FOR STANDARDS: The Functionality of Bromelain in Different pHs: Jell-O Lab Report

ABSTRACT

            To examine the effect of pH on the function of bromelain, an enzyme found in pineapple, we prepared 4 samples of lime Jell-O with fresh pineapple juice and different amounts of strong acids and bases.  Our control sample was Jell-O with 1 mL pineapple juice and a pH of 4.  Our other samples all had 1 mL of pineapple juice as well, but NaOH and HCl were added to the samples in order to obtain pHs of 1, 8, and 12.  They were left to freeze and after two days only one dish of Jell-O had set, the one with a pH of 12.  This means that the conditions were basic enough to denature the bromelain enzymes from the pineapple juice, and inhibit them from breaking down the gelatin, which helps the Jell-O solidify.  The other plates were still liquid, meaning the conditions were favorable for the enzymes, although the acid in the pH 1 dish should have denatured the enzymes as well.

INTRODUCTION

            Since different enzymes can thrive in different pH levels, the four samples of Jell-O with pineapple juice were prepared to find the optimum pH for bromelain.  Bromelain is an enzyme found in pineapples that breaks down proteins, like the ones found in gelatin.  Gelatin is made from the collagen from ground up animal parts, and pineapples have enzymes to break down collagen because animals are able to digest their seeds.  In order to create a next generation, the seeds ideally should exit the animal’s digestive system undisturbed.  By breaking down this collagen, the seeds can inhibit the digestion that would occur in the small intestine by disturbing the epithelium, which is made of collagen (Gregerson 2003).

For some enzymes, conditions that are too acidic or basic can denaturize them and inhibit their function, while others thrive in these extreme environments.  The primary function of enzymes is to catalyze reactions between substrates, or molecules.  This means it lowers the activation energy of the reaction, or in other words allows the molecules to combine or separate more efficiently than they would on their own.  However, certain external conditions, such as pH or temperature, can stop an enzyme from performing its action.  These factors alter the shape of the active site, the place where the substrates are received, so the enzyme can no longer accept molecules and catalyze their reactions.  This process is called denaturing, and it is permanent.  In this lab, the denaturing effects of pH specifically were examined, in normal, acidic, basic, and close to neutral conditions.

HYPOTHESIS

If the pH is 1 and very acidic, the enzymes will be denatured and the Jell-O will set.

If the pH is 4 like it normally is, the enzymes will function normally and the Jell-O will not set.

If the pH is 8 and close to neutral, the enzymes will function normally and the Jell-O will not set.

If the pH is 12 and very basic, the enzymes will be denatured and the Jell-O will set.

MATERIALS

4 petri dishes

1 beaker

6.67 grams of lime Jell-O mix

39 mL of boiling water

39 mL of cold water    

Stirring rod

Litmus paper

50 mL NaOH (only use what is needed to reach target pHs)

50 mL HCl (only use what is needed to reach target pHs)

4 mL fresh pineapple juice

3 pipets

Freezer

PROCEDURE

1. Pour Jell-O mix into beaker and add 39 mL of boiling water.  Stir until the powder is dissolved.

2. Add 39 mL of cold water.

3. Separate the mix into four equal parts, and then pour into four petri dishes.

4. Take the pH of one dish.  Add 1 mL of fresh pineapple juice.

5. Add HCl to the second dish.  Add in 1 mL increments and take the pH until it reaches a pH of 1.

6. Add NaOH to the third dish.  Add in .5 or 1 mL increments and take the pH until it reaches a pH of 7, or 8 if you overshoot.

7. Add NaOH to the fourth dish.  Add in 1 mL increments and take the pH until it reaches a pH of 12

8. Add 1 mL of pineapple juice to the final 3 petri dishes.

9. Label the petri dishes and place them in a freezer.

10. Check the dishes after anywhere from a couple hours to a few days later.  Observe which are still liquid and which have set.

RESULTS

	pH 1	pH 4	pH 8	pH 12
Did it set?	No	No	No	Yes

 In the sample with pH 1, the Jell-O was liquid, meaning that the bromelain was able to maintain its shape and function in the very acidic solution.  However, the enzymes should not have been able to function in a solution of such low pH, meaning that the pH reading may have been wrong.

In the sample with pH 4, the Jell-O was liquid, so the bromelain was able to maintain its shape and function in the regular pH of Jell-O.

In the sample with pH 8, the Jell-O was liquid, indicating that neutral or slightly basic solutions do not inhibit bromelain’s function.

In the sample with pH 12, the Jell-O set, which indicates that the conditions were too basic and denatured the bromelain, inhibiting it from digesting the collagen in the Jell-O.

CONCLUSION

            With the exception of the dish of pH 1, our hypotheses matched our results.  Our control, the unaltered pH 4 plate, did not set as expected, indicating that the bromelain functions in slightly acidic solutions.  Our pH 8 plate showed the enzyme can also work in slightly basic solutions, but our pH 12 plate confirmed that very basic solutions denature the enzyme.  The anomaly was the pH 1 plate, which in theory should have set as well because the solution was incredibly acidic and able to denature the bromelain, but for some reason the enzymes still functioned properly.  This might be due to an incorrect pH reading, since we did not always properly mix our solutions before testing their pH.  If the pH was taken in a more acidic part of the solution, the reading would be lower than it would be for the overall solution, and the pH would not affect the enzymes.  Another mistake we made was probably with one of our two constants, which were the amount of Jell-O and pineapple juice in the petri dishes.  Since we just added one pipet squirt of pineapple juice to each dish, the exact amount probably varied from dish to dish, and there could have been more in the pH 1 dish, and with the improper mixing maybe some of it was not denatured.  If the amount of pineapple juice was not a true constant, another constant was the amount of time the Jell-O was left in the freezer.  While we definitely made one error in this experiment with the pH 1 plate, the rest of our results confirm what we know about enzymes and our hypotheses, and show that bromelain works best in solutions with pHs closer to neutral.

CITATION

Gregersen, Hans. Biomechanics of the Gastrointestinal Tract: New Perspectives in Motility Research and Diagnostics. London: Springer, 2003. Print.

Hey there! This is on my website as well for standards SP1, 3, and 6. Check it out?
https://sites.google.com/site/michelleshonorsbio/webb-science-practices-standards/1-asking-questions-and-defining-problems
https://sites.google.com/site/michelleshonorsbio/webb-science-practices-standards/3-planning-and-carrying-out-investigations
https://sites.google.com/site/michelleshonorsbio/webb-science-practices-standards/6-constructing-explanations-and-designing-solutions