The objective of the present study was to test two extraction methods including solutions with different ionic strengths on the extraction and separation of the myofibrillar proteins from meat and fish muscles of different species. Meat samples from
Muscle proteins are grouped into three categories based on location in the skeletal muscle and solubility as sarcoplasmic, stromal, and myofibrillar proteins. Myofibrillar proteins are the main component of the skeletal muscle accounting for about 50% of total proteins and are mainly constituted by myosin and actin, involved in muscle contraction. Due to their structure and localization [
Proteomics techniques have been extensively applied to separate, characterize, and identify proteins in animal food products [
All reagents used in the experiment were of analytical grade. Potassium chloride, disodium phosphate, monopotassium phosphate, urea, thiourea, dithiothreitol, cholamidopropyl dimethyl hydroxy propanesulfonate (CHAPS), IGEPAL® CA-630 NP 40, glycerol, and tris were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acrylamide, bis-acrylamide, ammonium persulfate (APS), N,N,N,N-Tetramethylethylenediamine (TEMED), sodium dodecyl sulfate (SDS), tris(hydroxymethyl)-aminomethane, glycine, bromophenol blue,
Phosphate buffer (pH 7, 0.003 M), KCl phosphate buffer (pH 7.5), and Tris-HCl (pH 8, 20 mM) were freshly prepared. Ultrapure water was obtained in the laboratory using a Water Purification System Barnstead™ Pacific TII (ThermoFisher Scientific, USA).
Meat samples from
The flowchart of the extraction of muscle protein fractions from different species analyzed is shown in Figure
Flowchart of the extraction of sarcoplasmic and myofibrillar protein fractions from different species.
Two different extraction methods were carried out for myofibrillar proteins using denaturing and nondenaturing solutions. The extraction of myofibrillar proteins with non-denaturing solution is based on the method reported by Hashimoto et al. [
For comparison, myofibrillar proteins were extracted using denaturing solution according to Marino et al. [
For each species, all myofibrillar extracts obtained with the different methods were quantified using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Absorbance was measured at 580 nm by the spectrophotometric assay (Power Wave XS, Biotek, UK), with a bovine serum albumin (BSA; >98% pure, Sigma-Aldrich) standard curve.
The fifteen myofibrillar extracts of each species obtained by the denaturing or nondenaturing method were pooled and resolved by SDS-polyacrylamide gel electrophoresis in a gradient gel 8–18% [
Protein concentration and electrophoretic data were analyzed using the GLM procedure of the SAS statistical software [
Solubility is an indicator of protein extractability; indeed, a solubilized protein could be easily extracted into a solution from muscle fibers or myofibrils [
Protein solubility of myofibrillar proteins from the muscle of beef, lamb, chicken, sole, European hake, and sea bass using denaturing and nondenaturing solution (D = denaturing; ND = nondenaturing;
On the contrary, the extraction capacity of the denaturing solution seemed to be more efficient in lamb, chicken, and sole with an amount of myofibrillar proteins extracted of about 30% in lamb and of about 10% in chicken and sole higher than nondenaturing solution. It is known that the extraction of protein from skeletal muscles is a complex phenomenon that is influenced by the parameters of the extraction, by the tissue structure, and by the
The densitometric profile and SDS-PAGE of myofibrillar fraction extracted with denaturing and nondenaturing solutions from meat and fish species are showed in Figures
Densitometric profile and SDS-PAGE of a pool of 15 myofibrillar extracts obtained from beef, lamb, and chicken samples extracted with denaturing (a) and nondenaturing (b) solutions (1 = myosin heavy chain; 2 = 180–110 kDa; 3 =
Densitometric profile and SDS-PAGE of a pool of 15 myofibrillar extracts obtained from sole, European hake, and sea bass samples extracted with denaturing (a) and nondenaturing (b) solutions (1 = myosin heavy chain; 2 = 180–110 kDa; 3 =
The densitometric profile of SDS-PAGE revealed that the use of the denaturing solution led to a more complex profile in terms of number of bands and fragments extracted (30, 32, and 32 vs 26, 27, and 28 bands in the nondenaturing profile of meat samples and 35, 29, and 30 vs 31, 26, and 28 bands in the nondenaturing profile of fish samples) while the use of the nondenaturing solution revealed a major intensity for most of the myofibrillar protein analyzed.
The percentage of the main myofibrillar proteins extracted using nondenaturing and denaturing solutions from meat and fish species are reported in Figures
Percentage of the main myofibrillar proteins from beef, lamb, and chicken samples extracted with denaturing and nondenaturing solutions (D = denaturing; ND = nondenaturing; MHC = myosin heavy chain; a-act =
Percentage of the main myofibrillar proteins from sole, European hake, and sea bass samples extracted with denaturing and nondenaturing solutions (D = denaturing; ND = nondenaturing; MHC = myosin heavy chain; a-act =
It is known that myosin mainly contributes to the tensile strength of the muscle, while
The pH value of the solution in the salt-soluble method seemed to be favourable for protein extraction. Accordingly, Chen et al. [
The use of denaturing solutions led to a major extractability of myofibrillar proteins with low molecular weight (under 45 kDa) as actin (
These results could be due to the compounds such as urea, thiourea, CHAPS, and DTT of the denaturing solution. It is known that urea is a chaotropic agent, efficient in the rupture of hydrogen bonds, denaturing proteins by breaking the noncovalent and ionic links between amino-acid residues [
Nondenaturing and denaturing extraction methods were efficient to solubilize the main muscle proteins. Proteomic analysis revealed a good separation of proteins with well-defined bands without any contamination for all samples analyzed. The extraction method using nondenaturing solution lead to a major extraction of myofibrillar proteins with high molecular weight; on the contrary, the denaturing method provided good extractability of proteins and fragments with low molecular weight for the most meat and fish samples.
The nondenaturing extraction method showed several advantages such as easy to carry out, less invasive, and minimal use of toxic and polluting agents.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there are no conflicts of interest regarding the publication of this article.
This work was supported by the University of Foggia (FPRA: Fondi per Progetti di Ricerca di Ateneo) within the project “Improving meat tenderness via eco-friendly strategies”.