Tenderizing enzymes generally come from one of three sources: plant, fungal, or bacterial. Plant sources have the longest history, with fungal and bacterial being more recent developments. Several of the tenderizing enzymes have been around for a number of years and have been used in basic marinades for their ability to make tough meat more palatable, especially in the case of middle meat cuts from older, cull, or dairy animals. These proteases are available from different suppliers under different trade names, some containing combinations of enzymes, which can make it difficult to determine the actual enzyme activity.

The USDA has approved the following enzymes for use in tenderizing meat products: papain, bromelain, ficin, protease preparations from Aspergillus oryzae and Aspergillus niger , protease preparations derived from Bacillus subtilis and protease produced from Bacillus subtilis var. amyloliquefaciens (Table 8.1 ; USDA-FSIS, 2007) . This chapter will concentrate on those currently approved by the USDA for inclusion in meat products and mention those that have potential for future use.

Tenderizing Enzymes from Plants

The primary, exogenous enzymes that are of economic importance in meat processing include papain (from papaya), bromelain (from pineapple), and ficin (from figs ). Most were accidentally discovered to have tenderizing properties and all are isolated from plants. Papain, bromelain, and ficin all belong to a group of enzymes termed thiol, cysteine, or sulfhydryl proteases because they contain a cysteine residue in their active site. This class of enzymes is similar to those naturally found in meat (calpain, cathepsin B, and some caspases), and it is logical that they would be appropriately used for tenderizing purposes. Other plant enzymes have been more recently studied and will be discussed briefly.

Use of Tenderizing Enzymes in Meat Products

The proper use of enzymes in meat tenderization effectively involves understanding their strength and specificity, combined with the method of delivery and the total time they will be active in the particular product. Note that worker safety is also a very important aspect of using these products.

Enzyme Strength and Specificity

Most plant tenderizing enzymes are nonspecific when they start to degrade a protein. It is important to note that because of differences in potency or activity, the effectiveness of plant enzymes can vary from manufacturer to manufacturer. Each tends to standardize the enzymes to different levels. In many cases, the analysis method can also be different so it becomes very difficult to compare enzymes. It is very important to understand the strength of the particular protease to be used. The plant enzymes are typically analyzed for their ability to clot milk (Balls & Hoover, 1937) and are standardized based on an activity measured as a Milk Clot Unit (MCU). The assay involves mixing a known amount of milk powder with water and adding a known amount of enzyme. The amount of time that is required to clot the system is used to gauge the strength of the product and is reported in MCU. In this way, the strength of the enzyme can be standardized so that the reaction is a bit more targeted and less likely to become over-active. Typical use levels of botanical proteases for meat tenderization require 1,000 to 5,000 MCU per pound of treated meat. The amount of MCUs per pound of meat will vary with hold time and cooking conditions.

Bacterial and fungal enzymes are typically more specific in their targets and the degree of reaction is easier to control. Their methods of standardization typically differ from the plant enzymes and use different methods and associated activity units to determine the strength of the powdered product. This might involve their ability to degrade one of several different proteins which may or may not contain meat proteins. Because all of these enzymes have particular reactivity sites, these analyses may not indicate how they will react in a meat system. With most of the current standardization methods there is an inherent flaw in applying this information to meat, as pointed out by Fawcett and McDowell (1986) ; the substrates used to access the enzyme activity are not meat components and therefore do not predict the ability and the extent of tenderization very accurately. However, these are at least somewhat indicative of the basic activity of these crude enzyme preparations and can be used as a guide for dosage.

Method of Delivery

The tenderizing enzymes have traditionally been added by several different methods. Sprinkle applications have been the most widespread and have resulted in the availability of low-strength preparations for retail use. The enzyme is generally mixed with a suitable carrier like salt, flavorings, or other components and the consumer simply sprinkles the mixture on the surface of the meat. Time for the enzyme to react may or may not be allowed for prior to cooking. Within the meat industry, tenderizing enzymes are typically incorporated by any of the following methods: (1) inclusion in marinade or injection brine, (2) topical application immediately before freezing, or (3) injection into the animal prior to slaughter. All of these methods involve some sort of dose level determination based on the reaction/ storage temperature, length of storage, method of cookery, and other factors. It is important that the enzymes be applied evenly so that localized over-tenderizing does not cause inconsistent texture. Injection and tumbling will give more even distribution than tumble marination, depending on the thickness of the items being processed, with thicker items having more variability in tenderness because of slower diffusion of enzyme.

Worker Safety

It is important to remember that enzymes are proteins and, as such, they can eventually result in sensitivities in workers who handle them frequently (Dransfield, 1994) . Over exposure can result in workers developing allergies or other health issues. Particular care must be taken to avoid protease inhalation and skin contact through the use of personal protective equipment, including dust masks, gloves, and clothing that covers exposed skin. It is worth reviewing the safe handling practices for enzymes of the Enzyme Technical Association (ETA, 2008) .