3-Methyl-2-benzothiazolinone hydrazone and 3-dimethylamino benzoic acid as substrates for the development of polyphenoloxidase and phenoloxidase activity by zymograms.

In the present study, a sequential staining process of polyphenoloxidase and phenoloxidase enzymes was designed by the zymography technique. As a first step, electrophoresis was carried out under native conditions, and later, first staining was carried out with a revealing solution of 3-methyl-2-benzothiazoline hydrazone (MBTH)-3-dimethylamino benzoic acid (DMAB) that allowed the visualization of polyphenoloxidase enzymes, and later and using the same gel, we proceeded to the differential staining of phenoloxidase, adding a solution of H2O2. The technique was standardized using commercial enzymes of laccase (T. versicolor) and horseradish. The technique was used to identify polyphenoloxidases (laccases) and phenoloxidases (lignin peroxidase) of crude extracts obtained from the growth of the basidiomycete Lentinus strigosus on Pinus radiata. The technique showed great sensitivity to detect the different enzymatic activities (1.56 Activity Unit/mL minimum) in the same gel without interference between the enzymes and the solutions used. On the other hand, the efficiency of the technique was compared with the substrates that are commonly used for the detection of this type of activities such as 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and guaiacol, observing greater sensitivity and minimal interference, so that the present method will allow in the same gel, and visualize polyphenoloxidase and phenoloxidase activities simultaneously facilitating expression studies.

Review: Production and functionality of active peptides from milk.

In recent years, research on the production of active peptides obtained from milk and their potential functionality has grown, to a great extent. Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions, and they may ultimately have an influence on health. Individual proteins of casein or milk-derived products such as cheese and yogurt have been used as a protein source to study the isolation and activity of peptides with several applications. Currently, the milk whey waste obtained in the production of cheese also represents a protein source from which active peptides could be isolated with potential industrial applications. The active properties of milk peptides and the results found with regard to their physiological effects have led to the classification of peptides as belonging to the group of ingredients of protein nature, appropriate for use in functional foods or pharmaceutical formulations. In this study, the main peptides obtained from milk protein and the past research studies about its production and biological activities will be explained. Second, an analysis will be made on the methods to determinate the biological activities, the separation of bioactive peptides and its structure identification. All of these form the base required to obtain synthetic peptides. Finally, we explain the experimental animal and human trials done in the past years. Nevertheless, more research is required on the design and implementation of equipment for the industrial production and separation of peptides. In addition, different authors suggest that more emphasis should therefore be given to preclinical studies, proving that results are consistent and that effects are demonstrated repeatedly by several research human groups.