Carbohydrate, Photosynthesis and Enzyme Tests Show Property Differences in Bananas During the Ripening Process

 

THE PROTOCOLS:

Erika Boerman

Nick Gorbach

Mikala Mondelli

Anne Rolecki

 

 

ABSTRACT:

How does the ripening process affect the chemical properties of bananas? To answer this question we set up a series of tests to analyze the three stages of the process; unripe, ripe, and overripe. We made banana extracts from the banana and peel for each stage. Then we did carbohydrate, photosynthesis, and enzyme testing on bananas using the extracts. We identified carbohydrates such as those used in the known sugars lab with Benedict’s, Bial’s, Selivanoff’s, Barfoed’s, and Iodine tests. From these tests we discovered that different carbohydrates were present in each stage of the ripening. The results for unripe peel and fruit were contradictory because some tests suggested the sugar was a disaccharide and a reducing sugar, which cannot be true. The ripe peel contained a sugar with the same properties as fructose, and the rest of the fruit and peels had the same unidentified sugar. The presence of pigments like chlorophyll a and b, xanthophylls, and carotene was tested with chromatography and absorption. The photosynthesis tests showed no identifiable pigments at any stage of the ripening process for both the fruit and peel. Finally, from enzyme testing, we determined that PPO was present at all 3 stages, and that altering temperature, pH, or adding an inhibitor all changes its effectiveness by bringing the conditions away from the normal and optimal range of a neutral pH and medium temperatures. We determined that increasing heat and pH help PPO catalyze the reaction, while an inhibitor prevents it from doing so.

 

 

Table 1. Carbohydrate Tests on Bananas. Below are the results of the numerous carbohydrate tests
we did on the peel and fruit of bananas during three stages of ripening.

Stage	Part of Banana	Benedict's	Barfoed's	Selivanoff's	Bial's	Iodine
Unripe	Peel	Little Precipitate	Orange- Red Precipitate	1 minute	Olive	Rusty Orange
Unripe	Fruit	Moderate Precipitate	No Change	45 seconds	Brown	No Change
Ripe	Peel	Moderate Precipitate	Orange- Red Precipitate	50 seconds	Olive	Brownish Orange
Ripe	Fruit	Much Precipitate	Orange- Red Precipitate	37.5 seconds	Brownish	No Change
Overripe	Peel	Much Precipitate	Orange- Red Precipitate	47.5 seconds	Brownish	Brown
Overripe	Fruit	Much Precipitate	Orange- Red Precipitate	35 seconds	Brownish	No Change

DISCUSSION:

In this experiment we tested bananas at three different stages of the ripening process to answer the question of whether or not there is any chemical difference between the bananas at the three stages. The three stages were unripe, when the bananas were green, ripe, when the bananas were yellow, and overripe, when the bananas were brown and even sometimes black.
Since bananas taste differently depending on which stage of the ripening process they are eaten, we hypothesized that there would be a difference in chemical properties of the bananas at the three different stages. Our group predicted that more kinds of sugars are present in the overripe stage because riper bananas taste sweeter, but there are some sugars present throughout all three stages of the ripening process. We thought that the most chlorophyll would be present in the unripe stage since the peel is green versus the ripe stage where the peel turns yellow. We also thought that through the three stages of ripening we would find three different pigment combinations of green, yellow, and brown to give us the colors of the peel. In the enzyme testing, within a normal range of approximately pH 6-8 (close to neutral), we thought the reactions would proceed faster, while outside the approximately neutral range reactions would be slower. We predicted that reactions would proceed faster the higher the temperatures rose until about 70°C, when reaction rates would drop. Overall, we predicted that extreme conditions are detrimental to the rate of reaction.

Upon completing the series of experiments listed in the methods section of this paper, our group collected data that confirmed our hypothesis that there would be chemical differences between the bananas at the three different stages of the ripening process.

Carbohydrate Testing
After performing Benedict's test on the extracts from bananas and their peels we found that our precipitate was an orange-red color thus indicating that the carbohydrate contains a free aldehyde group. For both the peels and the actual fruit the amount of precipitate visually increased from unripe to ripe to overripe. The unripe peels and fruit showed less precipitate than the ripe peels and fruit, which in turn showed less than the overripe peels and fruit. Since our extract did show a positive result it agrees with our prediction that one of the sugars present in bananas is structurally similar to fructose. We cannot tell if sucrose is present from this test because sucrose did not react under the conditions provided (Table 1).

Our results from Barfoed's test showed that the unripe fruit was the only one that did not change color and therefore contained poly- and disaccharide sugars. The rest of the fruit solutions and all the peel solutions showed a change in color, indicating that they contained monosaccharide sugars (Table 1). These results do not confirm our prediction that there would be more complex sugars present in the later stages as compared to the unripe stage of development. As the fruit and the peels ripened the sugars became less complex, thus the reason for the absence of poly- and disaccharide sugars in the later stages.
After performing Selivanoff's test we verified that the sugars in the unripe, ripe, and overripe banana's fruit were monosaccharide ketoses. The ripe and overripe peels also tested positive for monosaccharide ketoses, while the unripe peel tested positive for disaccharide ketoses (Table 1). Since Selivanoff's test and Barfoed's test provided results that slightly contradicted each other in the unripe peel and fruit, we are not sure about the accuracy of our tests. Barfoed's test gave us results that signified that poly- and/or disaccharides were present, while Selivanoff's test signified monosaccharides were present. We had performed two different trials for each test and obtained almost the same results each time. Therefore we did obtain consistency, yet the two tests still contradicted.

After completing Bial's test we were able to determine which type of furanose rings were present during the different stages of the ripening process. The peel of the unripe and ripe bananas turned the solution an olive color, thus indicating that the sugars were in the shape of a pentose-furanose ring. The peel of the overripe banana and the fruit at all three stages turned the solution brown, indicating that the sugars were in the shape of a hexose-furanose ring. These results show that the sugar's structures change slightly in the peel throughout the ripening process but remain the same in the actual fruit (Table 1).
Our results from the Iodine test showed that starch is not present in all three stages of the ripening process for both the banana's peel and fruit (Table 1). The solutions for the fruit at the unripe, ripe, and overripe stages darkened slightly and took on the yellowish cast of the iodine, but none of them turned the bluish-black color that would indicate starch.

Although it was not possible to figure out exactly which sugar(s) were present at each stage of development from looking at the results of just one test, the combined results gave us a clearer picture of the carbohydrates present. The results for the unripe peel and unripe fruit were the only ones that gave contradicting results between tests. For both cases, the contradiction came between the results of Benedict's and Selivanoff's tests. Although a sugar cannot be a monosaccharide and be a disaccharide, these extracts gave results that claimed both were true in those cases. However, for the other extracts, the structure of the carbohydrate was determinable from the results of the tests. For the ripe peel, the results described a reducing sugar that was a mono-ketose, not starch, a monosaccharide, and a hexose-furanose. These criteria exactly match the characteristics of fructose, the structure of which we found in the CEM 351 textbook (Jones, 1174). For all the remaining tests, we consistently found that the extracts contained a carbohydrate that was a monosaccharide ketose in pyranose, which was not one of the known sugars tested in the first carbohydrate lab.

Photosynthesis Testing
After completing our pigment identification we were not able to identify the different pigments in the fruit and peels of bananas during the stages of the ripening process. We performed two tests for each of the six solutions, one for the peel and one for the fruit at each stage, and each test showed no pigments in our extracts. Since both times we performed the test we obtained the same results, we had consistency but our consistency did not tell us what pigments were present. Our paper chromatography test only tested for the pigments chlorophyll A and B, xanthophyll, and carotene. Since our tests gave no results this could have happened due to the fact that bananas do not have these pigments in large enough quantities to produce a result or that they do not contain these at all. In this case, to obtain more accurate results we should have tested for all pigments. Our lack of pigments could also be because we were unable to successfully make a suitable extract to test for banana pigments, as we used a method that works for spinach but is unproven to work for bananas. Since our test did not produce us any results we were not able to calculate the Rf values.

We graphed the wavelength of light versus the corresponding absorbance levels (Figure 9). Our graph shows that for each solution the absorbency was highest in the lower wavelengths and steadily decreased as the wavelengths increased. This is concurrent with our predictions because we assumed that the bananas would have greater amounts of chlorophyll in the unripe stage and decrease as the banana ripened. We assume that more absorbance means that there more photosynthesis taken place because light is a key component to photosynthesis.
Although our paper chromatography testing did not produce the results we would have liked, our absorbance spectrum test gave ample evidence to support our hypothesis.

Enzyme Testing
Each banana peel and its respective fruit turned the litmus paper a shade of orange. This informs us that the pH levels were about 4. Bananas and peels in all stages of the ripening process reacted to catechol, so all of them have polyphenoloxidase (PPO).
Upon completing the tests for the effects of heat and inhibitors, both the heated solutions and the inhibited solutions were lighter than the control, but the heated solutions were darker than the inhibited solutions. This data shows that enzymes in heated solutions catalyze faster than enzymes in inhibited solutions (Table 3).

After testing the effects that different pH's had on the absorbances of banana extracts, we concluded that enzymes in bananas catalyze more the closer they got to neutral conditions, around pH 7 (Table 4). The banana extracts tended to absorb less the further away from neutral conditions, in both directions. This confirms that the rate of the reaction was catalyzed most effectively under neutral conditions.

Weaknesses that were present in our experiment were mainly due to the time constraint. There were two weeks in between our carbohydrate testing and our photosynthesis testing and then another two weeks in between the photosynthesis testing and enzyme testing. The lengthy amount of time in between the different tests would have caused our samples to spoil and so we had to attain new materials for each test. We could not have used the spoiled samples because obviously then we would not have had bananas at all three stages of the ripening process. Instead we would have just been testing the overripe stage over and over again. We assumed since we received the bananas from the same location every time that essentially they were the same.
One problem that arose during our experiment was the fact that we were not the only group to use the lab equipment and perform an experiment. Numerous groups were constantly moving in and out of the lab and possibly exposing unpredicted chemicals into our specimen. Many other groups were working with fruits too and as explained in our introduction, this may have caused more ripening to occur then was planned. Also in the adjacent laboratory there was another biology class performing breeding experiments on the Drosophila melanogaster or the common fruit fly. The lab groups even sometimes came into our lab and used our fume hood. Some of the flies could have managed to infiltrate our specimen and thus cause some contamination. Regardless of these complications, our hypothesis was overall confirmed from the tests conducted and the data collected.