Measuring quantitative proteomic distance between Spanish beef breeds.

Estimates of quantitative proteomic distance between populations have not been reported to date. Here, quantitative proteomic distances between three Spanish bovine breeds (Asturiana de los Valles, AV; Retinta, RE; and Rubia Gallega, RG) were estimated from two-dimensional electrophoresis profiles of meat samples of longissimus thoracis muscle at 2 h post-mortem. Statistically significant distances were detected between AV/RG and the most genetically different RE breed, using the novel QD measure of quantitative proteomic distance. In total, 18 differentially abundant myofibrillar and sarcoplasmic proteins/isoforms contributing to proteomic distances between breeds were confidently identified by tandem mass spectrometry. The fast skeletal myosin regulatory light chain 2 followed by other five interacting proteins exhibited the most pronounced relative change between breeds. In addition, most differentially represented proteins could be associated with variations in meat tenderness. Therefore, they could be candidate biomarkers for molecular breeding programs and authentication of the three Spanish beef breeds.

Lactobacillus sakei CRL1862 improves safety and protein hydrolysis in meat systems.

AIMS: The capacity of Lactobacillus sakei CRL1862 to prevent the growth of pathogens and its ability to degrade sarcoplasmic and myofibrillar proteins in pork meat systems was evaluated. In addition, basic safety aspects of Lact. sakei CRL1862 such as production of biogenic amines and antibiotic susceptibility were addressed. METHODS AND RESULTS: The bacteriocin-producing Lact. sakei CRL1862 showed respectively bactericide and bacteriostatic effect against Listeria monocytogenes and Staphylococcus aureus in beaker sausage assay during 9 days of storage at 22 °C. The hydrolytic effect of Lact. sakei CRL1862 on protein extracts was evaluated by SDS-PAGE and reverse phase HPLC. A more pronounced proteolysis was evidenced in inoculated sarcoplasmic proteins compared with myofibrillar extracts with the generation of predominantly hydrophilic peptides and increase of total free amino acids concentration. Lactobacillus sakei CRL1862 produced neither histamine nor tyrosine and exhibited no resistance to the antibiotics assayed. CONCLUSIONS: Lactobacillus sakei CRL1862 effectively controlled the growth of L. monocytogenes and Staph. aureus; moreover, it was able to hydrolyse pork meat extracts generating peptides and amino acids, which may improve hygienic and sensorial attributes of fermented meat products. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of an integrated approach to evaluate the major traits of Lact. sakei CRL1862 showed it can be applied as an autochthonous functional starter in meat fermentation.

Small peptides released from muscle glycolytic enzymes during dry-cured ham processing.

Glycolytic enzymes are a group of sarcoplasmic enzymes responsible for the extraction of the energy available from carbohydrates. The glycolytic pathway consists of 10 enzyme-catalyzed steps. Fragments identified in this study, within the range 1100-2600 Da, correspond to glycogen phosphorylase enzyme, which catalyzes the rate limiting step in the degradation of glycogen, enzymes that catalyze steps 6-10 of glycolysis (glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase, respectively), and lactate dehydrogenase, which catalyzes the interconversion of pyruvate and lactate. A total of 45 specific fragments of these enzymes resulting from the processing of dry-cured ham are reported for the first time in this work. This study evidences the intense proteolysis occurring in the sarcoplasmic fraction of dry-cured ham as well as facilitates the choice of the most adequate tools in the identification of naturally generated peptides through comparison between Paragon and Mascot search engines, together with UniProt and NCBInr databases.

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.

Bovine muscle 20S proteasome. III: Quantification in tissue crude extracts using ELISA and radial immunodiffusion techniques and practical applications.

The 20S proteasome is a large complex (700kDa) that exhibits endo- and exo-peptidase activities with wide specificity. In postmortem muscles, several sets of evidence suggest a possible significant contribution of proteasome to meat tenderisation. Hence, an accurate and rapid quantification procedure is needed to attest that new function during the ageing of meat. In the present work, we developed an ELISA test enabling the quantification of nM concentrations of the 20S proteasome. We further tested the radial immunodiffusion (RID) technique described as a more simple method that can quantitatively determine the concentration of an antigen in a complex mixture. The ELISA test allowed us to quantify the 20S protesome in tissue homogenates and fluids with a recovery of 100%, a coefficient of variation lower than 5% and a detection limit of 9ng/ml. Quantification of the 20S proteasome in various bovine tissue by ELISA showed the highest concentration in liver followed by spleen and kidney, with muscles exhibiting the lowest concentrations. In addition, measurement of the proteasome concentration in eight different bovine muscles with various metabolic profiles led to the conclusion that the relationship between muscle metabolic properties and proteasome concentration is rather complex. Nevertheless, heart muscle exhibited the highest proteasome content (331µg/g wet tissue) whereas the lowest values were found for M. Tensor Fascia Latae (213µg/g wet tissue), a fast twitch white muscle, M. Supraspinatus (209µg/g wet tissue), a slow twitch red muscle and M. Pectoralis profondus (203µg/g wet tissue), an intermediate muscle. As compared to other endogenous peptidases, muscle tissue contains relatively high amounts of proteasome. Hence this complex can be quantified using the RID, which allows quantification of protein in the µg range. Plotting the concentration values determined with both methods for all bovine tissues tested gave a straight line with a correlation coefficient of 0.99.

Serine peptidase inhibitors, the best predictor of beef ageing amongst a large set of quantitative variables.

For consumers, tenderness is the most important sensory attribute of beef meat and, though to a lesser extent, of pork. Tenderness is therefore by far the most common cause of its unacceptability. The major challenge for the beef industry is to evaluate the toughness of the meat as soon as possible after death. In this context, the aim of the present work was to develop an equation to predict the myofibrillar ultimate resistance of raw meat. The study was done on the Longissimus muscle from twenty three 19 months-old Charolais bulls grown in the same INRA farm. Muscles excised within 1h post-mortem were vacuum packed and stored at 15°C during 24h and then transferred to 4°C until used. The activities of lactate dehydrogenase, citrate synthase and myofibrillar Mg-Ca dependent ATPase, the levels of lactate dehydrogenase enzyme, myoglobin, myosin types I, IIa and IIb, cysteine and serine peptidase inhibitors, the pH, the osmolarity, the expressible juice, µ-calpain, m-calpain and calpastatin and meat toughness were measured. According to the physical method used here, the force measured on raw meat represents the resistance of the myofibillar structure. Stepwise linear regression was used to determine the best equation (p<0.05) for predicting toughness at 6 days post-mortem. A 6-variables predictive equation including serine peptidase inhibitors (partial R(2)=0.4), the rate (partial R(2)=0.25), and the extent of pH decline (partial R(2)=0.03), the at death LDH activity (partial R(2)=0.24), the extent of increase in osmotic pressure (partial R(2)=0.13), and the rate of µ-calpain activity loss (partial R(2)=0.09), explained 70% of the variability in meat toughness at 6 days post-mortem. This equation was developed from 20 animals and the other 3 animals, chosen randomly, were used to validate it. The absolute need for a predictive model of meat toughness and the nature of the serine peptidase inhibitors together with their potential target enzymes are discussed.

Purification of bovine cathepsin B: proteomic characterization of the different forms and production of specific antibodies.

Cathepsin B (EC 3.4.22.1) has been highly purified (14,225 fold) from bovine kidney by a rapid procedure that included the preparation of an enriched lysosomal extract, a selective fractionation with ammonium sulphate, size-exclusion chromatography, two cation-exchange chromatographies, and anion-exchange chromatography on diethylaminoethyl-Sephacel. After the last purification step, two hydrolytic peaks against Z-Phe-Arg-AMC were separated from each other, a minor peak corresponding to the cathepsin B single-chain form and a major one representing the double-chain form of cathepsin B. The single-chain form showed a molecular mass of 31 kDa on sodium dodecyl sulphate - polyacrylamide gel electrphoresis (PAGE) under reducing conditions, whereas the heavy chain of the double-chain form appeared as a doublet with molecular masses of 23.4 and 25 kDa, respectively. The identity of the different cathepsin B isoforms and the quality of the final enzyme preparation were confirmed by using two types of antibodies, one against a synthetic peptide sequence and one against purified cathepsin B. The proteomic study confirmed the identity of the different SDS-PAGE protein bands as cathepsin B isoforms and allowed the comparison and study of some structural differences between them at the level of their primary structures.

Partial purification and characterisation of dipeptidyl peptidase II from porcine skeletal muscle.

The purification and study of biochemical properties of dipeptidyl peptidase II (DPP II; EC 3.4.14.2) from porcine skeletal muscle have been carried out in the present work. The purification included ammonium sulphate fractionation and two HPLC chromatographic separations using a Resource-Q anion exchange column. The enzyme was purified 1270 fold, with a 1.6% recovery and was completely separated from DPP IV activity. The pure enzyme displayed one main protein band with a Mr of 58 kDa on SDS-PAGE. Maximum activity was reached at pH 5.5 and 65°C. Those synthetic substrates containing Pro in N-penultimate position were the most efficiently hydrolysed, whereas in the case of peptides, DPP II efficiently hydrolysed both X-Pro- and X-Ala- peptides. The serine peptidase inhibitors PMSF and Pefabloc SC suppressed DPP II activity in a high degree, whereas 3, 4-DCI and cysteine peptidase inhibitors exerted little effect. Alkaline metal salts inhibited the enzyme activity according to the size of the cation, and among the assayed divalent cations, only Cu(2+), Fe(2+) and Hg(2+) showed significant inhibition of the activity. This is the first time that porcine muscle DPP II has been purified and its biochemical characteristics studied. So, these results contribute to improve the knowledge in relation with the proteolytic chain and the generation of flavour characteristics in meat products.

Purification and biochemical properties of dipeptidyl peptidase I from porcine skeletal muscle.

Dipeptidyl peptidase I (DPP I; EC 3.4.14.1) was purified from porcine skeletal muscle after several steps such as heat treatment, ammonium sulfate fractionation, gel filtration chromatography, and HPLC anion exchange chromatography. The purified enzyme showed a native molecular mass of approximately 200 kDa on Sephacryl S-200 column chromatography. Two protein bands of 65 and 42 kDa were obtained by SDS-PAGE, indicating its oligomeric nature. Maximum activity was reached at pH 5.5 and 55 degrees C. DPP I shared some common substrate specificities, both on synthetic derivatives and on real peptides, with porcine muscle DPP III. The enzyme required reducing agents for full activation, although the halide requirement was not proved. DPP I was inhibited by the assayed cysteine peptidase inhibitors except p-CMB. The serine peptidase inhibitor 3, 4-DCI also inhibited the enzyme as did the divalent cations Co(2+), Mn(2+), and Zn(2+). On the basis of its properties, DPP I may contribute to the generation of dipeptides during the processing of meat and/or meat products, including cooked ham.