Enzyme Action of Malus domestica and Malus sylvestris
Using Lemon Juice as an Inhibitor


By Stacey Buffa, Melanie Depoy, Nicole Dickerson, Kami Kovacs


Abstract

        At the molecular level, different assortments of the species Malus domestica, which are hybrids of Malus sylvestris , were hypothesized to possess similar chemical components, including carbohydrates, skin and leaf pigments, and enzyme action.  Of the Malus domestica species Greening, Grimes Golden, and McIntosh apples were tested along with the Crab apple, an apple of the species Malus sylvestris. The apples’ chemical components did not vary on a molecule level due to the similarities in carbohydrates and photosynthetic pigments in the leaves and enzyme action. Each apple variety did however vary in the pH level and skin pigments. To test the likeness of carbohydrates in each apple, Bial’s and Benedict’s tests were performed. It was determined that the sugars present in all four apples were that of a hexose (or higher)-furanose after conducting Bial’s test, which resulted in varying shades of a deep, reddish brown. The presence of reducing sugars, such as a free or potentially free aldehyde or ketone group, was determined after performing Benedict’s test. The Crab, Greening, Grimes Golden, and McIntosh all revealed characteristics confirming the existence of fructose, which is a monosaccharide. In effort to determine how much fructose was present in each apple, diluted juices from each apple were placed in the spectrophotometer and readings were taken at a wavelength of 350nm. To uncover the similarities in pigments between both the apple skins and leaves, two tests were conducted. Pigment identification was accomplished using paper chromatography. The spectrophotometer was then used to create an absorption spectrum, which represents the relative absorption of different wavelengths of light by a pigment. We compared the absorption spectrum of each apple leaf chloroplasts and found the results to be strikingly similar. To determine the presence of polyphenoloxidase (PPO) in each variety of apple, the pH was recorded along with the addition of catechol. Each apple revealed a brown surface after the addition of catechol. To further our research on enzymes, we performed an experiment using lemon juice, which is known to counteract PPO, in attempt to measure the amount of PPO in each apple. It was assumed that the more PPO present in each apple, the less effective the lemon juice would be. It was concluded that, the higher the pH of the apple, the smaller the quantity of PPO.


Discussion

        The comparisons of the varieties of Malus domestica with that of Malus sylvestris were verified to possess similar qualities at the molecular level.  The presence of carbohydrate components, such as a ketone and aldehyde group, furanose or hexose ring structure was hypothesized to be similar, along with each consisting of fructose.  We also questioned if pigments contained in the leaves would have any effect on the pigments found in the skins.  As for enzyme action, we hypothesized that the application of lemon juice would inhibit PPO. We predicted that the results would vary slightly according to the pH of each apple.
        As predicted, the carbohydrate test performed showed no significant difference.  This was shown through Bial’s test, in which the McIntosh, Crab, Greening, and Grimes Golden apples all varied in shades of reddish brown.  Fructose was the only control used, which was determined to be present in all of the apples. However, apples also consist of glucose and sucrose. Our experiment lacked in not using all three of these carbohydrates as a control. Fructose appeared as a shade of olive/brown. It was concluded that the mixtures appeared a reddish brown instead of simply olive/brown because fructose is not the only carbohydrate present.
      Through Benedict’s test, we were able to deduce that there were free aldehyde and ketone groups in the McIntosh, Crab, Greening, and Grimes Golden apples, as predicted.  This was concluded through the results exhibiting a red precipitate formation for each apple. In this experiment, the aldehyde is oxidized while the copper is reduced. The reduced copper then precipitates and the original blue colored solution changes to an orange/red. These results exhibit the presence of a free or potentially free aldehyde and ketone groups (Maleszewski et al., p.66).
        It was hypothesized that the sweeter the taste of the apple, the higher the concentration of fructose. To test the levels of fructose present in each apple, we used the spectrophotometer at 350nm.  As a result, we found that with varying concentrations of each type of apple at 33%, 66%, and 100%, the absorbance readings were all very similar, all reading at an average of 2.41 % absorbance.  This means that the apples may not vary in levels of fructose, but it is possible that they vary in the levels of other sugars present, such as sucrose, which contains both fructose and glucose. For our photosynthesis experiments, we questioned if pigments contained in the leaves have any effect on the pigments found in the skins of McIntosh, Greening, Grimes Golden, and Crab apples.  We predicted that the pigments contained by the leaves had a direct effect on the skins, causing the pigments found in the leaves to be present in the skins as well.  Additionally, we also predicted that each apple, and their leaves, would contain the following: chlorophyll a, chlorophyll b, carotene, and xanthophyll, because they are all constituents that give the leaves color. However, upon concluding the paper chromatography experiment, it was evident that our prediction was wrong.  Although our prediction that the leaves of McIntosh, Greening, Grimes Golden, and Crab all contained chlorophyll a, chlorophyll b, carotene and xanthophyll was correct, these pigments were not all found in each apple skin. These results led us to conclude that our prediction about the correlation of pigments in the skin and leaves was false.
Chlorophyll a and b were found only in Greening and Grimes Golden apples’ skins due to their nature of being green in color.  We have concluded that the lack of the color green in McIntosh and Crab apples result in a lack of chlorophyll a and b.  Carotene, however, was found in the apples' leaves and skins of each apple variety.
        We also formed the prediction that each individual absorption spectrum of leaves would compare favorably to each other and also expected that the absorption spectrum of apple skins would follow the same pattern.  After measuring the absorption spectrum of all four leaves and skins, it was clearly evident to see that our prediction was false.  Both the leaves and skins absorption spectrum compared strikingly different from one another.  At certain wavelengths their absorbency values were largely different. However, the absorption spectrums all followed a similar pattern.  The absorbency of each apple would start out high at a wavelength of 400nm and slowly decrease to a much smaller absorption at 700nm. The leaves of all four apples had similar absorption spectrums due to their similarities in color.  Due to their nature of being green in color, they all possess similar abilities of absorbing light at different wavelengths.  We see the color green because green is the color most reflected by the pigments that the leaves contain.  In this case, chlorophyll a, b, and xanthophyll, which are all present in the leaves of the apples, all reflect green light.
        Although the patterns of absorbency for the four apple skins are similar, their own absorption spectrums vary at different wavelengths.  Greening and Grimes Golden have high absorbencies between 500 and 600nm due to their presence of both chlorophyll a and b, and xanthophyll.  However, Crab and McIntosh have high absorbencies between 400 and 450nm, as well as between 650 and 750nm, due to their pigments resulting in the color of their skin being red.  This is a direct result of their pigments reflecting more light at these wavelengths, which then result in the color red being seen.
        It was hypothesized that PPO would be present in all species of Malus domestica including Malus sylvestris, which was verified by the brown color revealed after cutting the apple. The degree of browning depends on the concentration and nature of the phenolic compounds present (Sanders et al.).  We hypothesized that the higher the pH of the apple, the lower the concentration of PPO, therefore, the slower it will take for the apple to appear brown. Our hypothesis was verified with the apples which were simply exposed to air. McIntosh, having the highest recorded pH of 5, took the shortest amount of time to exhibit the presence of PPO. Crab, having the lowest recorded pH of 2, took the longest amount of time to exhibit the presence of PPO.
        The hypothesis that lemon juice would inhibit PPO was also verified through the testing of both exposed apples saturated with lemon juice and concealed apples saturated with lemon juice. Of the apples saturated with lemon juice and left exposed to air, only Greening showed any sign of PPO. The McIntosh, Crab, and Grimes Golden apples were observed as very white and dry. Of the apples saturated with lemon juice and concealed, Grimes Golden revealed the presence of PPO, while all of the other apples were observed as shriveled and dry. In both cases, it was concluded that the juices dried up before browning could occur. This can be verified by acknowledging that as ascorbic acid is acting to reduce PPO, it is being oxidized (Sanders et al.). Therefore, the ascorbic acid and lemon juice were being used up in providing protection, leaving the apple dry.
        It was also hypothesized that the apples exposed to air would exhibit the presence of PPO at a relatively faster pace than those concealed. This was verified when the apples that were concealed took several hours to turn brown while the apples exposed to air ranged from twenty-one to fifty-eight minutes before PPO was revealed.  It has been concluded that the reactions were delayed as a result of little exposure to molecular oxygen, which is the cause of browning.
        The browning of the Green apple, which was exposed in air and saturated with lemon juice, may have been caused by the use of a blunt knife instead of a sharp knife.  A blunt knife would have caused more damage, opening more cells, and exposing the two compartments of PPO and phenolic compounds (Sanders et al).  This may have also occurred with the Grimes Golden apple, which was concealed and saturated with lemon juice, since it was also the only apple to turn brown in this category.
        For future experiments, one may consider damaging an apple with three different instruments: a sharp knife, a blunt knife, and a blender. The observations can be used to determine the relationship between the browning of the apple and the method of preparation.  Also, different citrus fruits contain different amounts of ascorbic acid along with varying pH levels. An apple may be cut with the same instrument and placed into several different solutions, including water, orange juice, lemon juice, and a water-salt solution.  The pH of each solution should be recorded.  Observations should be taken periodically, for example, at intervals of 10 minutes for two hours.  The relevance of the pH and which solutions worked best in preventing browning could be discussed after the results were obtained.  The difference in taste at the beginning of the experiment, then after the solutions were added should also be recorded to see if any relevant changes occurred. If change in taste did occur, the structures of carbohydrates may have been altered. Experimentation of the structure of carbohydrates before and after could verify these results.


Apple Testing

Figure 10: The comparison of the apples exposed to air, one with lemon juice and one without. From top left, moving clockwise, the apples are: Greening with lemon juice, Greening, Grimes Golden with lemon juice, Grimes Golden, Crab, Crab with lemon juice, McIntosh, McIntosh with lemon juice. The apples saturated with lemon juice revealed no to very little PPO when compared to those without lemon juice.