Green and Red Grapes Have Similar Carbohydrate & Enzyme Activity, Supported by Benedict's Test, and the Presence of PPO Test.

By: Lindsay Haylock, Joey O'Connor, Diana Tuman, and Teresa Raies
Group: Tape Dispensers


Abstract

    In order to look beyond the superficial layers of Vitis vinifera and into the chemical reaction process, enzyme, and carbohydrate composition, a series of tests had to be conducted. The question investigated was: what are the differences and similarities between the carbohydrates, enzymes, and the photosynthetic process in red and green grapes (Vitis vinefera)? We hypothesized that both grapes would be the same in enzyme activity and carbohydrate concentration, but the photosynthetic process is higher in red grapes than in green.  The carbohydrate analysis included Benedict’s test, which determined whether or not carbohydrates contain a free ketone or aldehyde reducing group, and Selivanoff’s test, which is based on whether ketoses or aldoses are present within solution.  The results showed both grapes contain monosaccharide ketoses because an orangish/red precipitate formed. Iodine test was conducted to see if starch is present within the compounds, the results showed no starch was present.  The Pigment Identification test was used to determine the presence of certain pigments and their rates of low.  Absorbance Spectrum tested how much light was absorbed, while the Action spectrum tested for efficiency of each color as a fuel source for photosynthesis. Our results neither supported nor opposed our hypothesis.  The Environmental Effects of Enzyme Activity tested for the presence of PPO, the affect of heat inhibitors on the enzyme, and the affect of pH on enzymes.  The results showed a PPO presence in both of the grapes and similar enzyme activity.  Overall green and red grapes had similar results when testing for carbohydrates, phtotosynthesis, and enzymes.


 
Fig. 5. Absorption Spectrum of Green and Red Grapes.  This graph represents the average amount of light absorbed at different wavelengths between green and red grapes.  Two trials were done with each grape and then averaged.  Both cases resulted in an overall decrease in absorption as wavelength increased. This displays that green grapes absorb more light than red grapes.

Discussion

    The basis of our experiment was to figure out the differences and similarities in carbohydrates, photosynthesis, and enzyme activity in red and green grapes. Initially, we hypothesized that red and green grapes contain similar types of free sugars in terms of aldoses and ketones, both that would have polyphenoloxidase (PPO), and that red has a higher absorbency spectrum.  In order to test our theory, both grapes were subjected to a series of tests.  Each of these tests has attempted to discover the difference between green and red grapes. Benedicts was the first test conducted (refer to Fig.1) and used to detect specific types of carbohydrates. The presence of a red precipitate and a color change form blue to red indicated that copper was reduced, therefore, precipitated and an aldehyde was oxidized.  The test supported that both red and green grapes contain free reducing sugars with an aldehyde or ketone group. 
Selivanoff’s test was conducted second (refer to Fig2.).  It was used to differentiate between a monosaccharide ketone and a disaccharide aldehyde group.  A monosaccharide is a single sugar, which has a shorter reaction time, less than 1 minute.  The color turns red faster if a sugar is a ketose, again less than 1 minute.  Therefore, if a red color is seen in less than 1 minute it is safe to say that the grape contains a monosaccharide ketone.  That is exactly what we observed in both of our cases, when testing the red and the green grapes.  If the reaction time is greater than 1 minute one can conclude that the grape contains a disaccharide aldose.   The test verified that red and green grapes both contain monosaccharide ketoses. 
    The final test dealing with carbohydrate investigation was the Iodine test (refer to Fig.3).  It distinguishes the presence of starch.  Starch is a coiled polymer of glucose.  Iodine interacts with those molecules and the solution turns a bluish/black color.   A bluish/black color is a positive test for starch, while a yellow/brown color is a negative (Maleszewski et al., 2003).   Based on our observations, neither the red nor the green grape contained starch, since the solutions were a brownish/ yellow color.  The initial hypothesis dealing with carbohydrate structure in red and green grapes was put to test again and again.  At this point supporting evidence indicates that the hypothesis was correct.    
    Further testing had to be done in order to establish if there are in fact differences in photosynthesis processes between red and green grapes.  The Pigment Identification Test determined the rate of flow between the two grapes.  Our initial prediction was that both grapes absorbed an equal rate of flow.  The following elements should be considered when calculating the rate of flow: Chlorophyll b, which appears closest to the initial marked line, followed by chlorophyll a, xanthophylls, and lastly carotene.  However after concluding the test, these results were not obtained.  Four trials were conducted, at the end of which the chromatography strips were clear with no color pigment on them. This also means that we could not calculate a rate of flow for any of the pigments. These results can be seen in Fig.4. We obtained no visible results because we think that grapes do not actually undergo photosynthesis. The photosynthesis for grapes takes place in the leaves.
    The Absorption Spectrum of grape chloroplasts was used to test how much light each grape absorbed.  Our initial hypothesis was that green grapes would have a smaller absorption spectrum than the red. Green grapes absorb all color except its own; since that is what is seen to the eye, therefore, green is reflected.  Red absorbs all light and reflects red, which is what the observer can see looking at a red grape.  The test concluded that there was a slight difference between the red and the green grape.  Figure 5, indicates the results, that do not support our initial hypothesis. This may be due to the fact that we think the photosynthesis of grapes takes place within the leaves and not the actual grape. So the absorbance that was measured was only due to the color or the initial extract of each grape.
    The Action Spectrum of grape chloroplasts uses the Hill Reaction to determine the spectrum of photosynthesis.  It was used to test the efficiency of each color as a fuel source for photosynthetic reactions.  Our initial hypothesis was that red grapes with have a higher photosynthesis rate than the green grapes. We thought this would be correct due to the fact that all grapes start off green and then mature into red grapes.  A dark solution would indicate a high absorbency rate, since all the light is absorbed (red grape extract), and a low transmittance rate. Due to indophenol, the NADP reductase will no longer donate electrons to NADP and give them to the indophenol. In this process of accepting electrons the solution will become lighter as the reaction continues (Maleszewski et al., 2003).  A light solution would indicate a low absorbency rate (green grape extract), therefore, high transmittance.  However, these predictions and hypothesis were not supported by the results of our experiment.  The experiment concluded that both the green and the red grapes have equal efficiency in terms of photosynthetic reactions.  These results are illustrated in Fig.6 and Fig.5.     
  The final round of tests concentrated on the enzyme activity in red and green grapes.  The Environmental Effects of Enzyme Activity tested for the presence of PPO, the affect of heat inhibitors on the enzyme, and the affect of pH on the enzyme.  Our initial hypothesis was that both grapes contained PPO.  Polyphenoloxidase (PPO), is an enzyme that is found in plants usually responsible for browning of freshly peeled fruit or vegetables (Ross E. Koning, Feb. 3, 2003).  We found that both grapes contained PPO, since the inside of the grape turned brown in both cases when we added catechol as a substrate. The catechol makes the grape turn brown due to the fact that it reacts with PPO to make obenzoquinone (which makes the grape turn brown). The results can be seen in Fig. 7.  Following PPO we tested the temperatures at which the enzymes are still active.  We initially hypothesized that green grapes are more active than the red at all temperature levels (we thought this would occur since all grapes are initially green and then turn red), however we have found that prediction to be false after conducting the test.  Both grapes withheld the temperature equally; these results can be seen in Fig.8.  The final test in the enzyme sequence was the effect of pH.  We initially predicted that green grapes are more active than the red in all pH levels (again due to the fact that all grapes start off green and end up red).  However, once again we were proven wrong by the results of the experiment.  The results clearly indicated that both grapes were equally active in the pH levels ranging from pH of 5 to pH of 8.5.  Fig. 9 clearly illustrates the results.  After the tests were concluded several of our initial hypothesis were not supported by the data gathered.
    Our tests concluded that grapes contain monosaccharides ketones that require less digestion to be absorbed into the system. Monosaccharides occur naturally in foods such as fruits and honey (Cauldwell, 2003).  Those fundamental blocks provide both structure and taste to the element, making it needed for the human diet as well as easy to absorb. Since monosaccharides are monomers, they require less digestion and are absorbed shortly after they enter the small intestine (Cauldwell, 2003).  Seems like a small fact, however, that would make an enormous difference to a small child who needs all the nutrients he/she can get.
    Another benefit of grapes is found in something as unnoticed as its seeds. Grape seeds are a rich source of catechins and procyanidins, and they are included in red wine and grape juice.  The catechins and procyanidins are enzymes that are used as anitoxins. Based on our results both grapes contain enzymes, therefore both serve as antitoxins. These compounds act as antimutagenic and antiviral agents (Saito et al., 1998).  Phenolics in grapes and red wines have been reported to inhibit oxidation of human low-density lipoproteins (LDL) in vitro (Frankel et al, 1995)(Teissedre et al., 1996). Recognition of such health benefits of catechins and procyanidins has led to the use of grape seed extract as a dietary supplement (Laparra et al., 1979).                      
    There are a number of factors that could have contributed to incorrect data that was gathered during testing.  One of the biggest flaws with our experiment was the Pigment Identification test with the paper chromatography strip.  One of the reasons that no results were obtained could be due to human error.  The concentration of the solution could have been inefficient, or the different paper that was used for the experiment.  Another explanation for the lack of the results could be the fact that grapes themselves do not go through photosynthesis, but rather gather nutrients from the leaves and stems that transform CO2 into necessary products. 
Proper weighing of the specimens is always an issue at hand.  Improper dilution of the substrates could have greatly affected our results.  Figuring which grape had the presence of the PPO enzyme was a challenge.  Both grapes turned an undesirable color if left on the table, however red grapes would show it less due to its dark coloration, which does not mean that they have less of it, maybe in fact more than the green.     
    Testing our hypothesis allowed us to look beyond the color, taste, and texture of the grapes into its core carbohydrate structures, and the way photosynthesis carries out its light reactions.  The grapes were put through a series of tests that were described in detail in previous paragraphs.  To summarize our finding, it is easy to say that looking solely at the carbohydrate structure both grapes are very similar, even though they appear quite different on the surface.  Our initial hypothesis was incorrect when we predicted that the green and the red grapes would have major differences when looking at the photosynthesis and the observations from light reactions.  The data gathered from the experiments supported the fact that grapes very few subtle differences, but otherwise, they are very similar.   One subtle difference that we found from our data is that red grape absorbs more light, due to the fact that it reflects red and absorbs green which has a smaller wave lengths and greater absorption. In the action spectrum, the more light absorbed can be directly connected to the photosynthesis rate.  A young grape is white with a tint of green, however, as it grown and takes in the sunlight it becomes darker and richer in color, adding the nutrients to the taste.  In summary, our initial hypothesis was refuted by a collection of numerous data and gathered observations, however few differences were found between red and green grapes.