Discovered Using

Carbohydratic, Photosynthetic and Enzymatic Methods

By:

Katherine Kruse, Andrew McCoy,

Jacquelyn Ormiston, and Nicole Vanderstow

For - LBS 145 Fall 2002

Douglas B. Luckie, Ph.D.

Abstract

There are many differences inherent in black and green grapes; differences in sugars, enzymes, and pigment makeup. In the

present work, we used a series of tests to determine the differences between these two similar but distinctive types of fruit.

Through the use of carbohydrate identification procedures, it was determined that both grapes may contain, among other sugars:

free aldehyde or ketone groups, a monosaccharide ketose, and either a pentose or a furanose. Through chromatography tests,

pigments associated with each grape were found to be different; according to our experiments, black grapes probably contain

chlorophyll b. No other information can be discerned from the experimentally obtained data.

It was further discerned through spectrophotometry that the absorption spectra and action spectra were different for each

grape. Through investigation of the enzymatic activity in each grape it was found that both fruits contained PPO. It was further

revealed that PPO as found in these grapes was effected by the pH of its buffer. These findings reveal that there are many

similarities as well as differences between black and green grapes. These differences can begin to explain the variation in their

taste as well as their color.

Figures

Figure 1 – Selivanoff’s Test This test checked for the presence of ketose groups or aldose groups. When Selivanoff’s solution is added to the sample along with heat, a reaction will occur if either group is present. If monosaccharide ketoses are present, the sample will react in less than a minute. If the reaction time is around one minute, disaccharide ketoses are present. If the reaction takes more than a minute, aldoses are present. A shows the solutions without heat. B shows the reaction after 15 seconds of heat. C shows the reaction after 30 seconds of heat. D shows the reaction after 45 seconds of heat. E shows the reaction after one minute of heat. F shows the reaction after one minute and 15 seconds of heat. H shows the reaction after one minute and 30 seconds of heat. I shows the reaction after all heating had occurred and the changes had fully completed.

Discussion

While not every test works is every experiment, carbohydrate tests and grapes seem to get along quite well. Initial predictions

were that both kinds of grapes would react in ways that were somewhat different when put through several standard

carbohydrate tests. These predictions did not entirely hold true, even though there was a slight difference in the results of some

of the tests. Taste tests suggest a testable difference between the two grapes, which was not completely shown by our results.

This is because the tests we performed were qualitative, not quantitative. They showed that both grapes contained the same

sugars, not how much of each sugar each grape contained. The results of Benedict’s test, for instance, showed that green

grapes had a darker color than black grapes. These differences, while small, are the first step in differentiating between black

and green grapes.

Both solutions reacted in a positive manner in the Benedict’s test, indicating that there are free aldehyde or ketone groups

present. The green grapes, however, reacted slightly more than the black grapes. This darker solution was very comparable to

the positive control, which in this case was fructose. It may be inferred from these results that the levels of monosaccharides are

higher in the green grapes than in the black grapes. There was no reaction in the negative control, as expected.

Both grape solutions also went through color changes in their solutions in Barfoed’s test. Both solutions produced a precipitate

when mixed with Barfoed’s solution, although the black grapes produced slightly more precipitate. These results lead to the

conclusion that monosaccharides were present in both types of grapes, but there may be slightly more monosaccharides present

in the black variety. This means that at least one or both of the monosaccharides fructose and glucose may have been present in

the solutions.

This same glucose and fructose in the grapes may have caused the solutions to turn red in Selivanoff’s test, since fructose and

glucose are monosaccharides. Both kinds of grapes took less than a minute to react, indicating that there is a monosaccharide

ketose present. While other sugars more than likely are present, it cannot be reported that they are present based upon the

given data. Perhaps in the absence of monosaccharide ketoses, the reaction would have occurred over a longer period of time,

indicating either disaccharide ketoses or aldoses. Since monosaccharide ketoses were present, the reaction occurred quickly,

and all that can be accurately discussed is the presence of those ketoses.

The black grapes reacted slightly faster, therefore it is possible to infer the presence of a higher monosaccharide to disaccharide

ratio in the black grapes than in that of the green grapes. This difference, although tangible, is not extreme. It can be inferred

from this data that both types of grapes presumable have close to a 1:1 monosaccharide: disaccharide ratio.

We encountered positive reactions in Bial’s test, with all six grape test tubes turning olive-green. This shows that furanoses are

present. This does not mean that there aren’t six-membered rings and others, it only means the five-membered rings are present.

We can infer that hexose furanose rings are present because of the olive-green solution left after the reaction.

We believe that a more obvious difference in the sugar contents of each type of grape could be found using more sensitive

techniques. These techniques would involve some sort of purification of the grape solution using distilling or other apparatus.

This would allow each sugar to be separated and then its relative concentration and strength calculated. This would allow us to

exactly pinpoint which sugars are present as well as determine at what concentration they are present. This would have allowed

us to account for more of the flavor difference between the two types of grapes.

The grapes have visibly different colors, mainly due to the pigments found in each type. Utilizing paper

chromatographytechniques involving a petroleum ether and chloroform solvent, we found that green grapes had no identifiable

pigments using this test. Black grapes, however, had a pigment with an approximate Rf of .324 present on both of the

chromatography strips. It was previously discovered in LBS 145 lab that Chlorophyll b’s Rf is approximately .309. It is

therefore logical to infer that the pigment found in black grapes might be Chlorophyll b. Other pigments are more than likely

present in both types of grapes. Using our techniques, however, the only pigment that we possibly identified was Chlorophyll b.

Using more sensitive techniques, with different solvents, it might be possible to discover the presence of the other pigments that

are likely present in the grapes.

When the chloroplasts were removed from the grapes, their absorption spectra were relatively easy to find. Green grapes and

black grapes had the expected difference in absorption spectra, as their colors differ it was expected that they would absorb

different wavelengths of light. Our data found that the shortest wavelengths, corresponding to the portion of the visible spectrum

made up of purple and blue light were absorbed the most by both green and black grapes. This is somewhat counterintuitive,

since the color of a fruit is the light it is reflecting, one would conjecture that black grapes would absorb the least amount of light

on the darker end of the spectrum. This is not the case according to our data. Also, one could deduce that green grapes should

have a low absorbance near the green portion of the spectrum. This corresponds to an area near 550 nm. This was not the

case, as this was simply a midpoint in the absorption spectrum of green grapes.

There was a presence of PPO in both kinds of grapes, which had a pH of approximately 6.5. This was expected since the

enzyme polyphenoloxidase is responsible for catalyzing the oxidation of organic compounds found in the grapes. Inhibitors

change the active site and keep the enzyme from acting with its specific substrate. This was observed in our experiments. The

PPO solution in the tubes did not react in the presence of an inhibitor. High temperatures were also found to inhibit the enzyme.

The tubes that were heated showed no reaction when catechol was added to the tube. The results for this section of the tests

were essentially the same, with the same tubes reacting or not reacting, for both green and black grapes.

The reaction rate, as expected, changed with the pH of the solution. The optimum pH for the enzymatic activity of PPO is near

7.0 for green grapes, and near 7.5 for black grapes. This difference is almost negligible and could probably be discounted using

more sensitive techniques.

There was a presence of PPO in both kinds of grapes, which was expected since this enzyme is responsible for catalyzing the

oxidation of organic compounds found in the grapes. The reaction rate, as expected, chanced both with the pH of the solution

and with the concentration of Catechol and PPO in solution. The optimum pH for the enzymatic activity of PPO is near 7.0. It

was also discovered that the optimum concentration is around 1 part catechol to 3 parts dH20.

These findings begin to account for the differences between black and green grapes. While they may look very similar, they are

in fact different in several ways. These differences are key in the marketing and sales of these grapes. Some people prefer

sweeter grapes. While these people may not really care what sugars are present in their grapes or why these grapes are the

color that they are, these things are important to scientists that could be trying to develop a genetically altered, better grape. If

society as a whole enjoys green grapes for their color and black grapes for their sweetness, perhaps it is scientifically feasible to

combine these two great attributes into one product that would sell better that the two separate products combined.

There are differences between the grapes. Using our methods, we have discovered some small, minor differences. These

differences could be further studied using more sensitive and detailed methods and technologies. This would prove interesting as

it would be quite simple to challenge our findings with work involving more complicated method and using much more sensitive

equipment. It would be intriguing to find out which of our experiments was correct and which was incorrect simply because we

could not study in deep enough detail.