Optimization of the Electrophoresis Tricine-SDS-PAGE for Simultaneous Detection of Protein and Peptide in Artisinal Cheese

Abstract

The objective of this paper was to optimize an electrophoretic methodology (Tricine-SDS-PAGE) to monitor the degree of caseins hydrolysis during the maturation time of artisanal cheese. A cheaper and easier method was obtained using small samples and micro-quantities of reagents. Simultaneous detection of proteins and peptides (100 kDa to 1 kDa) in the same gel was another advantage of the method. Initially, protein extraction was performed with 2 mg of lyophilized cheese dissolved in 1.0 mL of sample electrophoretic buffer for 1 h under stirring. After that, the Eppendorf tubes of the samples were kept at -4 °C for 4 h with additional centrifugation at 5,433 ×g for 2 min. This defatting process using centrifugation/refrigeration promoted a good separation of proteins and peptides from the fat layer. After this step, 30 μL of the supernatant of the protein extracts was applied to the electrophoresis gel. The results revealed a clear image of protein and peptides in the polyacrylamide gels and allowed an excellent response of the distribution of casein bands (α, β and κ) and the exposure peptide in cheese. The utilization of artisanal cheese as a pilot study of molecular protein analysis could be helpful for further correlation of the fingerprint of protein-peptide profile with taste quality.