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.     

Glutathione peroxidase activity,tissue and soluble selenium content in beef and pork in relation to meat ageing and pig RN phenotype

Since glutathione peroxidase (GSHPx) may be important for meat quality, its activity was examined in relation to animal species (beef and pork), muscle type (oxidative and glycolytic), selenium content, meat ageing and RN phenotype in Hampshire crossbred pigs. The GSHPx activity in bovine M. Longissimus dorsi [LD; 1.9 (0.4) U/g, mean (S.D.)] was significantly higher (P<0.001) than in M. Psoas major [PM; 1.5 (0.5) U/g] and the activities in the two muscles were correlated (r=0.66; P<0.001). Pork LD had a several fold lower GSHPx activity [0.4 (0.2) U/g] than bovine muscles but, in contrast, a somewhat higher content of selenium [113 (15) ng/g] than bovine LD [106 (12) ng/g] and PM [95 (17) ng/g]. The proportion of soluble selenium in bovine LD (72%) tended to be higher than that in bovine PM (64%), and it was significantly lower in pork LD (52%). Significant correlations between GSHPx activity and soluble selenium were obtained in both beef and pork. Thus, GSHPx activity varied among single animals, with species and muscle type, whereas meat ageing and pig RN phenotype had no effect.     

Thermal properties of titin from porcine and bovine muscles

The thermal properties of titin isolated from porcine and bovine longissimus muscles were investigated by differential scanning calorimetry in the temperature range from 20 to 100?°C. A single peak with average maximum temperatures of 75.6 and 78.4?°C characterized porcine and bovine titin denaturation, respectively. The peaks were much broader than those from the other major muscle proteins. Titin denaturation enthalpy values (1.6-2.6 J/g) were only about half those of whole meat and also lower than those previously determined for myosin, actin, or collagen. The relatively high titin denaturation temperature suggests that it may be partially responsible for meat toughening when muscle tissue is heated above 60?°C.     

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.     

The significance of the activity of glycogen debranching enzyme in glycolysis in porcine and bovine muscles

The purpose of the study was to examine the activity of glycogen debranching enzyme, GDE, in porcine and bovine muscles differing in rate of contraction and oxidative capacity. The activity of GDE, the activity of phosphorylase, total glucose content, lactate content and pH were measured from meat samples taken 35min post-mortem and ultimate pH 24 or 48h post-mortem. Both GDE and phosphorylase are needed for the complete degradation of glycogen. In porcine muscles the activities of these glycogen degrading enzymes were higher than in bovine muscles. The activities were increasing with the increasing fast twitch and glycolytic character of a muscle of a given species. However, the increase in the activity of phosphorylase was greater than the increase in the activity of GDE. It was concluded that the GDE may restrict the rate of glycolysis in fast twitch muscles.     

肉制品中牛源性成分Taqman荧光定量PCR检测法的建立

本实验根据牛线粒体差异序列设计特异性PCR引物和Taqman探针,并基于16S rDNA内参基因设计通用引物及Taqman探针,绘制并通过标准曲线确定样品中总DNA浓度以及牛源性成分DNA浓度,采用相对定量法测定肉制品中牛源性成分的质量分数。并通过特异性、灵敏度实验,及混合肉样回收率及市售肉制品检测,对本方法进行验证。结果表明,方法特异性强,可对牛肉DNA进行特异性扩增。最低检出限为10 pg/μL,具有较高的灵敏度。并根据回收实验可知,平均回收率为98.62%。可以为肉制品中牛肉含量的测定提供技术参考。     

Development and validation of a nephelometric immunoassay for IgG1 in milk

A nephelometric immunoassay, with a detection range of 0.3 to 5 g IgG1/l, was (leveloped for the determination of immunoglobulin in bovine milk. The assay exhibited no significant cross-reactivity with alphas1-casein, alphas2-casein, beta-casein, K-casein or beta-lactoglobulin and 39% cross-reactivity with IgG2. The nephelometric assay was compared with ELISA and RID (24 h and 48 h incubations) assays using 105 duplicate milk samples covering IgG1 values ranging from 0.45 to 1.8 g. The results obtained from all assays showed good agreement with the exception of those obtained by the RID assay (24 h incubation) which gave lower results in samples containing more than 1.2 g IgG1/l. It was concluded that the nephelometric assay is a reliable, rapid and convenient method suitable for the quantification of IgG1 in milk. The assay can be configured for routine high-throughput milk quality assurance for IgG1 in dairy laboratories.     

Transaminases of Skeletal Muscle. 1. The Activity of Transaminases in Post-Mortem Bovine and Porcine Muscles

SUMMARY: The activity of glutamic-oxaloacetic transaminase (GOT) and glutamicpyruvic transaminase (GPT) of bovine and porcine muscle tissue and muscle press juice was determined. The total GPT activity of muscle tissue is about one tenth of the GOT activity. There are no remarkable differences in the activities of GOT and GPT between these slaughter animals and other species (rat, rabbit and man). The GOT activity of the longissimus dorsi muscle of pigs is significantly higher than that of the same bovine muscle. The mitochondrial (GOT_M) and sarcoplasmic isozymes (GOT_B) of GOT in skeletal muscles of cattle and pigs were determined after electrophoretic separation. The ratio GOT_M:GOT_S in skeletal muscle was found to be about 1:1. There is only a small decrease in GOT activty during storage of muscle tissue at 0 or +4°C for several weeks postmortem. The small activity of GOT_M in the muscle press juice does not substantially change during storage of muscle tissue under these conditions, indicating that there is no drastic change of the mitochondrial structure during aging of meat. Bacterial spoilage of meat, however, results in the release of GOT_M from the mitochondria.     

Fatty acid content and composition of english beef, lamb and pork at retail

We have determined the fatty acid content and composition of retail samples of meat and assessed them with respect to UK dietary recommendations. Fifty beef sirloin steaks, pork chops and lamb chops were purchased from four supermarkets on separate occasions. The percentage of muscle (boneless basis) in the samples was 84.4 ± 4.3, 69.8 ± 7.7 and 78.9 ± 7.1 for beef, lamb and pork, respectively, with fatty acid contents of 3.84 ± 1.3, 4.73 ± 1.66 and 2.26 ± 0.7 g per 100 g muscle, respectively. Adipose tissue fatty acid contents were 70.0 ± 8.2, 70.6 ± 8.6 and 65.3 ± 9.4 g per 100 g tissue. A range of C20 and C22 polyunsaturated fatty acids (PUFA) was present in the muscle of all three species and pork adipose tissue but their concentrations in lamb and beef adipose tissue were too low to measure. The mean P:S ratios for beef, lamb and pork muscle were (adipose tissue values in parentheses): 0.11 (0.05); 0.15 (0.09) and 0.58 (0.61), and the n−6:n−3 ratios were 2.1 (2.3), 1.3 (1.4) and 7.2 (7.6). We conclude that the muscles of red meat species are a valuable source of PUFA, particularly the C20 and C22 n−3 fatty acids, in the human diet and that, considered as part of a varied diet, the low P:S ratio of the ruminant muscle, the high n−6:n−3 ratio of pork and the total fatty acid contents do not detract significantly from the nutritional value of lean meat.     

Mineral Composition of Selected Bovine, Porcine and Avian Muscles, and Meat Products

THE MINERAL CONTENT of young and old avian muscles, in mature bovine and porcine tissues destined for processing, and in selected meat products and additives was determined. Inter- and intra-species variations in mineral composition were found among all the muscle tissues investigated. Muscle-to-muscle variation was found to be highly significant for all but one of the minerals analyzed in the avian (K) and bovine (P) muscle tissues and all but three (Fe, Ca, P) in the porcine tissues. The mineral content of three major meat products and four meat additives are reported. The least variability in tissue mineral content was found when the data was expressed on a dry weight, fat free basis.     

Development and application of a method for the analysis of two trichothecenes: deoxynivalenol and T-2 toxin in meat in China by HPLC-MS/MS

A reliable and sensitive method was developed and successfully applied for the determination of deoxynivalenol and T-2 toxin simultaneously in pig dorsal muscle, pig back fat and chicken muscle by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) analysis. Limit of detection of deoxynivalenol and T-2 was 0.02 μg/kg and 0.007 μg/kg, and limit of quantification of deoxynivalenol and T-2 was 0.07 μg/kg and 0.02 μg/kg, respectively. Sixty-six meat samples were analyzed and deoxynivalenol was detected in the samples of pig back fat, with concentrations lower than 0.5 μg/kg, and T-2 toxin was detected in the samples of pig dorsal muscle, pig back fat and chicken muscle, with concentrations lower than 0.5 μg/kg. The results of sample analysis show that only trace residues of deoxynivalenol and T-2 toxin were detected in the samples analyzed.     

Oxidative status, in vitro iron-induced lipid oxidation and superoxide dismutase,catalase and glutathione peroxidase activities in rhea meat

Rhea (Rhea americana) muscles Obturatorius medialis (OM) Iliotibialis lateralis (IL) and Iliofibularis (I), obtained from farmed animals, were evaluated regarding their oxidative/antioxidant status. The mean level of thiobarbituric acid reactive substances (TBARS) expressed as malonaldehyde (MDA) content was of 0.84 mg MDA/kg wet tissue for the three muscles. TBARS level was significantly higher in IL than OM and I, with the two latter showing similar levels. The mean level of carbonyl proteins expressed as dinitrophenylhydrazine (DNPH) was 1.59 nmol DNPH mg⁻¹. Carbonyl protein levels were significantly different (P < 0.05) between the three muscles (IL > OM > I). Iron-induced TBARS generation was not significantly different between the three muscles at any time, nor for each muscle during the 5 h of the experiment. Superoxide dismutase activity in IL muscle was significantly higher (P < 0.05) than in I muscle. However, the difference between IL and OM muscles was not significant. The differences between the three muscles became not significant when the results were expressed by mg of protein contained in the extract, instead by g of wet tissue. No differences were found for catalase (μmol of discomposed H₂O₂ min⁻¹ g⁻¹ wet tissue or by mg of protein contained in the extract) and glutathione peroxidase (μmol of oxidized NADPH min⁻¹ g⁻¹ of wet tissue or by mg of protein contained in the extract) activities between the three muscles.     

A Research Note AIM APPARATUS FOR MEASUREMENT OF CONTRACTILE PROPERTIES OF PORCINE SKELETAL MUSCLE

Describes the design of apparatus and development of a test procedure to measure the contraction time, one-half relaxation time, net twitch tension/g wet weight muscle, net tetanus tension/g wet weight muscle and twitch-tetanus ratio in muscle of porcine animals weighing up to 100 kg.     

Ochratoxin A in raw materials and cooked meat products made from OTA-treated pigs

The aim of this study was to determine ochratoxin A (OTA) concentrations in the raw materials and cooked meat products made from pigs sub-chronically exposed to OTA. The treated animal group (n = 5) was administered with 300 μg OTA/kg of feed for 30 days, whereas the control group (n = 5) was left untreated. OTA concentrations were quantified using immunoassay (ELISA) and high performance liquid chromatography with fluorescence detection (HPLC-FD). OTA concentration was the highest in the kidney, followed by the lungs, liver, blood, spleen, heart, and adipose tissue. As for the final meat products, the highest average OTA concentration was detected in black pudding sausages (14.02 ± 2.75 μg/kg), then in liver sausages (13.77 ± 3.92 μg/kg), while the lowest was found in pâté (9.33 ± 2.66 μg/kg). The results pointed out that a sub-chronic pig exposure leads to the accumulation of OTA in raw materials and consequently in meat products, whose level of contamination is directly dependent on OTA contents in raw materials used for their production.     

Evaluation of the capillary electrophoresis method for measurement of immunoglobulin concentration in ewe colostrum

Capillary electrophoresis (CE) is a technique routinely used in clinical laboratories that allows the separation and quantification of blood serum proteins in a rapid, precise, accurate, and inexpensive manner. Recently, CE has been proposed to separate and measure colostral proteins, but an evaluation of the agreement between CE and radial immunodiffusion (RID) method, currently used to quantify IgG in colostrum, is still lacking. The purpose of this study was to test the ability of a CE instrument, normally used in blood serum protein analysis, to realize the correct quantification of total Ig concentration in ewe colostrum, using RID assay as reference. Colostrum samples (n = 68) were collected from 35 multiparous Sarda ewes at first milking (n = 33) and at 24 h postpartum (n = 35). The mean ± standard deviation of IgG concentration measured by RID and whey colostrum total Ig concentration measured by CE were 54.76 ± 41.82 g/L and 54.70 ± 41.43 g/L, respectively. Lin's concordance correlation coefficient (r = 0.993; 95% confidence interval = 0.989 to 0.996) and linear regression analysis results (RID = 1.0022CE ? 0.063; R² = 0.986) showed an excellent agreement between these 2 methods. Bland-Altman analysis confirmed that CE method can be a suitable alternative to RID: the mean of the differences between CE and RID was ?0.055 ± 4.95 g/L (95% confidence interval = ?1.25 to 1.14 g/L) and the agreement limits were ?9.75 to 9.60 g/L (low limit 95% confidence interval = ?11.82 to ?7.68 g/L; high limit 95% confidence interval = 7.57 to 11.72 g/L). In conclusion, the current study indicates that CE method may be a reliable tool for the quantification of the total Ig concentration in ewe colostrum.     

Development of an HPLC–UV method for the simultaneous determination of tetracyclines in muscle and liver of porcine,chicken and bovine with accelerated solvent extraction

A simple and especially rapid method-using accelerated solvent extraction (ASE) and HPLC has been developed for the quantitative determination of oxytetracycline, tetracycline, chlortetracycline, minocycline, methacycline, demeclocycline and doxycycline in muscle and liver of porcine, chicken and bovine. Samples of muscle and liver were extracted with trichloracetic acid/acetonitrile using ASE instrument, parameters such as extraction temperature (40–80 °C) and pressure (45–85 bar) were investigated and the selected extraction (60 °C, 65 bar) was most effective. The limits of detection were lower than 10 μg/kg and limits of quantification no more than 15 μg/kg for all compounds in muscle and liver. The recoveries of tetracyclines spiked at levels of muscle 50–150 μg/kg, liver 150–450 μg/kg, averaged from 75.0% to 104.9% with the relative standard deviation values less than 10%. The method was applied to determine 30 real porcine livers. It is demonstrated that the new method is robust for detection and quantification of seven tetracycline residues in muscle and liver of porcine, chicken and bovine.     

Origin of variable extraction of myosin from myofibrils treated with salt and pyrophosphate

When isolated myofibrils are treated with solutions containing NaCl and pyrophosphate to imitate conditions in meat being cured, considerable dijfirences are observed between myofibrils in the concentration of NaCl required for extraction of the myosin-containing A-band. The diferences in extent of extraction at an intermediate concentration of NaCl are between bundles of myofibrils rather than within them, indicating that the variation derives from differences between muscle fibres. The extraction behaviour is determined by the myosin isoenzyme content of the muscle fibre. Thus, the proportion of myofibrils extracted at an intermediate concentration of NaCl in preparations from various bovine and rabbit muscles reflects the documented proportion of fast-contracting fibres in each muscle. Rabbit M plantaris myofibrils from fast white fibres are extracted in a low concentration of NaCl, whereas those myofibrils containing slow myosin are extracted only in a high Concentration of NaCl. Myofibrils probably from fast red fibres require an intermediate concentration of NaCl for extraction.     

Production of a horse-specific monoclonal antibody and detection of horse meat in raw meat mixtures by an indirect ELISA

A stable hybridoma cell line (DD3) has been produced secreting a monoclonal antibody specific for horse muscle proteins. The DD3 monoclonal antibody (mAb) did not show significant cross-reactivity when tested against beef, chicken, pig and soya proteins or bovine caseins, gelatine and bovine serum albumin. The DD3 mAb was used in an indirect ELISA format for the detection of defined amounts of horse meat (10–500 g kg-¹) in beef meat mixtures. Immunorecognition of monoclonal antibodies adsorbed to horse meat adsorbed onto the ELISA plate was made with rabbit anti-mouse immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymic conversion of the substrate gave clear optical density differences when assaying mixtures of minced beef containing different amounts of horse meat.     

Purification and properties of rabbit muscle proteasome, and its effect on myofibrillar structure

This paper describes the purification and properties of a multicatalytic proteinase complex, proteasome, from rabbit skeletal muscle, and its effect on myofibrillar structure. The purified proteasome gave a single band on polyacrylamide gel electrophoresis under non-denaturing conditions and gave eight bands under denaturing conditions, indicating that this enzyme comprises multiple hetero-subunits with low molecular mass. The purified proteasome was not activated by ATP and ubiquitin, and was markedly inhibited by Z-Leu-Leu-Leu-H (aldehyde). These data indicate that the purified proteasome is not 26S, but 20S. The proteasome degraded synthetic peptides maximally at pH 8.0. Relative to pH 8.0, activities were gradually decreased with the lowering of pH, but the degree of decrease was substrate-dependent, and the activity at pH 5.0 still retained about 30˜60% of the activity at pH 8.0, indicating the possibility that the proteasome is active in the muscle during conditioning. When the proteasome was heated at 60 °C for 20 min and treated in the presence of 0.005% SDS, the activity increased over 1.5 and 4.5 times, respectively. SDS remarkably increased the V_max value of the enzyme at pH 8.0. The proteasome was also activated by high hydrostatic pressure up to 100˜150 M Pa and gradually decreased at 200 MPa or higher. Electron microscopic observation revealed that obvious gaps between filamentous structure, the complete loss of M-line and partial loss of Z-line structure were caused by proteasome.