Enzymatic browning of apples experiment

Lab Exercise

Laboratory Exercise - Browning

Enzymatic Browning in Fruits and Vegetables Introduction: We have all witnessed this type of event or had it happen ourselves. Someone picks up an apple or banana as part of their lunch and as they inspect it, they notice a soft brownish area in the apple or a black spot on the banana. Additionally, while we call these events bruising, we understand that the bruises in the fruit are nothing like the bruises a person gets. What causes this bruising or browning of fruit and why does it happen? This bruising process in fruits and vegetables is referred to as enzymatic browning. This browning is catalyzed by a family of enzymes called diphenol or polyphenol oxidases. These enzymes convert aromatic compounds found in fruits and vegetable into a range of pigment molecules that are ultimately converted to melanin. This conversion requires that the enzymes be released from the fruit cells and that oxygen is present. These reactions are beneficial to fruits and vegetables growing naturally in the environment, but are a major challenge for the food and agriculture industries. For the growing fruit or vegetable, these reactions are part of a defense mechanism. When the cells in the skin or the tissue of a fruit or vegetable are damaged, the cells release both polyphenol oxidases (enzymes) and the aromatic substrates into their immediate surroundings. In the presence of oxygen, the browning reactions begin leading to the ultimate production of melanin. Melanin has both antimicrobial and antioxidant properties. This is important to the plant because the melanin prevents infections and inflammation which could destroy the fruit or even the entire plant. For the agriculture and food industries enzymatic browning is a major challenge. Any damage to fruits and vegetables during the picking, transportation, or food preparation process can initiate enzymatic browning. This browning reduces the quality of fruits and vegetables as it decreases their nutritional value as well as their flavor. Putting safeguards in place to prevent browning adds cost to the picking, shipping, and shelf life of fruits and vegetables. Likewise, in the food service industry, preventing browning of fresh fruits and vegetables increases both the preparation time and cost of the product. Since this bruising process is mediated by enzymes, many of the solutions to preventing browning come from our understanding of enzymes and how they function.

Laboratory Exercise - Browning

Figure 1. Enzymatic Browning of apples, potatoes and bananas.

In the following experiments we will:

• Use polyphenol oxidase from potato to study enzymes and the dependence of enzyme mediated reactions on temperature and pH.

• Use cut apples and potatoes as well as a series of solutions to identify ways to prevent browning when preparing a fruit.

Background Living cells perform a multitude of chemical reactions very rapidly because of the participation of enzymes. Enzymes are biological catalysts, compounds that speed up a chemical reaction without being used up or altered in the reaction. The material with which the catalyst reacts, called the substrate, is modified during the reaction to form a new product (see Figure 1). But because the enzyme itself emerges from the reaction unchanged and ready to bind with another substrate molecule, a small amount of enzyme can alter a relatively enormous amount of substrate. The active site of an enzyme will bind with the substrate, forming the enzyme-substrate complex. It is here that catalysis takes place, and when it is complete, the complex dissociates into enzyme and product(s). Enzymes are, in part or in whole, proteins and are highly specific in function. Because enzymes lower the energy of activation needed for reactions to take place, they accelerate the rate of reactions. Since enzymes are predominantly protein in nature, they therefore require an appropriate structure to function normally. Thus, any environmental factor that alters protein structure may impact enzyme function.

Laboratory Exercise - Browning

Substrate + Enzyme Enzyme – Substrate Complex Enzyme + Product

Enzymes have a characteristic reaction cycle: 1) a substrate or substrates molecules bind to the active site of the enzyme forming the enzyme-substrate complex. 2) The substrate is then converted to product. 3) The product then dissociates from the active site. 4) The active site is available for another substrate molecule to bind. In this way, enzymes function as catalysts that are not used up in the chemical reaction and are available to enhance the rate of future reactions. When evaluating enzyme function there are two basic ways to measure enzyme activity: 1) determine the rate of disappearance of the substrate, and 2) to determine the rate of appearance of the product. In this experiment we will investigate the appearance of product, benzoquinone, to investigate the activity of the enzyme polyphenol oxidase. Polyphenol Oxidase is one of the enzymes which catalyze enzymatic browning reactions. This exercise will investigate the result of polyphenol oxidase activity. In the presence of oxygen, polyphenol oxidase catalyzes the removal of electrons and hydrogens from catechol, a phenolic compound found in plant cells. Catechol is converted to benzoquinone, a pigment product. The pigment products are responsible for the darkening of fruits and vegetables, such as apples and potatoes, after exposure to air (Figure 2). In these experiments you will use visualize enzymatic browning in two ways. In the first series of experiments you will make observation of apples and potatoes slices treated in different solutions to alter the rate of browning. This experiment can easily be done in the kitchen at home or even a dorm room. In the second experiment you will extract polyphenol oxidase from a potato tuber to quantify the rate of oxidation of polyphenols and expand you understanding of enzyme function by designing your own simple experiments. These exercises are designed for a laboratory setting with access to specific scientific equipment.

Laboratory Exercise - Browning

Figure 2. The Oxidation of Diphenol Compounds. A. The basic structure of phenolic compounds including catechol which will be used as the primary substrate in these experiments. B. In the presence of polyphenol oxidase, diphenols such as catechol are converted to quinones such as benzoquinone which yield the brown pigments related to enzymatic browning. Pre-Laboratory Questions and Concepts: 1. Describe the basic levels of protein structure and explain how structure relates to function.

2. Outline the enzyme reaction cycle. If the enzyme being studied is a protein how does the

protein structure relate to the function of the enzyme.

3. Proteins can be denatured by a variety of changes in their physical environment. What does it mean for a protein to be denatured? What type of conditions lead to proteins being denatured?

4. Define the terms acid and base.

A. B.

Laboratory Exercise - Browning

5. Create a simple diagram of the pH scale. Include neutral pH and the relative concentrations of hydrogen ions and hydroxyl ions at neutral, acidic, and basic pH ranges.

6. How do pH changes impact protein structure and enzyme function?

7. What is the impact on reaction rate when you increase the amount of substrate (reactant) in an enzyme mediated reaction? What happens when you increase the amount of enzyme?

8. We will be using five solutions in this experiment (Vinegar, Lemon Juice, Baking Soda, Milk of Magnesia, and Water). What is the general pH range for each of the solutions?

Process of Science: This entire laboratory exercise is investigative in nature. You will be evaluating the enzymatic browning of fruits and vegetables using apples and potatoes as examples. The questions you have just answered should help you think about the two different experiments we will be doing in this laboratory exercise. At this time for the experiment just as you have seen in the previous laboratory exercise you should create:

1. Two key question being investigated in this exercise.

2. A hypothesis or proposed answer to the question asked.

3. A prediction for the outcome of the experiment based upon your hypotheses you developed. The prediction should written as an if/then statement and be specific to the measurements being made.

4. An explanation of your reasoning for each of your hypotheses and predictions.

Laboratory Exercise - Browning

Procedures: Experiment: Browning in Apples and Potatoes This experiment will allow you to observe the browning of apples and potatoes after treatment with solutions of varying pH. Note: These instructions are for the entire experiment. Remember that you need only do EITHER apple or potato and ONE of the test solutions. 1. Preparation of solutions for browning experiment.

A. Obtain and label 5 disposable plastic cups.

i. Vinegar ii. Lemon Juice

iii. Baking soda iv. Milk of Magnesia v. Water

B. Prepare the appropriate solution in each cup.

i. 50 mL or ¼ cup of vinegar in the first cup. ii. 50 mL or ¼ cup of lemon juice in the second cup.

iii. Add ½ teaspoon of baking soda to the third cup, then add 50 mL or ¼ cup water. Swirl to dissolve the baking soda.

iv. Add 50 mL or ¼ cup of Milk of Magnesia and then add 50 mL or ¼ cup of water to the fourth cup. Swirl to mix well. You may need a spoon to get this solution to mix evenly.

v. Add 50 mL or ¼ cup of water to the fifth cup.

2. Preparation and treatment of apple slices.

A. Prepare a container to hold you apple slices. A plate or plastic storage container will work for this.

B. Place labels on the container. C for control or untreated and then labels i to v for the different solutions.

C. Prepare 6 apple wedge samples of approximately the same size. i. The exact size of the samples is not important.

ii. Cut the wedges so that they will easily fit into the solution cups

Laboratory Exercise - Browning

D. Treat the apple wedges as follows: c. Control no treatment. Place on your container by the c i. Vinegar treatment

ii. Lemon juice treatment iii. Baking soda treatment iv. Milk of Magnesia treatment v. Water treatment

E. Immediately after each treatment observe the apple slices and record your

observations in the Data Table 1. It is usually helpful to take a photo of apples slices for comparison.

F. When making observations include: i. Color / extent of browning

white, lightly brown, very brown, pink/reddish ii. Moisture level

damp, dry, slimy iii. Texture

smooth, wrinkled

G. Make a minimum of two additional observations over the next 24 hour period. i. The exact time of the observations is not important, but the approximate

spacing is. ii. Make the first observation 2 to 3 hours after preparation of the apples.

iii. Make the second observation 20 to 24 hours after preparation of the apples iv. Record the time of observation in the Data Table 1.

3. Preparation and treatment of potato slices.

A. Repeat steps A through G above this time using potato slices. B. You can use the same solutions for the potato slices that you did for the apple slices. C. Record the data for the potato slices in Data Table 2.

4. If you are interested in adding an experimental variable to these observations, student can

use different types of apples or potatoes and compare rates of browning.

Laboratory Exercise - Browning

Results: Experiment: Browning in Apples and Potatoes Data Table 1. Apple Browning Observation

0 Hours _______Hours

______ Hours

Color Moist Texture Color Moist Texture Color Moist Texture

C

i

ii

iii

iv

v

Data Table 2. Potato Browning Observations

0 Hours _______Hours

______ Hours

Color Moist Texture Color Moist Texture Color Moist Texture

C

i

ii

iii

iv

v

Laboratory Exercise - Browning

Conclusions and Discussions: Experiment: Browning in Apples and Potatoes Answer the following questions for the browning observations with both the apples and potatoes.

1. How does the color change in the samples dipped in solution compare to the color change in the control samples?

2. Which of the five experimental samples of the apple and potato had the greatest change in color? The least change in color?

3. Rank the color change from least to greatest for the five different solutions.

4. Did the acidic or basic solutions impact color change the greatest?

5. Propose a mechanism for acids or bases changing the rate of browning of the apple and potato.

Process of Science Questions and Conclusions: Earlier, for this experiment you created a key questions, hypotheses, predictions, and explanations for this prediction. Based upon your data and the questions you have answered related to this exercise you should be able to complete the process of science questions and conclusions. Answer the following questions. 1. Did your data support or falsify your hypothesis? 2. How did you come to this conclusion? 3. Did these results change your thinking about this topic? How? 4. What changes would you make to your hypothesis based on this new data? 5. What changes would you make to the experiments to better clarify your results?