Abstract
The objective of this work was to demonstrate how the extraction method affects the reliability of biomarker detection and how this detection depends on the biomarker location within the cell compartment. Different extraction methods were used to study the sarcoplasmic and myofibrillar fractions of the Longissimus thoracis et lumborum muscle of young bulls of the Asturiana de los Valles breed in two quality grades, standard (Control) or dark, firm, and dry (DFD) meat. Protein extractability and the expression of some of the main meat quality biomarkers—oxidative status (lipoperoxidation (LPO) and catalase activity (CAT)), proteome (SDS-PAGE electrophoretic pattern), and cell stress protein (Hsp70)—were analyzed. In the sarcoplasmic fraction, buffers containing Triton X-100 showed significantly higher protein extractability, LPO, and higher intensity of high-molecular-weight protein bands, whereas the TES buffer was more sensitive to distinguishing differences in the protein pattern between the Control and DFD meat. In the myofibrillar fraction, samples extracted with the lysis buffer showed significantly higher protein extractability, whereas samples extracted with the non-denaturing buffer showed higher results for LPO, CAT, and Hsp70, and higher-intensity bands in the electrophoretic pattern. These findings highlight the need for the careful selection of the extraction method used to analyze the different biomarkers considering their cellular location to adapt the extractive process.
Highlights
The objective of this work was to demonstrate how the extraction method affects the reliability of biomarker detection and how this detection depends on the biomarker location within the cell compartment
Different extraction methods were used to study the sarcoplasmic and myofibrillar fractions of the Longissimus thoracis et lumborum muscle of young bulls of the Asturiana de los Valles breed in two quality grades, standard (Control) or dark, firm, and dry (DFD) meat
Samples extracted with the lysis buffer showed significantly higher protein extractability, whereas samples extracted with the non-denaturing buffer showed higher results for LPO, catalase activity (CAT), and Hsp70, and higher-intensity bands in the electrophoretic pattern
Summary
Variations in meat quality depend on the specific changes that occur at the muscle cellular structure and metabolism levels, which rely on metabolic pathways triggered during the post-mortem conversion of muscle into meat. The extraction of the meat proteome is influenced by the interaction of multiple factors such as the extraction method, the protein solubility, the protein location, and the post-mortem changes that occur during the transformation of muscle into meat [3] To address this complexity meat scientists commonly divide the whole proteome in two fractions, sarcoplasmic and myofibrillar, which require different extraction methods due to their different extractabilities and water solubilities. Considering the above, we hypothesized that the analysis of biomarkers of the conversion of muscle into meat and the ultimate meat quality may be significantly affected by the muscle extraction method Extraction conditions, such as buffer pH, ionic strength, type of salt, extraction volume, and homogenization, influence muscle protein extractability [8,9,10]. We aimed to compare the reliability of protein extraction for a meat of standard-quality grade (Control) with that for a type of defective meat (dark, firm, and dry (DFD)), which exhibits alterations in the post-mortem muscle metabolism that produce a dark color and poor processing characteristics, such as higher water-holding capacity, unstructured texture, and higher spoilage [11,12,13]
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