Abstract
Among the biochemical changes that occur in cheese ripening, proteolysis is considered the most complex phenomenon, and it plays a crucial role in the development of the characteristics of many cheese varieties. Proteolysis leads to changes in cheese texture through hydrolysis of the protein matrix, increases water holding capacity due to hydrolysis of peptide bonds and consequent release of carboxylic and amino groups, and increases cheese pH due to ammonia release. The peptides formed by protein hydrolysis have a significant impact on the taste of ripened cheeses, and the amino acids released are precursors of complex catabolism reactions that lead to the formation of volatile compounds. In addition to contributing to sensory aspects, many peptides released during cheese processing and ripening are also recognized as bioactive peptides and may have several biological functionalities such as antimicrobial, antioxidant, immunomodulatory, opioid, mineral carrier, and antihypertensive activities. Due to the importance of proteolysis for the formation of desirable characteristics in ripened cheeses, several methodologies have been used in the study of proteolytic changes that occur during cheese ripening. Non-specific traditional methods include determination of soluble nitrogen in different solvents such as water, pH 4.6 buffers, trichloroacetic acid, NaCl solution, and ethanol, as well as the quantification of reactive groups. These methods provide results that allow the analysis of the extent of proteolysis in general but do not allow the characterization of the specific peptides formed and the consequent study of the mechanisms involved. With the advancement of analytical techniques, specific methods that allow the identification of the hydrolysis profile of caseins, and peptides formed such as electrophoretic, chromatographic, and mass spectrometry techniques have been widely used to characterize and understand the mechanisms involved in cheese proteolysis. This chapter will address the proteolytic changes that occur during cheese ripening, aspects related to the formation of bioactive peptides, and different analytical techniques applied in the evaluation of proteolysis in ripened cheeses. Different methodologies will be discussed, from the traditional non-specific methods to the sophisticated analytical methods that are allowing the elucidation of the mechanisms involved in proteolysis and the effect of proteolytic agents on the formation of casein degradation products in the cheese ripening process.