Analytical Tools for Rapid, Sensitive, Quantitative Identification of Potential Meat Quality Markers

Abstract

Conversion of muscle tissue to meat, including tenderization, is a complex process. As the consumer considers meat tenderness to be the most important criterion for meat quality, this conversion should be elucidated on a biomolecular basis. With such a biochemical background, specific sensitive analytical tools could be developed which would correlate physicochemical parameters with the degree of tenderness.Various events take place at the level of the myofibrils which have been correlated with meat tenderness. These include Z-disk weakening [1], degradation of desmin or titin, leading to fragmentation or weakening of myofibrils, the disappearance of troponin-T and simultaneous appearance of 28 -32 kDa proteins or detection of a 110 kDa protein fragment, as monitored thoughout the conditioning period by electron microscopic studies and SDS polyacrylamide gel electroAbstract. Myofibrillar extracts from bovine Musculus longissimus dorsi (MLD) were subjected to SDS-PAGE, electroblotted and fragments of the 30 kDa band determined by internal and N-terminal Edman sequencing, giving unequivocal proof, troponin-T (TNT) to be the origin of this band. Based on the N-terminal primary sequence of the 30 kDa band, a peptide with high antigenic sites was synthesized, conjugated to keyhole limpet hemocyanin (KLH), antibodies were generated and an enzyme-linked immunosorbent assay (ELI-SA) was developed for the determination of TNT concentrations in meat samples. For routine determinations of meat quality markers it seems more convenient to analyse soluble meat extracts, produced by trichloroacetic acid (TCA) or HCl treatment. In the supernatants of TCA-treated MLDs, prominent peptide fragments from glyceraldehyde-3-phosphate Keywords: Capillary electrophoresis, Immunoassays, Peptides, Proteins, Meat quality markers phoresis, respectively [2 -7]. In particular, the disappearance of troponin-T and the concomitant appearance of 28 -32 kDa proteins seem to be good indicators of post-mortem proteolysis. However, the origin of the 30 kDa fragments was speculative and not determined on a structural basis [8 -10]. Therefore, we recently applied the technique of internal microsequencing to provide for the first time unequivocal proof for the origin of the 30 kDa protein [11]. The 30 kDa protein transferred to ProBlott and Immobilon membranes was digested with trypsin, the peptide mixture separated with HPLC, selected peptides isolated and sequenced. Eight peptides, varying from 5 to 13 amino acid residues, displayed between 50 and 100% homology to rabbit troponin-T, according to our protein homology search. As SDS-PAGE operations are tedious, inconvenient and time-consuming, we suggest in this communication could be separated by HPLC and identified by Edman degradation. Both fragments were found to increase with ageing and might become useful indicators of meat quality. After HPLC separation and structure elucidation of MLD HCl extracts, fructose-biphosphate aldolase, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase and myogl...

ELISA Based Quantification of Chicken Specific Troponin-T Peptide in Skeletal Muscle TCA Extracts

“…Based on our previously developed competitive ELISA for the detection and quantification of TnT (16-31) fragment in trichloracetic acid (TCA) soluble beef skeletal muscle extracts [6,14], as a marker of meat tenderness, an ELISA was similarly/respectively developed for the detection of the 21 aa TnT fragment EPAPPPEEKPRIKLTAPKIPE, which was reported to be present, in chicken skeletal muscle extracts [8].…”