What Do Enzymes in Pineapple Juice Do to Milk?

Background Information for Teachers

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.

Image Credit: Svenja Lohner, Science Buddies / Science Buddies
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.

Image Credit: Svenja Lohner, Science Buddies / Science Buddies
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).

Additional Background Links

• Chemical Reactions, from Ducksters
• Enzymes, from the Royal Society of Biology
• Enzyme Deficiencies, from the National Stem Cell Foundation
• Milk Protein, from Milk Facts
• Cottage Cheese, from Allrecipes

Prep Work (20 minutes)

1. Make or buy fresh (unpasteurized) pineapple juice. To make the juice, take a fresh pineapple and cut off the rind on a cutting board. Cut the pineapple into smaller pieces and grate it. Alternatively, you can place it in a juicer or blender.
2. Then, wrap the grated fruit in a piece of cheesecloth and squeeze the juice into a microwavable container.
3. Pour half of the juice into the second microwavable container and put this into the microwave. Heat it long enough to get it boiling. When it starts to boil, carefully take it out of the microwave and let it cool down. If you have a microwave available in the classroom, you can also let students boil the pineapple juice during class. You can also boil the juice on a stove.
4. Seal both containers and keep them at room temperature until class starts. If you need to store the juice longer than a couple of hours, keep it in the fridge and bring it back to room temperature before your class starts. Make sure to label the containers so you know which one contains the microwaved pineapple juice.
5. You should have enough juice in both containers so that each student group can have at least one teaspoon of each for their experiments (1/2 cup is about 24 teaspoons).
6. Take the milk out of the refrigerator before class, so that it is at room temperature for the experiment. The milk should be as fresh as possible.
7. Note: All the solutions can be disposed of in the sink at the end of the lesson.

Engage (15 minutes)

1. Show students the milk and the cottage cheese. Name the two items and then ask them,

   Did you know that this cottage cheese is made from milk? How do you think milk is turned into cottage cheese?

   Listen to students' replies. Students might, for example, say that something needs to be added to the milk that makes it become cottage cheese. Elicit responses that mention that some kind of chemical reaction has to happen in order to turn the milk into cottage cheese.

2. If students have not done so before, point out that in order to make cottage cheese from milk, the milk has to undergo some kind of reaction. State that, today, students will learn about chemical reactions.

   What are examples of chemical reactions that you know?

   Have students mention 2-3 examples of chemical reactions they know. For example, they might know that burning, rusting, and photosynthesis are chemical reactions.

   What is a chemical reaction and what usually happens during a chemical reaction?

   Have students share their ideas.

3. Use students' replies to define a "chemical reaction" as a process in which one or more substances are transformed into one or more new substances that have a different chemical composition. You can write a general reaction equation [reaction substrate(s) → reaction product(s)] on the board to demonstrate what a chemical reaction equation looks like. The new chemical composition and different properties of the new substances [the reaction product(s)] are the results of the rearrangement of the original substance's molecular structures. Let students write that definition into their student worksheets.

Explore (30 minutes)

1. Tell students that today they will explore a specific chemical reaction. They will mix two substances, pineapple juice and milk, and then observe what happens. Their task while doing this experiment will be to collect data as evidence that a chemical reaction really happened. Briefly discuss:

   How can we know that a chemical reaction really happened?

   Listen to students' replies. If they have difficulties, remind them of the definition of a chemical reaction: a chemical reaction requires a chemical change of the original substances, which usually includes a change in their properties. Indicators of such chemical changes include a change in color, the formation of bubbles, a noticeable odor, or the formation of a precipitate (a solid that is formed inside a solution).

2. Divide the class into student pairs.
3. Have students label their cups milk, milk + juice, milk + heated juice, juice, and heated juice.
4. Go from group to group and add one tablespoon of milk to the milk, milk + juice, and milk + heated juice cups. Explain to students that this is regular milk that they can buy in the supermarket. Have students swirl the milk cup slightly and observe.

   How does the milk look?

   What properties does the milk have?

   What happens if you gently swirl the milk in the cup?

   Do you notice anything unusual?

   Have students list their observations in their worksheets. The milk should look just like regular milk (see Figure 3).

   Image Credit: Svenja Lohner, Science Buddies / Science Buddies
   Figure 3. Regular milk in a mini cup.
   
   
5. Go from group to group and add one teaspoon of the unheated pineapple juice to the juice cup. Explain to students that this is fresh pineapple juice that you prepared before class by blending and juicing a pineapple.

   What do you think will happen when you mix the pineapple juice with the milk?

   Have students share their predictions with their partners.

   Instruct students to pour the contents of the juice cup into the cup labeled milk + juice. Have students gently swirl the cup, stir with the spoon, and observe what is happening for about five minutes. While swirling, they should pay attention to what is happening on the walls of the cup. Have students share their observations.

   What do you notice after adding the pineapple juice to the milk? Use all your senses to make observations.

   Do the properties of the milk change? If yes, how?

   How does the milk and pineapple juice mixture (in the milk + juice cup) compare to the plain milk (in the milk cup)?

   Have students write their observations on their worksheets. The milk should start to curdle and may take on a slightly yellow color because of the pineapple juice. The curdling does not happen immediately but is noticeable within 1-5 minutes. The curd should be especially visible on the cup walls and when spooning up the milk, as shown in Figure 4. If you let the milk + juice cup sit long enough, the curd will settle down at the bottom, whereas the liquid at the top should become almost clear (but still yellowish).

   Image Credit: Svenja Lohner, Science Buddies / Science Buddies
   Figure 4. Milk that has been curdled by adding fresh pineapple juice.
   
   
6. Discuss the findings with your students.

   What happened to the milk after mixing the two substances?

   What evidence can you collect that supports or contradicts the claim that "a chemical reaction took place"?

   Do you think that a chemical reaction happened when the milk and the pineapple juice were mixed?

   How does your curdled milk compare with the milk in the milk cup?

   Can you explain your results?

   Have students share their observations and explain why they think these observations support or contradict the idea that a chemical reaction happened. For example, they might state that as they mixed the milk with the pineapple juice a new substance (the curd) with new properties formed. This is an indicator that a chemical reaction has happened.

7. Allow children to touch the curd that has formed. Let them feel its texture and have them describe what it looks like.

   What does the curd look like? Does it remind you of anything?

   Have students share their thoughts. They will probably describe the curd as small white clumps that feel soft to the touch. Point out that the curd is actually cottage cheese. They just performed a reaction that results in cottage cheese! It is made by curdling milk and then separating the curd from the liquid.

8. Tell your students that in the next step they will repeat the same experiment but that this time they will use pineapple juice that has been heated in the microwave. Go from group to group and add one teaspoon of heated pineapple juice to the heated juice cup. Tell students that this is the heated pineapple juice. If you have access to a microwave in the classroom, you can also let students heat the juice themselves.
9. Then have students predict what happens if they mix the heated juice with the milk.

   When adding the heated pineapple juice to the milk, do you expect the same results as before? Why or why not?

   Have students make their predictions. You can ask them to raise their hands to show whether they think they will get the same result or a different result.

10. Have students pour the heated pineapple juice to the cup labeled milk + heated juice. Let them swirl the cup or stir the milk with a spoon. Again, they should observe what is happening for about five minutes. Have students share their results.

    What happened to the milk after mixing the two substances? Use all your senses to make observations.

    What evidence can you collect this time that supports or contradicts the claim that "a chemical reaction took place"?

    Do you think that a chemical reaction happened when the milk and the heated pineapple juice were mixed?

    How does the milk in the milk + heated juice cup compare with the milk in the milk cup now?

    Can you explain your results?

    Have students write their observations on their worksheets. They most likely noticed that this time the milk did not (or barely did) curdle. It still might look a little yellowish because of the pineapple juice, as shown in Figure 5. Based on their earlier discussion, students will most likely correctly infer that this time no chemical reaction happened, as the milk did not change and there was no precipitate (curd) formed when it was mixed with the heated pineapple juice. Note: A little curdling still might happen with the heated pineapple juice, although much less than with the unheated juice, as some bromelain enzyme might still be active. If the milk still curdles a lot when adding the heated pineapple juice, the milk might have been too old or the pineapple juice might not have been boiled long enough.

    Image Credit: Svenja Lohner, Science Buddies / Science Buddies
    Figure 5. When using heated pineapple juice, the milk does not (or barely does) curdle.

Reflect (20 minutes)

1. Summarize the results with your students. Have your students look at all three milk cups. As you reflect on the results of the experiment together, ask students to fill out the respective part in their worksheets.

   How do all of your results (within the three milk cups) compare?

   Only the milk with the unheated pineapple juice should have formed curd (Figure 6).

   Image Credit: Svenja Lohner, Science Buddies / Science Buddies
   Figure 6. Comparison of the results in all three milk cups (left: regular milk, middle: milk with pineapple juice, right: milk with heated pineapple juice).

   Why would a chemical reaction happen with fresh pineapple juice but not with heated pineapple juice?

   Let students speculate why this happens and make their hypothesis. Encourage them to use observations and prior knowledge to support their hypothesis. Then explain that chemical reactions sometimes need a little help because they do not happen all by themselves under the conditions present. All chemical reactions need some energy to get started (the activation energy). If this energy is too low, the reaction will not happen. In this case, the reaction happened with fresh juice but not with heated juice. This means that there must be something in the unheated (but not in the heated) pineapple juice that helps get the curdling reaction started.

2. Introduce enzymes to your students. Tell them that in nature many chemical reactions have little helpers called enzymes. Point out that many reactions in our bodies, for example, food digestion or energy production, are done with the help of enzymes. Explain that pineapple juice has an enzyme in it called bromelain. This enzyme (a protease) chops up proteins that are present in the milk (the casein) into smaller compounds. This is why the milk started curdling when you added the pineapple juice. Note: You can show your students the schematic diagram of the protease reaction while you explain the reaction.
3. Discuss enzyme activity with your students.

   Can you tell how the bromelain enzyme helped the milk change to curd?

   Listen to students' replies. Remind them that bromelain is an enzyme that chops up proteins. When added to the milk, it will break up the casein proteins that are inside the milk. Breaking the casein proteins causes the smaller pieces to clump together. The result is the curd they see. The curdling chemical reaction is due to the enzymatic activity of the bromelain enzyme.

   What do you think happened to the bromelain enzyme when the pineapple juice was boiled?

   Listen to students' replies. Remind them of what they observed during the reaction with heated pineapple juice. Guide students to conclude that when the pineapple juice is boiled, the enzyme bromelain is inactivated, which means it does not work anymore. This is why no curdling chemical reaction happened with the heated pineapple juice.

4. Finally, explain to your students that even though an enzyme is present, it does not have to be active. The activity of an enzyme describes how efficient it is in performing its chemical reaction. If the conditions are not optimal for an enzyme or if the enzyme is defective, it will not be able to perform very well. Heating or boiling an enzyme usually inactivates it, which means it is no longer functional.
5. If a student points out that lemon juice or vinegar can also curdle milk, lead them through thinking how they could test whether this was also due to enzymatic activity (lemon juice or vinegar tested at room temperature vs. after boiling) and challenge students to try it at home. For more details, see the Background Information section.
6. Come back to your original question. Show students the milk and the cottage cheese again. Then ask,

   Based on what we have learned in this lesson, can you now answer how the milk is turned into cottage cheese?

   Have students reiterate that the milk is turned into cottage cheese with the help of enzymes. Explain that in reality, this enzyme is not bromelain but rennet, which is also a protease but is derived from animals.

7. Optional: Have the students imagine what would happen if an enzyme inside their bodies was inactive or not working properly, similar to the inactivated bromelain in the pineapple juice.

   What do you think would happen if an enzyme inside our bodies, for example, an enzyme for digestion (such as lactase), was inactive, missing, or not working properly?

   Guide students to conclude that enzymes are vital for our body functions. Missing or deficient enzymes can have negative effects on our health. For example, if we have an inactive digestive enzyme, we are not able to digest our food properly. You might know of someone who is lactose intolerant; this is the result of the enzyme lactase not working properly. Lactase is a digestive enzyme that breaks down milk sugar (lactose). People who are lactose intolerant have an inactive lactase enzyme and thus cannot digest dairy products. They can take pills that contain active lactase enzymes if they want to eat dairy products to temporarily give their body the enzyme to digest the diary. There are many other disorders (allergies are not part of such disorders) that are caused by an enzyme not working properly inside our bodies.

Make Career Connections

Discussing or reading about these careers can help students make important connections between the in-class lesson and STEM job opportunities in the real world.

Chemist

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Career Profile

Everything in the world is composed of chemicals. Chemists investigate the properties, composition, and structure of matter. For example, they could create new products by exploring new chemical reactions or improve processes that make existing products such as rubber, plastics, or foods. Read more

Biochemist

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Career Profile

Biochemists study the chemical composition of living things. For example, they try to find out how all the chemical reactions in our bodies, such as growth or digestion, work. They also study enzymes and their activities, as well as how they could be used to make useful products such as cheese or other foods. Read more

Lesson Plan Variations

• With your students, test how milk curdles with acids such as vinegar or lemon juice. How does the pH affect the rate at which milk curdles? Do heated vinegar or lemon juice curdle milk?
• Temperature can affect the rate at which a chemical reaction takes place. Determine how temperature affects the rate of milk curdling by proteases. Heat or cool your initial milk sample before you add the fresh pineapple juice. Does the milk curdle faster when it is warm or cold?
• Test how concentration changes the rate at which a chemical reaction takes place. Change the amount of pineapple juice (proteases) added to the milk. How does this change the milk curdling process?
• Find a cottage cheese recipe online and make it with your students.
• Test whether freezing the pineapple juice makes the enzyme inactive.