecules, and appears as a biphasic process throughout the disorganisation of post mortem muscle in fish cold-stored above 0 degree C.

Protein changes in post mortem sea bass (Dicentrarchus labrax) muscle monitored by one- and two-dimensional gel electrophoresis

This study was devoted to the identification of specific peptides and proteins which can be used as indicators of freshness in fish. The post mortem evolution of protein patterns in farmed sea bass muscle was monitored by Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and two‐dimensional electrophoresis (2‐DE) after 0, 2, 4, and 6 days cold storage. SDS‐electrophoresis, of total proteins and proteins soluble in low‐ionic‐strength solutions, revealed the gradual disappearance of a protein band of 16 kDa immediately after fish death. 2‐DE allowed the classification of fish samples according to post mortem time. Three spots of interest, which disappeared progressively, were identified on the 2‐DE patterns. Further research is required to establish the identity of these polypeptides and to evaluate their expression and post mortem evolution in another fish species.

Neutral calcium-activated proteases from European sea bass (Dicentrarchus labrax L.) muscle: polymorphism and biochemical studies

Calcium-dependent proteinases or calpains were studied in fish muscle. Hydrophobic chromatography, followed by anion-exchange chromatography of the soluble fraction of sea bass white muscle proteins, resulted in three peaks of calcium-dependent protease activity at neutral pH (A, B and C). They are all neutral cysteine calcium-activated proteinases and can, therefore, be classified as calpain-like enzymes. From the Ca2+ concentration required for activity, A is a mu-calpain, and B and C are m-calpains. They share many properties with calpains from other vertebrate cells but differ in native mass, subunit composition, and the unusual numbers in which they are present. Their specific pattern of expression throughout the year could be of great importance to the resulting rate and extent of degradation of fish flesh after death.

Proteolytic potential in white muscle of sea bass (Dicentrarchus labrax L.) during post mortem storage on ice: time-dependent changes in the activity of the components of the calpain system

The variations in the amounts of milli-calpain and its specific inhibitor in the white muscle of sea bass (Dicentrarchus labrax) during storage at 4 °C for up to 7 days were determined after separation by hydrophobic chromatography on a Phenyl Sepharose gel. There was a significant decline in postslaughter m-calpain activity with an important inter-individual variability in the rate of decrease of the total activity. In contrast with the calpastatin of mammalian post mortem muscles, calpastatin remained constant within fish muscles after death. The initial levels of protease and inhibitor activities, and their behaviour through post mortem storage, are discussed and implications for the mechanism of tenderisation of fish muscle are suggested.

Low mislabeling rates indicate marked improvements in European seafood market operations

Over the span of a decade, genetic identification methods have progressively exposed the inadequacies of the seafood supply chain, revealing previously unrecognized levels of seafood fraud, raising awareness among the public, and serving as a warning to industry that malpractice will be detected. Here we present the outcome of the latest and largest multi-species, transnational survey of fish labeling accuracy to date, which demonstrates an apparent sudden reduction of seafood mislabeling in Europe. We argue that recent efforts in legislation, governance, and outreach have had a positive impact on industry regulation. Coordinated, technology-based, policy-oriented actions can play a pivotal role in shaping a transparent, sustainable global seafood market and in bolstering healthier oceans.

Milli-calpain from Sea Bass (Dicentrarchus labrax) White Muscle: Purification, Characterization of Its Activity and Activation In Vitro

Abstract: A calcium-activated neutral cysteine protease was purified to homogeneity from Dicentrarchus labrax white muscle using three steps: hydrophobic interaction, anion exchange, and gel filtration chromatographies. The purified enzyme showed a native molecular weight of 124 kDa with an oligomeric structure (large subunit of 80 kDa and small subunit of 24 kDa). It has been classified as a milli-calpain from its calcium sensitivity. Activity was maximal at pH 7.0, 24°C in Tris buffer without NaCl as determined by means of a two-level experimental design and response surface methodology. Sea bass calpain is neither glycosylated nor phosphorylated and shared some common cleavage specificities and activation and autolysis mechanisms with other typical mammalian or invertebrates calpains. Calcium-induced activation and autolysis of calpain has been characterized together with the effect of the strontium cation acting as a calcium analog. On the basis of its in vitro properties, the contribution of the sea bass milli-calpain to the process of postmortem deterioration of fish muscle is discussed, even though further information such as in vivo regulation or in vitro effects on myofibrils is required.

Isolation and properties of white skeletal muscle α-actinin from sea-trout (Salmo trutta) and bass (Dicentrarchus labrax)

Abstract Fish a-actinin purified from sea-trout and bass white muscle by means of two different extraction procedures was used to investigate the eventual presence of different muscle isoforms in Z-disks. These fish α-actinins have the same apparent molecular weight (100 kDa) and the same isoelectric point (pI = 5.6), and also have a total antigenic identity towards anti-bass and anti-chicken α-actinin antibodies, suggesting a single molecular species. The role of fish α-actinin as an anchorage site for thin actin filaments and elastic titin filaments in Z-bands was studied. Despite conservation of the actin-binding site, fish α-actinin has a better actin-binding ability (kD = 0.3 μM), than chicken smooth muscle a-actinin (kD = 1.6 μM). Several other structural and functional characteristics of fish α-actinin were also studied: conservation of sequence and domain structure, the role of divalent ions (Ca2+, Mg2+) and the dielectric constant of the medium in α-actinin-actin interaction. Although the reason for fish white muscle a-actinins close affinity to actin was not clearly established, our results suggested that the physicochemical environment of the Z-filaments in Z-disks might be crucial.

Restriction Fragment Length Analysis of the Cytochrome b Gene and Muscle Fatty Acid Composition Differentiate the Cryptic Flatfish Species Solea solea and Solea aegyptiaca

Overlapping external morphometric characters easily confound the flatfishes Solea aegyptiaca and Solea solea (Soleidae) in areas of the Mediterranean Sea where both species live in sympatry. This leads to uncertainties in the fisheries and marketing of the species, in addition to misinterpretations in biogeography and conservation studies. This paper describes a simple restriction fragment length-based diagnostic test that differentiates S. solea from S. aegyptiaca, as well as from other species of the Soleidae family. Furthermore, the two species living in sympatry in the Gulf of Kavala (North Aegean Sea, Greece) present significant qualitative differences in muscle fatty acid composition, a property that can also be used to distinguish the two cryptic species.