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...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 L 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.展开更多
文摘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 L 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.
