Bovine muscle 20S proteasome. II: Contribution of the 20S proteasome to meat tenderization as revealed by an ultrastructural approach

The role of the 20S proteasome proteolytic effects was revisited using an ultrastructural approach with the aim to explain some particular structural changes identified in type I muscles and in high pH meat. In both types of meat, major changes observed after ageing are an increase in the thickness of the Z-line followed by the appearance of an amorphous protein structure spreading out over the I-band. This was followed by a total degradation of this amorphous structure and of the Z-line. Partial transversal fragmentation of the myofibrils within the I-band can also be detected. The data reported clearly demonstrate that the 20S proteasome was able to mimic these sequential structural changes, a feature never obtained with either calpains or cathepsins. It is the first time that a direct implication of this complex in postmortem muscle is postulated.     

Bovine muscle 20S proteasome: I. Simple purification procedure and enzymatic characterization in relation with postmortem conditions

Over the last decade, several sets of evidence support a possible contribution of the 20S proteasome to the meat tenderizing process. This assumption was emphasized by recent investigations demonstrating that the 20S proteasome was active in the absence of activators and exhibited endo- and exoproteolytic activities, a status often strongly debated before. In the present work, we developed a new rapid and simple purification procedure for muscle 20S proteasome and revisited the physicochemical properties of this complex in relation with the postmortem muscle environmental conditions, i.e. temperature, pH, osmolarity, etc. From a crude extract obtained from freshly excised muscle tissue, reasonable amounts of highly pure proteasome were prepared within a maximum of 4 days using only three chromatography steps. This purified proteasome was used to investigate the effect of pH, temperature, ionic strength and sodium dodecyl sulphate (SDS) on the major hydrolytic activities of this complex, i.e. trypsin-like (TL), chymotrypsin-like (CL) and peptidylglutamyl peptide hydrolase (PGPH) activities. Taken together, the data obtained suggest that the 20S proteasome constitutes a high hydrolytic potential in postmortem muscle conditions. To attest this finding, the 20S proteasome was further quantified by ELISA in at death and postmortem muscles including Longissimus, Rectus abdominis, Diaphragma pedialis and Tensor fascia latae bovine muscles. The primary conclusion was that time course changes in proteasome concentrations were not dependent on the kinetics of the pH fall. Secondly, the proteasome concentration in conditioned meat was in good agreement with previously reported proteolytic activity. Furthermore, the decrease in the muscle proteasome concentration can be considered as slow and this is particularly true in type 1 muscles for which the decrease in the amount of this complex did not exceed 7% during the first three days postmortem. This would suggest that the 20S proteasome was relatively stable during meat conditioning, a feature supporting a potential role in the meat tenderizing process.     

Lipoamide dehydrogenase, citrate synthase and beta-hydroxyacyl-CoA-dehydrogenase in skeletal muscle. I. Determination of activity in tissue extracts

It is to be expected that changes in the subcellular distribution of the mitochondrial enzymes lipoamide dehydrogenase (LIPDH), citrate synthase (CS) and beta-hydroxyacyl-CoA-dehydrogenase (HADH) in the muscle tissue give information on the type and the extent of damage of mitochondria during storage and treatment of meats; such changes may be also used as basis of methods for the differentiation between fresh and frozen/thawed meat. Standard methods for the determination of the activities of LIPDH, CS, and HADH in tissue extract and muscle press juice are described. The influence of enzyme concentration, pH and temperature on the enzyme activities in muscle extract was investigated. Furthermore the error in the enzyme analyses by the standard methods was determined.     

Screening for the coccidiostats halofuginone and nicarbazin in egg and chicken muscle: development of an ELISA.

Nicarbazin and halofuginone have been widely used as coccidiostats for the prevention and treatment of coccidiosis in poultry. It has been shown that accidental cross-contamination of feed can lead to residues of these compounds in eggs and/or muscle. This paper describes a direct competitive assay for detecting halofuginone and nicarbazin, developed as qualitative screening assay. In an optimized competitive ELISA, antibodies showed 50% binding inhibition at approximately 0.08 ng ml(-1) for halofuginone and 2.5 ng ml(-1) for dinitrocarbanilide (marker residue for nicarbazin). Extraction from the matrix was carried out with acetonitrile followed by a wash with hexane. The assay's detection capability (CCbeta) for halofuginone was < 0.5 microg kg(-1) in egg and < 1 microg kg(-1) in muscle. For dinitrocarbanilide, the CCbeta was estimated at < 3 microg kg(-1) in egg and < 10 microg kg(-1) in chicken muscle.     

Effect of increasing amounts of a linoleic-rich dietary fat on the fat composition of four pig breeds. Part III: Triacylglycerol composition in muscle and fat tissues

Here, we study the triacylglycerol (TAG) profile of four different tissues of the pig (backfat, abdominal fat, and muscles trapezius and longissimus thoracis et lumborum). For this purpose, 48 pigs of four breeds (Landrace, Large White, Duroc and a crossbreed Landrace × Duroc) were given one of four diets containing increasing amounts (0%, 2%, 4% and 8%) of a fat blend rich in linoleic acid. The effects of dietary fat and breed on TAG were tested separately for each tissue, and the results are presented using five TAG composition markers, PLL, PStO, PStL, PStSt and OOO. The increasing linoleic acid content provided by diets 1–4 showed a positive effect on the levels of TAG related to this dietary supply (here represented by PLL), and on those of PStL. This increase was at the expense of TAGs containing mainly fatty acids from de novo synthesis (represented by PStO) and in PStSt and OOO. A comparison of the relative % of change for the five selected TAG markers in the distinct tissues indicates that PLL and PStL show much higher increases in muscle than in adipose tissues, whereas PStO and PStSt show similar percentages of decrease in all tissues. OOO showed a higher % of decrease only in trapezius. Results indicate that the breed has a null or scarce effect on the levels of PLL and PStO. For the remaining TAG markers, Large White showed a higher synthesis of saturated TAG (PStSt and PStL) in fat tissues, but not in muscle. Large White also had the lowest levels of OOO in all tissues, being the breed most susceptible to the changes in dietary linoleic acid content. Moreover, the Landrace showed enhanced deposition of monounsaturated TAG in trapezius muscle and abdominal fat.     

Different changes in mastication between crisp grass carp (Ctenopharyngodon idellus C.et V) and grass carp (Ctenopharyngodon idellus) after heating: The relationship between texture and ultrastructure in muscle tissue

In order to study special mastication of Crisp grass carp (CGC), the relationships between the texture and ultrastructure in raw fresh and heated CGC and grass carp (GC) were studied. In raw samples, all textural results of raw CGC (RCGC) were higher than those of raw GC (RGC). From the ultrastructural results, short muscle fiber diameter, dense fiber density, large collagen amounts, narrow intermyofibrillar spaces and gaps determined the texture of fillets. In the heated samples, the hardness, chewiness, resilience and springiness of heated CGC (HCGC) were higher than in heated CG (HCG). Compared to raw samples, the hardness, fracturability, springiness and chewiness of HCGC were higher than those of RCGC. However, all textural results of HGC were significantly reduced. The ultra-structural results showed that the better mastication of HCGC was connected with denaturation and coagulation of myofibrillar and sarcoplasmic proteins, the low ratio of shrinking in inter-muscular connective tissue, large interstitial material, much less fiber–fiber detachments and myofiber–myocommata detachments.