FOS 4310L EXPERIMENTAL FOODS (Spring) Lab #8: Enzymes

Enzymes in General
By definition, enzymes are complex, globular protein catalysts that accelerate chemical reaction rates by factors of 1012 1020 at ambient temperatures.1 Enzymes increase the rate of specific chemical reactions but are not used up or destroyed in the process. Therefore, they are not a part of the final product. Because the enzyme is physically unchanged, it can move on to interact with other compounds. Within the human body, every cell contains thousands of types of enzymes with specific purposes. Some break down carbohydrates, fats and proteins so they can be absorbed into body cells. Others act as triggers for reactions to build muscle and tissues. Some are a part of reactions for chemical processes and basic cellular, muscular and neurological functions.2 Many of the foods we ingest from plant and animal sources contain enzymes. The fact that they are proteins means that they are subject to the same conditions that affect all proteins. Enzymes can be denatured by excessive temperature and pH fluctuations. This denaturation will cause enzyme inactivation the enzyme is not longer able to catalyze reactions. This fact is very useful in food preparation. In one case, the enzyme needs to remain active to give in the desired effect in the final product. In another case, the enzyme must be inactivated to prevent product degradation.1, 3 Whenever enzymes are being used in food preparation, both the pH and the temperature of the food must be considered if the desired results are to be achieved.3

Effects of Temperature and pH on Enzymes

Temperature The effect of temperature on enzymes can be variable. In general, enzymes function very slowly at subfreezing temperatures. Their activities increase as the temperature increases. Most enzymes have an optimal activity within the range of 30 400C and begin to denature above 450C.1 Treatment of a food product will depend on the desired state of the enzymes present. For example, vegetables to be frozen are blanched (heat treated) to inactivate (denature) the enzymes that could continue to cause deterioration during storage. On the other hand, having an active enzyme may enhance the usefulness of the food. Enzymes are added after heating to some dairy products such as yogurt or to milk for those who are lactose intolerant.3 Effects of pH Enzymes generally show maximum activity at a particular pH value called the pH optimum. For most enzymes this optimum is within the range of pH 4.5 8.0. Extremes of pH generally inactivate enzymes by denaturing them to the point that they cannot reform to an active state.

For these reasons, within the food industry, the pH is carefully controlled to maximize, prevent or inhibit (slow down) enzymatic activity. 1

Effect of Proteolytic Enzyme

[Bromelain Enzyme]

Gelatin is a gel-forming agent which has the ability to form stable gels over a wide pH range without the addition of specific ions or other chemical substances. 4 It is acquired from the collagen of animals. 5 Gelatin hydrates in warm water then forms the gel framework during cooling. However, solidification of the gel matrix can be disrupted by some enzymes. Natural meat tenderizers use enzymes to degrade muscle and connective tissues. The three enzymes most commonly used are papain (from papaya), ficin (from figs) and bromelain (from pineapple). In combination, these three are active during the heat of cooking to decrease the dense nature of the meat resulting in a tenderer product when eaten. Fresh pineapple contains active bromelain enzyme. Just as it degrades meat connective tissue, active bromelain will degrade gelatin molecules preventing solidification of the gel matrix. However, like most enzymes, bromelain can be inactivated by the heat treatment of canning. For this reason, when preparing gelatins, it is advised that fresh, unprocessed pineapple never be used. This information is printed on packages of Jell-O and other similar products.
Other references: Jamesen (page 120 Gelatin) and Handout: Gelatin Information

Fruit Discoloration due to Enzymatic Oxidation

[Polyphenoloxidase Enzyme]

Enzymes play an important role in the maturation of fruits and vegetables. They catalyze reactions that convert starch into sugar thereby increasing fruit sweetness. Enzymes contribute to ripening and eventual softening in some foods. They are also responsible for the pigments that produce characteristic colors associated with some vegetables and fruits. Some fruits and vegetables develop a brown discoloration when peeled, cut, bruised or allowed to stand for long periods of time. The browning develops when the under surfaces are exposed to air (oxygen). This kind of discoloration is called enzymatic browning because the initial reactions are enzyme catalyzed.6 The enzyme responsible is polyphenoloxidase (or PPO). Browning reactions can be hindered and, in some cases, reversed by the addition of antioxidants and certain processing and storage methods. Enzymatic browning can be controlled by the use of several methods: 1. Heat inactivation moderate heat treatment of vegetables 2. Chemical inactivation inhibitors or chelating agents 3. Reducing agents convert the o-quinones back to phenolic compounds to prevent final product formation 4. Oxygen exclusion block enzyme exposure to oxygen in the air 5. Other enzymes enzymes to attack PPO 6. Treatment with honey honey has some inhibitory compounds
Other references: Jamesen (page 141) and Handout: Enzymatic Browning: Kinetics of Polyphenoloxidase

Effect of Blanching on Enzyme Activity

[Peroxidase Enzyme]

Another enzyme found in a variety of plant and animal tissues is peroxidase. In plant products, peroxidase activity can lead to the development of off-flavors during frozen storage. Compared to other enzymes, peroxidase has been found to be stable to moderate heat treatments. The primary method used to inactivate any enzyme prior to dehydration or freezing is blanching. Heat denatures the enzyme causing it to unfold and become inactive. Some enzymes, such as peroxidase, can become active again after heat treatment.1 Due to its thermal stability and ability to reactivate, peroxidase activity is used as an overall indicator of the adequacy of blanching in vegetables. One test of peroxidase activity is the reaction of the enzyme with guaiacol reagent. The enzyme must bind with 4 molecules of guaiacol. Once a sufficient amount is bound, a visible color complex is formed. The longer the blanching time, the more enzyme is inactivated and the less is available to react with guaiacol. The less enzyme available the longer it takes for color formation.

References
1

Fennema, Owen R. 1985. Food Chemistry, 2nd edition, Chapter 6. Marcel Dekker, Inc. New York, New York.
2

Insel, Paul, R. Elaine Turner and Don Ross. 2001. Nutrition, pp. 70 and 186. Jones and Bartlett Publishers, Sudbury, MA.
3

McWilliams, Margaret. 2005. Foods Experimental Perspectives, 5th edition, pp. 309 - 310. Pearson Prentice - Hall Inc. Upper Saddle River, NJ.
4

Bennion, Marion. 1980. The Science of Food, pp. 364 366. Harper & Row Publishers, Inc. New York, New York.
5

Jamesen, Karen. 1998. Food Science Laboratory Manual, pp. 120. Prentice Hall, Inc. Upper Saddle River, NJ.

File: Lab 8 Enzymes Notes