Research Question: At what temperature is the enzyme ...



Research Question: At what temperature are the enzymes within bromelain denatured?

Introduction: On every package of gelatin powder, such as Jello, there is a warning stating: “Do not use fresh or frozen pineapple, kiwi, gingerroot, papaya, figs, or guava. Gelatin will not set[1].”. After seeing this I got curious as to the reason why the gelatin would not set. For experimental purposes I chose to research pineapple more in depth because it was a less costly fruit to purchase, which would maximize the amount of trials that can be done. After very little time of searching pineapple, I found the answer to why the gelatin would no set: bromelain[2]. Bromelain is a chemical within raw pineapple that has two enzymes that digest protein. The protein that is necessary for gelatin to set, or change from a liquid to a solidified gelatin state, is consumed by the bromelain when raw pineapple is put into gelatin. This is the reason why you cannot put raw pineapple into gelatin. This still leaves the question: Why can canned pineapple be added to gelatin? Again, the answer was easy to find. Canned pineapple is heated as a part of the canning process to preserve the food[3]. But, what temperature is the bromelain within pineapple denatured? This is question important because knowledge of the ideal temperature would allow people to use fresh pineapple in their gelatin, and not rely on canned pineapple. This could allow more people an option of using fresh pineapple in their gelatin. Although fresh pineapple and canned pineapple tend to have the same nutritional value[4], people may want to use fresh pineapple for it’s taste and texture[5], or maybe just because they have extra from another recipe.

AIM: To find the best temperature to denature bromelain within pineapple to allow for use in gelatin.

Hypothesis: Bromelain will be denatured around 75 degrees Celsius.

Null Hypothesis: Bromelain will not be denatured around 75 degrees Celsius.

Controls: Raw pineapple in gelatin, pineapple soaked in room temperature water for 5 minutes, and gelatin

Independent Variable: Heat of water

Dependent Variable: Hardened Gelatin

Materials:

1. Raw pineapple

2. Water

3. 400ml beaker

4. 100ml beaker

5. Bunsen burner

6. Razor or knife

7. Unflavored gelatin

8. Test tubes

9. Test tube rack

10. Graduated cylinder

11. Refrigerator

12. Thermometer

Method:

1. Make the unflavored gelatin. To make the unflavored gelatin, boil 280ml of water. While waiting for the water to come to a boil, pour the gelatin packet into 140ml of room temperature water. When the water is boiled, mix both the boiled water and gelatin water together.

2. Label test tubes according to the temperature being tested (i.e. Room temperature control, 25*, 50* ect)

3. Measure 5ml of gelatin using a graduated cylinder, then pour the 5ml of gelatin into each test tube.

4. Cut the pineapple into similar sized pieces small enough to fit into the test tubes being used.

5. Make the three controls.

a. Place one piece of raw pineapple into each of the test tubes filled with gelatin. Label this rack ‘raw pineapple’ and place in refrigerator to allow hardeneing.

b. Place the pineapple peices in room temperature water for 5 minutes. Once time has passed, pour the water out and place one pineapple piece in each designated test tube. Label this rack ‘room temperature pineapple’and place in refrigerator to allow hardening.

6. Pour 5ml of gelatin into test tubes.

7. Next make first heat trial. Boil water at 50*C. Place the pineapple pieces into the boiling water for five minutes. Use the thermometer to ensure the heat remains constant throughout the time. Once five minutes has passed, pour out the water. Place one piece of pineapple into each of the designated test tubes.

8. Repeat steps 6-8 until all heat trials have been completed.

9. Place each test tube rack into the refrigerator and allow hardening overnight.

10. Check and record how many tubes have hardened into a gelatin state and how many remain liquefied. To ensure all tubes are being correctly recorded, turn the test tubes upside down, over a sink. Completely hardened gelatin will not move and remain in the test tube, while liquefied gelatin will pour out of the test tube.

Results:

|Trials |Total Number |Total Number |Total Number Hardened—Trial 3 |Average Trials Hardened|

| |Hardened—Trial 1 |Hardened—Trial 2 | | |

|Gelatin |3/3 |3/3 |3/3 |9/9 |

|Raw Pineapple |0/3 |0/3 |0/3 |0/9 |

|Room Temperature Water |1/3 |0/3 |1/3 |2/9 |

|50* Water |1/3 |1/3 |1/3 |3/9 |

|75* Water |3/3 |2/3 |3/3 |8/9 |

|100* Water |3/3 |3/3 |3/3 |9/9 |

Table 1. This table shows the amounts per trial of hardened gelatin and the averages of each trial.

Photo 1. This is a photo of gelatin, containing a denatured piece of pineapple, completely hardened within a test tube. Any completely hardened test tubes could be turned completely upside down without any liquid leaving the test tube, as shown here.

Conclusion:

The data supports the hypothesis; bromelain was denatured at 75 degrees Celsius. This conclusion was reached by averaging the all the trials seen within Table 1. Bromelain was denatured 8/9 times in the 75 degree Celsius trial. This was shown through the hardening of the gelatin in eight of nine test tubes, each filled with 5ml of gelatin and a piece of pineapple cooked for 5 minutes in 75 degree Celsius water. The gelatin would not have hardened if the bromelain was not denatured because of the protein-eating nature of the enzyme. Because bromelain eats the proteins that allow the gelatin to set, it can be seen that any test tubes that remain in a liquid state have been affected by the enzyme bromelain. Table one shows how many test tubes set, or hardened, versus how many remained liquid. Photo 1 shows how each test tube was only counted if it was completely hardened. This method allowed for an complete confirmation of whether the test tube was hardened or liquefied. The 100 degree Celsius trials show complete hardening, which supports the hypothesis that bromelain was denatured at 75 degrees Celsius. This supports that hypothesis because it would be expected that at any degree higher than 75 degrees Celsius there would be 100% hardening of the gelatin, which is what the results showed. The results of the 50 degree Celsius trials also support the hypothesis because only 1/3 of all the test tubes actually hardened. This shows that 50 degrees Celsius was not enough to denature even half of the test tubes. The control, gelatin by itself, shows that the gelatin alone hardened in all of the test tubes, which shows there was nothing wrong with the type of gelatin used or the method in which it was made. Also, the control of raw pineapple support the assertion that raw pineapple’s bromelain would eat the proteins within gelatin because 10% of the test tubes remained in a liquid state. All of the data as seen in table 1shows support for the hypothesis and rejects the null.

Although the data supports the hypothesis and rejects the null hypothesis, there are still anomalies within this experiment. In Control #3, room temperature water, there was the most fluctuation in data. Each trial resulted in a different amount of hardened gelatin. This can be explained in that the water could have diluted the strength of the pineapple juice, therefore diluting the power of the bromelain within each piece. This would not show up in any of the heated trials because the enzyme is being denatured and therefore most of the gelatin should not harden.

Evaluation:

The size of each piece of pineapple could have lead to differences between liquid and solid state seen. The pieces of pineapple were not cut to any standard or regulated size. They were cut to a relatively similar size by sight. This could be a reason why within the second 75 degree trial only 2/3 of the test tubes hardened instead of 3/3 as seen in the other two trials of that heat trial. This might also be the reason why in the control trials of room temperature water there are 2 hardened test tubes. If the pieces were smaller than all the others, and placed in water for 5 minutes they are more likely to be diluted in the power or amount of bromelain present within the pineapple piece. This does not affect the conclusion drawn because the other trials clearly support the hypothesis. Also, the slight fluctuation of 2/9 is not a substantial enough of a number to conclude that those results are an indication of anything other than human error.

Human error could be avoided more thoroughly in this lab in multiple ways. The major change that would be necessary to make is the measurement each pineapple piece. If this lab were repeated, the size of the pineapple piece would have to be regulated to ensure the same amount of bromelain is getting into each test tube. This lab could also be made better by the inclusion of more trials of each heat level to see trends of each trial better and make more firm conclusions of which heat is best for denaturing bromelain. More time would be necessary for the inclusion of more trials, and could also allow for the inclusion of multiple different heats to be tested. The intervals of 50, 75, and 100 were chosen, as they are substantially different heats that increase by 25 degrees. For more clear results, a smaller range of heats closer to the 75 degree Celsius range could be used to more accurately predict a more precise range of which heat the enzyme is denatured.

This experiment could be expanded to test the other ingredients such as kiwi, papaya, or figs, the other ingredients listed on gelatin packets, to see if they contain a protein similar to the bromelain found within pineapples. Knowledge of which fruits cannot be used with gelatin is important to people that cook at lot or are trying to remain in a more fresh, natural, raw fruit lifestyle. This lab, with minor adjustments, could be easily expanded to test the other fruits, which would be valuable knowledge to anyone working with gelatin. This is important knowledge so that anyone wanting to use pineapple in gelatin knows what temperature is necessary to heat pineapple to, to be able to use it within recipes involving gelatins.

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[1] Jones, Marilyn. "February 23." Goatview Farm. N.p., 23 Feb 2005. Web. 11 Jan 2013. .

[2] Scheve, Tom.  "Why do pineapple enzymes tenderize steak -- and your tongue?"  04 August 2008.  .

[3] Helmenstine, Anne. "Pineapple and Jell-O." . About.. Web. 12 February 2013. .

[4] Hitti, Miranda. "Canned Fruits, Veggies Healthy, Too." Web MD. Web MD Health News, 16 Mar 2007. Web. 15 Jan 2013. .

[5] "Canned vs. Fresh Fruits and Vegtables." Fit Day. Fit Day. Web. 12 Feb 2013. .

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