Endogenous Enzymes As Processing Indicators

Enzymes present in food materials may be used as diagnostic indicators of processing steps that would be dif ficult to assess otherwise. The following are several examples of the use of endogenous enzymes as indicators of the effectiveness of a processing step, often involving heat treatments to inactivate organisms or enzyme activity.

Pasteurization of Milk

The purpose of pasteurization is to destroy potentially harmful microorganisms in the milk through a process of rapid heating to about 85°C for a very short time (several seconds). It is sometimes difficult to determine whether the processing has effectively eliminated the targeted microorganisms. One way to assess the effectiveness of the treatment is to test the viability of the organisms. This, however, is a lengthy process not well suited for remedial heating. It happens that endogenous milk alkaline phosphatase (EC is heat inactivated in a temperature range similar to that required to destroy the potentially harmful organisms. Thus, a simple colorimetric test (5) for alkaline phosphatase can quickly indicate whether the temperature of the milk has been raised sufficiently to inactivate the enzyme and, by inference, the microorganisms.

Blanching of Vegetables

Vegetables are often placed in boiling water to inactivate certain hydrolytic enzymes that would otherwise cause detrimental effects in terms of flavor and/or texture. Although certainly less a problem in the home, the proper degree of heating required to inactivate these enzymes, while not adversely affecting texture is critical to industries involved in the longer-term storage of such products in cans or bottles. Inadequate blanching in this case can result in seriously degraded product by the time the consumer opens the container for consumption of the product. Thus, it is important to know when a sufficient amount of heating has been achieved. A convenient way to assess the blanching process is to look at the activity of endogenous peroxidase (EC While complete inactivation of peroxidase is not desirable, the proper degree of blanching is achieved when about 5 to 10% of the peroxidase activity remains. The assay method usually utilized for this test involves the use of gulacol and is based on the colorimetric determination of the enzymatic product tetragulacol (6). This methodology has been used to assess the degree of blanching in products such as peas, corn, beans, and other vegetables.

Oat Rancidity

Oats contain lipolytic enzymes, the action of which can lead rapidly to rancidity. In this case, as discussed for vegetables, heat inactivation of those lipolytic enzymes is often used to extend the shelf life of oats and products formulated with oats. Since the assay of the lipolytic enzymes is problematic, endogenous oat peroxidase activity serves as a more convenient indicator of sufficient thermal inactivation of the lipolytic enzymes.

Soybean Treatment

Soybeans are very high in protein and are therefore used to supplement other foodstuffs that are lower in protein quantity as well as quality. However, soybean meal is usually heated to alter the bitter flavor and to inactivate some antinutritional components such as trypsin inhibitors as well as lipoxygenase activity. Applying too much or too little heat in this process can produce undesirable effects. The endogenous urease enzyme (EC is used as a convenient assay (7) to indicate the correct degree of thermal treatment.


Several types of chemical reactions can affect color and flavor in food products. The Maillard reaction is a nonenzy-matic reaction between a sugar and an amino function, often one on an amino acid. Although the reaction itself can proceed without the participation of an enzyme, the reaction is influenced by the concentration of the reactants, which in turn may be influenced by the presence of exo-proteases and exo-amylases. Thus, endogenous enzymes such as /i-amylase, a carboxypeptidase, or a leucineami-nopeptidase can contribute to Maillard browning in food products, baked goods in particular. These reactions lead to significant color formation (evidenced by the color of the crust on a loaf of bread) as well as many flavor characteristics.

Enzymatic Browning

Another source of color in foods comes from a reaction usually referred to as enzymatic browning. This reaction is mediated by polyphenol oxidase (EC This enzyme is able to interact with a large number of phenolic compounds and thus had been referred to by several names including tyrosinase, catecholase, polyphenolase, and cre-solase. The basic oxidation reaction normally results in the formation of an unstable quinone, which then proceeds through a number of steps to form melanins. Melanins are brown to black in color and are responsible for undesirable spots on many fruits and vegetables such as bananas, apples, mushrooms, and potatoes. Because of the widespread presence of polyphenol oxidase and the damage it causes in many food products, numerous methods have been developed to try to prevent this reaction from occurring. Clearly, eliminating as much oxygen as possible will help. In addition, reducing agents such as ascorbic acid, sodium sulfite, and thioi compounds can also help by reacting with the quinone compound. These reactions do not, however, inactivate the enzyme itself, so when the oxidizing agents are depleted, the browning reaction may still proceed. Under the proper conditions, ascorbic acid can inactivate polyphenol oxidase reacting with an active-site histidine residue. Likewise, thiol compounds can also affect the enzyme activity by chelating the required metal cofactor (Cu++) (1).

Other Flavor Reactions

Members of the plant genus Allium, which includes onions, shallots, garlic, and leeks, have strong, often irritating redolence that is the result of endogenous enzymatic activity. The characteristic odor of these plants is generally unnoticeable in the whole, undamaged bulb because the enzyme involved, allinase, is compartmentalized and unable to catalyze the odor-causing reaction. When tissue damage occurs, such as slicing the onion or garlic bulb, the enzyme is released and proceeds to react, producing the thiol compounds responsible for the offending (to some) aroma. For more detailed discussion of similar reactions in related food materials, the reader is referred to Reference 8.

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