Bial’s Test Reveals Differences in Taste of Lemons and Limes May Be Closely Related to Structure of Furanose Ring

By

Carly Gratopp, Emily Miller, Jude Pagel, Sahil Parikh 

“The SahilEmilyCarlyJude Team”

    LBS 145

Sec. 5

T.A. Phil, Ryan

Feb. 4th

Abstract: (Written by Jude Pagel, revised by Sahil Parikh and Carly Gratopp)

            After realizing the numerous similarities between lemons and limes, we were fascinated with finding out what exactly it is that sets the two fruits apart from one another. Why is the taste so different?  To determine this we used a few different tests on sugars, enzymes and photosynthesis.  Results from Bial’s test were interesting, showing that juices from lemons had hexose-furanose rings, while juices from the limes had pentose-furanose rings. The results for Benedict’s, Barfoed’s, Selvinoff’s, and the Iodine test showed similar results between the two fruits. Absorption spectrum testing was used to test for photosynthetic differences.  Two lemons and two limes were tested.  Results from the experiment show incredible similarities in data.  The results for the effect of pH on the juices show very similar results as well.  In testing for PPO, we found that the acidity of the lemons and limes inhibits PPO.  While the enzyme could be present, our available techniques could not detect it.    Based on our results, we have concluded that the difference in taste in lemons and limes could come partially from the difference in rings.  The hexose-furanose ring of lemons gives a different taste than the pentose-furanose rings of limes.  The photosynthetic process most likely does not play a role because the lemons and limes show very similar absorption of light. Results also show that pH probably has the same effect on lemons and limes.  The ring structure of the fruits must be a distinguishing factor.

Discussion: (Written by Sahil Parikh, Revised by Jude Pagel and Emily Miller)

            If a young child is asked to distinguish between a lemon and a lime, they will simply reply one is green and one is yellow, one is sweet and one is sour. Lemons and limes are of essentially the same size and shape. In fact, many people today still consider lemons and limes to be interchangeable. So, how exactly are these fruits different aside from obvious color variance? Why is one sweet and one sour?

            The human tongue is covered with taste buds. With in each taste bud are tiny taste receptors that respond rapidly to chemical stimulus. Food tastes sour if it contains acid (Beidler, 1952). However, lemons and limes both contain high levels of citric acid (Sinclair, 1984). Again, the question of why the drastic taste difference is posed.

            To answer this question, the group attacked it from several angles, attempting to find any sort of difference in carbohydrate groups (specifically sugars), photosynthesis and color, and enzymes, therefore causing differences in taste. The SahilEmilyCarlyJude Team believed that lemons and limes are composed of similar sugars, have the same photosynthetic qualities, and regulated enzymes much in the same way, and that sugars would place the most importance on the taste difference between the two fruits. In short, the group expected to find only very small and subtle differences in the composition of the fruit. Yet, the only way to ascertain whether there was validity to this statement was to perform a wide array of tests covering all three categories in question. For the sugars, five various carbohydrate tests were done; two tests were done for photosynthesis; finally, for the testing of enzymes two tests were performed.

            A series of carbohydrates tests were performed in order to identify the properties of the sugars and to see if there is starch in the lemons and limes (Table 1). In the first test, Benedict’s test, the juice of the two fruits was tested in order to test for the presence of reducing sugars. During the test, for both lemons and limes, a red precipitate was formed (Figure 1). This color change indicates that both fruits have reducing sugars, meaning they both have a free aldehyde or ketone group (Maleszewski, 2003). Secondly, the Barfoed’s test was performed to test for the presence of monosaccharides. Using a stock solution of fructose as a control to confirm the validity of the experiment, neither the lemon juice nor the lime juice produced any results for this test (Figure 2). This is curious, as the group assumed that both lemons and limes contain fructose, which is a monosaccharide. The results of this test do not support that hypothesis. Thirdly, the group executed the Selvinoff’s test. In this test, time is significant in discovering more about the sugars. If the solution turns red in under one minute, it is a monosaccharide ketose. If the solution turns red at approximately one minute, it contains a disaccharide ketose. Lastly, if the solution turns red in over one minute, the solution has an aldose. Lemons changed red in approximately 40 seconds and the limes approximately 55 seconds, indicating both are monosaccharide ketose (Figure 3). Because lemons turned red faster, the group hypothesizes that lemons have a higher concentration of monosaccharide ketoses. Using lab methods, it is impossible to prove this. The next test performed was Bial’s test. Here, the specific color change of the solutions reflects information about the sugars. An olive green color establishes a pentose-furanose ring while a muddy-brown color verifies a hexose-furanose ring. For distinguishing sugar differences between lemons and limes, Bial’s test was the most interesting and notable. The lemon juice produced a muddy-brown color, indicating it is a hexose-furanose ring. On the other hand, limes produced an olive green solution displaying it is a pentose-furanose ring (Figure 4). The last test performed was the iodine test. As the group expected, neither solutions turned black when the iodine was added, therefore confirming there is no starch present in either fruit (Figure 5).

            In order to compare photosynthesis in the two fruits, two tests were carried out. The first test was paper chromatography, which exhibits the pigments in each fruit by which light initiates the process of photosynthesis. When testing the rinds of the two fruits, two different methods were carried out. Unfortunately, both tests were inconclusive. No pigments appeared on any of the strips (Figure 6). This inconclusiveness is a result of one or more possible mishaps. It is probable that the paper chromatography in its current form is not the proper method by which to test the specific pigments in lemons and limes. Also, lemons and limes resemble the seed of a plant. If it had been possible to get a hold of the leaves of the lemon and lime plants, most likely more results would have occurred due to the fact that most photosynthesis takes place in the leaves of the plant, instead of the seeds. However, according to a previous study, significant amounts of carotene and xanthophyll are present in the rinds of the lemons and limes (Calvert, 1982). This leads to the conclusion that perhaps too much white pulp that connects the peel to the flesh inside was present in the mixing of phosphate buffer and rind, disguising the pigments normally found. The other photosynthesis test was absorption spectrum. Using both the boiling and blending methods and recording data every 15nm, it is evident that the absorbencies for lemons and limes show similar trends. Both fall consistently until around 500nm, where they both level off. Even the actual values for absorbencies are extremely similar between lemons and limes (Tables 2 and 3). Both the photosynthesis tests show the same results proving that photosynthesis most likely has little impact on the taste difference of the two fruit.

            The last category of tests completed was the enzymes test. A fruit or vegetable left on the kitchen counter for too long tends to ripen and eventually die. This is due to the breakdown of living enzymes in the food (Freedom You Nutrition Center, 2003). PPO is an enzyme found in these foods that helps to oxidize and break down the food. PPO is organic and has environmental limitations such as temperature and pH. Lemons and limes have such a high pH that PPO is not likely to play such a high role in decomposition. A normal potato covered in catechol, to speed up the reaction, turns brown where the catechol is present as the PPO transforms it to O-benzoquinone. However, when the normal potato is cover with catechol and either lemon or lime juice, this browning does not occur (Figure 9). When catechol is added directly to lemons and limes, again no browning occurs. The acidic juices of the lemons and limes inhibit the PPO from reacting with catechol and forming O-benzoquinone. It is possible PPO is present in both lemons and limes; however, it cannot be detected because the catechol cannot function correctly under citrus conditions. If there is indeed PPO in the fruits, it is very small portions because the pH range is outside of that found in the structured lab. 

            The group made a large effort to minimize error by repeating experiments multiple times.  Still, there is always room for procedural error, as well as certain constants, such as poorly made solutions, that would confound an experiment repeatedly.  The team is fairly confident that there were no errors in the sugar tests, as the results were consistent with controls monitored during both the independent lab as well as the previous weeks’ structured lab. 

For the photosynthesis experiments, there are several possible sources of error.  In the paper chromatography experiment, the pigments could not be extracted in a concentrated enough fashion to yield sufficient amounts of pigments to show up on a chromatograph. (Fig6)  The group suspects that in the boiling method, this is due to the acetone solution not being potent enough to extract sufficient amounts of pigment, and in the blending method, it is suspected that the white of the rinds diluted the pigments of the skins.  It is important to note that the group realizes that there are indeed different pigments in the skins, however with the resources available it was not possible to extract them in a measurable fashion.  Given more time and better equipment, the group would have liked to perform additional experiments, such as THC chromatography, and skinning the lemons and limes as opposed to using the whole rind in order to minimize the damage caused by the non-pigmented portion.

            The enzyme experiment with PPO was inconclusive, as the presence of PPO could be neither confirmed nor rejected by the method set forth in the structured lab, as we proved with our experiment designed to test the inhibition of PPO due to the acidity of the lemons and limes.  There was little room for error here, and we believe there was none.  We would have liked to be able to test different enzymes using different methods, but again were limited by equipment constraints.

            The goal of the group was set to determine whether we were correct in assuming there were slight differences in lemons and limes with regard to carbohydrates, photosynthesis, and enzymes, and to determine which of these categories holds the biggest weight in taste difference of the fruits. Operating within the constraints of time and equipment we have been limited to, the group found it difficult to adequately support this hypothesis.  Our photosynthesis experiments could have been improved upon in many ways, and our enzyme tests could have looked at different enzymes through different methods.  The group can however, use the results of the sugar tests, specifically the Bial’s test, to conclude that the two fruits do in fact contain different sugar compounds, and therefore infer that different sugars mean different tastes.

Figure 9: The presence of PPO. To the potato on the far left has been added only 1ml of catechol and has turned brown, indicating the presence of PPO. The middle left potato has been covered in lemon and then 1ml catechol was added. The potato on the middle right has been covered with lime juice and then had 1ml catechol added. Neither of these browned like the control on the left. They are the same as a plain potato (far right). This shows that lemon and lime juice both inhibit the catechol from reacting and turning the potato brown.