Exogenous Enzymes Research into the use of exogenous proteolytic enzymes has been conducted for over 60 years and has investigated countless enzymes from plant, bacteria, and fungal sources. Exogenous enzymes added to meat to enhance tenderness react differently to the myofibrillar and connective tissue portions of the meat. Currently, just five of the many exogenous enzymes that have been studied have been classified as ‘Generally Recognized as Safe’ (GRAS) by USDA’s Food Safety Inspection Service (FSIS) and come from varying plant, bacterial, and fungal sources (Table 1). Consequently, only these five can be added to meat for the purposes of enhancing tenderness. Additional, promising sources of exogenous enzymes have been identified. In particular, enzymes have been isolated from kiwi fruit (actinidin) and ginger that show potential for future inclusion in meat systems. Temperature is probably the most controllable factor influencing enzyme activity. Most of the exogenous enzymes used to tenderize meat have an optimal activity in the 50-70°C range (Table 2). As a result, most of the activity takes place during the cooking process and to a lesser degree during cooler storage. Product pH will also influence enzymatic activity. Most of the enzymes have an optimal pH in the normal range of meat, but some function best at a more acidic or alkaline pH for optimal protein degradation. The addition of salts can have positive or negative effects on enzymatic activity. Many of the enzymes do not penetrate meat, causing the method of application to play an important role in uniform distribution into the meat product. The proper identification of enzyme, time, temperature and pH will be dependant upon the processing system and desired product. An overview of optimal pH and range of activity is found in Table 3. Enzymes are often sold on the basis of activity; however, the assay depends upon which enzyme is being tested. Papain, bromelain and ficin activity is often measured in milk clot units. Enzymes from Aspergillus oryzae are measured in hemoglobin units of tyrosine. Enzymes from Bacillus subtilis are measured by a proteolytic test on casein. Assay details are found in Table 4. The relationships among assays are not strong, making it difficult to directly compare the activities of enzymes. The same enzyme can show differing results in activity levels and optimal pH and temperature ranges depending upon the substrate. The milk clot assay relies on a subjective determinate of clotting time while the others provide results using a spectrophotometer. The assay used is partly based on which one conforms to the optimal activity level for each enzyme, depending on pH, temperature and substrate.