What Do Enzymes in Pineapple Juice Do to Milk?

Chemical reactions are essential for life. A chemical reaction involves two or more substances that, when mixed together, result in a new product or new products. The new product, which often has different properties than the original substrates, is formed by the breaking and making of interatomic bonds. Many such chemical reactions are happening inside our bodies. Some of these reactions involve breaking down compounds into smaller ones or destroying waste products that the body does not need anymore. When our bodies digest food, for example, the food is broken down into smaller compounds that our bodies can use to make energy, such as the breaking up of dietary proteins in meat, eggs, and fish (long chains of amino acids) into their smaller parts, the amino acids. Other reactions make new compounds that the body needs. Amino acids, proteins, and fatty acids are some examples of compounds that our bodies make.

Figure 1. Proteases are examples of digestive enzymes that help break down proteins into smaller fragments called amino acids.

To perform these reactions, our bodies have helpers that are like little machines, called enzymes. Enzymes are proteins that the body makes to speed up chemical reactions and help with breaking compounds apart or putting new ones together. An example of a protease enzyme is shown in Figure 1. Each enzyme has a specific function, which means that not all compounds can be broken apart or built by the same enzyme. This is why our bodies make over a thousand different enzymes—all of which are needed for different reactions inside our bodies. These reactions might be part of food digestion, cell division, energy production, blood clotting, cell repair, etc. Because enzymes are responsible for so many different reactions, they are important to the human body. In turn, this means that a malfunctioning or deficient enzyme can have detrimental effects on our health. For example, lactose intolerance, which means that your body cannot digest cow's milk, is caused by a deficiency of the enzyme lactase that splits lactose (milk sugar) into smaller sugar molecules (glucose and galactose). Scientists have thus developed drugs, such as Lactaid® tablets, that contain active lactase enzymes as a supplement for people with lactose intolerance.

An enzyme's activity tells you how well that enzyme performs its function. If an enzyme is very active, it is very efficient in performing its reaction. An enzyme is considered inactive if it does not work anymore. Each kind of enzyme has a certain environment in which it works best, or in which it is most active. Enzymes in our bodies, for example, are most active at body temperature (about 37°C or 98.6°F). If you heat an enzyme too much, it will usually be destroyed. This process is called protein denaturation (Figure 2). It means that the enzyme loses its three-dimensional structure as the applied heat breaks bonds that hold the enzyme together. The unfolded protein is usually not able to function anymore.

Figure 2. Heat treatment usually unfolds a proteins complex three-dimensional structure, a process called protein denaturation, which leads to the protein's inactivation.

Bromelain is a protease enzyme found in pineapple juice. Proteases such as bromelain break down proteins into peptides or amino acids (see Figure 1). Such enzymes can be found in all living organisms and they break down proteins that are folded the wrong way, proteins that we take up with our food, or proteins that are not needed anymore. In this activity, students will study how bromelain reacts with milk. Milk mostly consists of water, lactose, fat, protein, and minerals. The fat is suspended in liquid as fine droplets or globules, which makes milk an emulsion. At the same time, there are also proteins in milk, mostly whey and casein. Because casein is poorly soluble in water, casein proteins build spherical structures, called micelles, which allow them to stay in suspension as if they were soluble. With both the fat and the proteins in suspension, the milk is a white liquid as we see it. However, the casein micelle structures can easily be disrupted or changed. Once chemical reactions break the intermolecular bonds, the micelles cannot be reformed. The micelle structure is holding the casein proteins in suspension, and without it, the casein proteins will clump together and come out of solution (precipitate). The result is a gelatinous material, called curd.

If you add bromelain to milk, the protease enzyme will catalyze a chemical reaction that breaks up the milk protein casein into its individual amino acids. As a result, the milk will curdle and clumps will form. This allows for a great visualization of how mixing two substances results in a new substance. Whereas bromelain is active at lower temperatures, it will become inactivated at temperatures above 80°C or 176°F. When inactivated bromelain is added to milk, the curdling reaction will not happen. Experimenting with room temperature and heat-inactivated bromelain and milk, and observing the effects, allows students to discuss the role of enzymes in chemical reactions and to directly observe the consequences of a deficient enzyme.

It should be noted that milk can be curdled by methods other than adding enzymes. Some students might be aware that milk spoils when left too long, which also results in milk curdling. This is also due to the action of proteases that break up the casein. These proteases can come from several sources, such as the milk itself or bacteria from the air. Also, acids such as vinegar or lemon juice are able to curdle milk. The reason for this is that the strong acids (pH 2 or less) in these solutions react with the casein proteins, similar to how the bromelain enzymes break up the milk protein. However, the pH of a solution usually does not change when it is heated up. This means boiled lemon juice or vinegar will still make milk curdle. This is why boiling a solution is a great control experiment to conduct in order to find out if the milk curdles due to enzymatic activity or other (non-biological) factors. Pineapple juice is slightly acidic but not acidic enough to make milk curdle. This is why heated pineapple juice does not curdle milk significantly.

In this lesson, students will explore chemical reactions by mixing pineapple juice and milk. Students will observe if, and if so, how the properties of milk change during the reaction and then infer from their results whether a chemical reaction happened. They will know a chemical reaction occurred if the composition of the reactants (milk and pineapple juice) has changed after mixing them, which is usually a result of breaking or forming new chemical bonds. Indicators of chemical changes include a change in color, formation of bubbles, a noticeable odor, or formation of a precipitate (a solid that is formed inside a solution).