Introduction
In our IB biology class, we’ve learned about enzymatic reaction and we’ve done the experiment on that unit. The experiment was about the rate of enzymatic reaction of lactosemilk by using an enzyme called lactase at different temperatures. At different temperatures, the rate of reaction changed. I was interested in this topic because there were more factors, not only temperature but also hydrogen ions concentration, presence of inhibitor, and concentration of substrate, which affect the rate of an enzymatic reaction.
When I saw cut apples in my childhood, they were brown after certain times and it didn’t look fresh. At that time, my mother used to dip cut apples in salt water and this action prevented them from turning to brown. I was curious to know how salt water slows down this browning reaction. Through the IB biology class, I now know that it was due to an enzymatic browning reaction and also, I could realize that the different levels of acidity(hydrogen ion concentration) have different speeds that can prevent the enzymatic browning reaction. Therefore, this experiment was selected to investigate the browning reaction in detail and to measure the reaction in relation to the hydrogen ion concentration in order to find out the different degrees of slowing down the reaction.
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Research Question
To what degree do different levels of hydrogen ion concentration have an impact on the rate of enzymatic browning reaction of cut apples?
Hypothesis
The lowest pH level(pH 1) will slow down the enzymatic reaction the most and at the highest pH level(pH 13), the enzymatic reaction will work the fastest which will catalyze more catechols by polyphenol oxidase and cause more browning on cut apples.
Background Information
All enzymes are proteins. Therefore, enzymes are easily broken even at a high temperature and lose the catalytic ability, and even if the hydrogen ion concentration changes slightly, the function is lost. In particular, enzymes maintain the tertiary structure of the enzyme protein, and the non-covalent bonds such as hydrogen bonds belonging to the chemical bonds are very weak bonds, so they can be easily broken even by slightly heating or slightly changing the hydrogen ion concentration around them, resulting in protein denaturation. According to Robinson, “As the pH changes, the ionization of groups both at the enzyme's active site and on the substrate can alter, influencing the rate of binding of the substrate to the active site. These effects are often reversible.”, and they are also called denaturation. Denaturation refers to a non-active state of an activation site, and once it is deprotonated by temperature or pH, the catalyst function is lost. Thus, all enzymes have a pH that is suitable for the reaction.
In this experiment, the effect of the different levels of acidity on the rate of enzymatic browning reaction was investigated. The reason why the browning reaction occurs in fruits is the enzymatic reaction of the enzyme called polyphenol oxidase. An apple also has this enzyme, so browning occurs naturally. Polyphenol oxidase or phenolase reacts with oxygen when exposed to air. Then this enzyme catalyzes a factor called catechol into a substance called o-quinone. As this reaction further going on, brown melanin is produced which causes the color to change in an apple.
This experiment does not require any harmful chemicals. However, students should wear a lab coat in order to avoid the possible harmful chemicals from another experiment performed by other students in the same lab. Do not eat cut apples and the leftover apples that were used in the experiment. They could be exposed to harmful chemicals used in the lab.
Observation
As the concentration of hydrogen ion approaches pH 7, darker colors were extracted from cut apples and there were many colors close to brown. Also, browned part appeared 2 minutes after apples were cut. By comparing each result of average browned part, the rate of enzymatic browning reaction slowed down at the highest pH level(pH 13) and the lowest pH level(pH 1). However, cut apples at pH 1 had less reaction than at pH 13. pH 7 had the highest and fastest browning reaction among 5 different pH levels which was the closest to apple’s optimum pH(pH 5.5 ~ pH 6.5).
Overview of Processing
The raw data was processed with 5 different colors that were collected at each pH level and the result turned into a table and graph. The graph represents the average of each group within the five cut apples because this shows a general idea of the rate of enzymatic browning reaction. The data presents the browning reaction of cut apples after 1 hour taking out of a beaker. Each value was obtained with a program called Image Color Summarizer.
Data Analysis
Colors extracted from the experiment and each color’s occupying percentage the average percentage of the browned part. In order to find out the average results of the browned part, each color that is considered as a brown, sandy color, sandy brown, and reddish-brown, were added with a unit of percentage. The results were observed from the website called Image Color Summarizer which shows the color extraction with an occupying percentage. The amount of browned part of cut apples was compared to determine what pH solution is effective to prevent the browning reaction. The concentration of hydrogen ion near pH 7 has darker colors extracted from cut apples. Also, there are many colors closer to brown at pH 4, pH 7, and pH 10. They contain sand color and sandy brown whereas pH 1 contains only light beige. This returns that there are more catechols that are catalyzed by polyphenol oxidase in the pH levels near pH 7. Furthermore, it is clear that pH 1 slows down the enzymatic browning reaction. It is evident that pH 7 is the closest to the optimum pH of cut apples. In pH 7, the cut apples contain reddish-brown which is the darkest color extracted from this experiment. There is also the highest percentage of the browned part(23.51%). This result represents enzymatic reaction works the fastest at pH 7 which indicates that optimum pH is near pH 7.
pH 1, pH 4, and pH 13 had a lower percentage than the result gathered without pH. As they had a relatively lower percentage on browning reaction, it is evident that they slow down the enzymatic reaction. Even Though the hypothesis didn’t support the result of this experiment, the result was accurate when compared to results gathered by other scientists. J Lachman, V Pivec, M Orsák, and J Kučera, who wrote the Czech Journal of Food Sciences in 2000, they participated in a similar experiment and found the same result. They recorded the conclusion that the optimum pH of PPO activity(polyphenol oxidase activity) is 7. The hypothesis of this experiment shows that pH 13 will be the optimum pH for PPO activity as it states there will be the fastest catalyzing reaction of catechol by polyphenol oxidase. However, after the experiment, the result was different and showed that pH 7 had the fastest enzymatic reaction.
Suggestion for Improvement
For this experiment, more trials should be done in order to come up with a more precise conclusion. Each group of the cut apples had different colors extracted from its image and it was difficult to compare. In order to compare them, only 5 colors were extracted from each image which made results less precise. Moreover, as the cut apples were dipped into the pH solution, the color of the apples came out into the solution for pH 10 and pH 13. This possibly had an impact on the color of the cut apples. If more colors were compared and cut apples were dipped for less time, the result would be more accurate and possible error would be avoided.
Conclusion & Evaluation
The results gathered in this experiment could clearly answer the research question which was: “Does different level of hydrogen ions concentration have an impact on the rate of enzymatic browning reaction of cut apples?”. After obtaining the data and comparing it, it was difficult to come to a conclusion to the original hypothesis. As in the average percentage of browned part in each group, the data was inconclusive because the cut apples in a low pH level and in a high pH level were respectively lower than the cut apples in a middle pH level in terms of their average percentage of browned part. Even though the low pH level showed the lowest value of browned part, we could not conclude that the reaction works the fastest in higher pH level. Throughout this experiment, there were the fastest and highest enzymatic browning reaction at pH 7 and the lowest reaction at pH 1. At pH 13, there were lower reaction happening, however, it was higher than the reaction at pH 1.
As a result, I was able to understand higher acidity(lower pH level) is more efficient in preventing the cut apples’ enzymatic reaction than lower acidity(higher pH level). Also, it clearly explained why do people use salt water to slow down browning on fruits because salt water is lower in pH which is higher in acidity.
Bibliography
- Robinson, P. K. (2015, November 15). Enzymes: Principles and biotechnological applications. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692135/
- Sandhyarani, N. (2018, February 22). Effect of pH on Enzymes. Retrieved from https://biologywise.com/ph-effect-on-enzymes
- Lachman, J., Pivec, V., Orsák, M., & Kučera, J. (2000). Enzymatic browning of apples by Polyphenol oxidases. Czech Journal of Food Sciences, 18(No. 6), 213-218. doi:10.17221/8352-cjfs
- Ashish. (2017, September 20). Tricks To Prevent A Freshly-Cut Apple From Turning Brown. Retrieved from https://www.scienceabc.com/eyeopeners/why-do-apples-turn-brown-and-how-can-you-prevent-it.html
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