Journal of Agricultural Science and Technology B 9 (2019) 423-427 doi: 10.17265/2161-6264/2019.06.006 Optimization of the Electrophoresis Tricine-SDS-PAGE for Simultaneous Detection of Protein and Peptide in Artisinal Cheese Juliana de Oliveira Carneiro 1 , Marilia Penteado Stephan 2 , Izabela Miranda de Castro 2 , Ana Carolina Sampaio Doria Chaves 2 , Alexsandro Araújo dos Santos 2 , Tatiana de Lima Azevedo 2 and Maria Gabriela Bello Koblitz 1 1. Laboratory of Biochemistry, Federal University of the State of Rio de Janeiro, Rio de Janeiro, Cep 22290-240, Brazil 2. Laboratory of Biochemistry, Embrapa Food Technology, Rio de Janeiro, Cep 22210-085, Brazil 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. Key words: Artisanal cheese, electrophoresis, maturation time, proteolysis. 1. Introduction The stage of cheese maturation involves many physical, chemical and biochemical reactions. The time can vary from a few days to more than two years depending on the type of cheese. Proteolysis is the most complex and important primary event that occurs during cheese ripening and it plays a vital role in the development of texture and flavor. Hydrolysis of caseins leads to the formation of large and intermediate-sized peptides, which are degraded into smaller peptides by enzymes produced by starter culture and non-starter bacteria or fungi [1]. This will result in peptides of differentiated molecular mass as already described by studying artisanal cheeses using Corresponding author: Marilia Penteado Stephan, DSc, research field: biochemistry. proteomic analysis [2]. It is important to emphasize that others authors [2] used a very expensive proteomic and peptidomic methodology of analysis as well as the laborious procedure of protein extraction. Many studies show the application of capillary electrophoresis, liquid chromatography coupled with Tandem mass-spectrometry (MS/MS), and ultra-performance liquid chromatography to study proteolysis in cheese [3]. It is very important to say that electrophoresis in gel of polyacrylamide is a simple technique that can be also used to study proteolysis of cheese. Three kinds of electrophoresis could be applied for this study, SDS-PAGE [4], Native PAGE [5] and Tricine-SDS-PAGE [6]. However, using only the procedure with Tricine-SDS-PAGE [6], it was possible to identify peptides with molecular weight up to 1 kDa. The D DAVID PUBLISHING