The Higher Dietary Value of Uncooked vs. Cooked Brassica Olecacea
based on Carbohydrate, Pigment and Enzyme Tests

by Amanda Bale, Kristen Burgess;
David Heimberg, Ashudee Kirk;
 Rachel Parry

       Broccoli is well known for its nutritional value, but does the cooking process remove some of the nutrition?  Using a series of experiments, we tested if there was a difference in the presence of carbohydrates, such as glucose, sucrose and starch, in cooked broccoli as compared to uncooked broccoli.  The tests we used for carbohydrates were Barfoed’s, Benedict’s, Selivanoff’s and an Iodine test.  We also tested for a reduction in certain photosynthetic pigments in the uncooked and cooked broccoli. Using paper chromatography we were able to test for the rate flow of these pigments.  Finally, we tested for the extent of enzyme activity in the cooked and uncooked broccoli.  More specifically, we were looking for the enzyme polyphenoloxidase (PPO).  We found that the majority of carbohydrates were reduced in the cooked broccoli in contrast with the uncooked broccoli.  We also found that cooking caused a loss in the amounts of certain photosynthetic pigments including chlorophyll a, chlorophyll b, and carotene.  The activity of the enzyme PPO was observed as lower in the cooked broccoli than in the uncooked broccoli as well.  These findings support the idea that the cooking process can cause a reduction in nutrients contained in broccoli, therefore reducing its nutritional value. 

Discussion:

           We begun with a question:  would fresh broccoli contain more nutritional properties as compared to cooked broccoli, more specifically would uncooked broccoli contain higher amounts of carbohydrates, photosynthetic pigments, and enzymes than cooked broccoli?  Remembering what our mothers told us about how cooking drains foods, we thought that there would be a significant difference.  We began by testing for certain carbohydrates: reducing sugars, monosaccharides, ketoses and aldoses, and starch (Table 1). 

            To test for reducing sugars, we ran the Benedict's Test.  This test determines whether the carbohydrate in question contains a free aldehyde or ketone group.  After boiling the Benedict's reagent and broccoli solutions, we found that the uncooked broccoli contained a yellow precipitate, which was unexplainable in comparison to our controls (Figure 1). The xylose control does have a slight yellow to brown precipitate which may be an indication that xylose may be but no direct correlation was made. The possible reason for this precipitate could have been an error made by us with either incorrect measurements or just not correctly and efficiently removing the entire solid broccoli from our solutions. The cooked broccoli had an orange red precipitate present, which means that the aldehyde or ketone group present in a carbohydrate in this solution was being oxidized. This is an indication of the presence of reducing sugars.

            To distinguish monosaccharides from di- or polysaccharides in the broccoli samples, we used the Barfoed's test.  In this test, a rusty orange red precipitate would show monosaccharide presence, and no precipitate present indicates di- and polysaccharides.  After 2 minutes in the boiling water bath, the tubes containing the uncooked broccoli contained both a red precipitate and a green precipitate, indicating that monosaccharides were present. The green precipitate also leads back to the theory that the solid broccoli was not correctly and efficiently removed from our solutions. This occurred in all three of our uncooked trials. The tube containing the cooked broccoli contained no precipitate (Figure 2). This indicates that no monosaccharides were detected within the cooked broccoli solution. We believe that this occurred because the monosaccharides present in the fresh broccoli diffused into the water while the broccoli was boiled. This lowered the amount of monosaccharides to the extent that they were undetectable by the Barfoed’s test.  This further supports our prediction that the cooking of broccoli reduces its nutritional value.

            In the Selivanoff's Test, a reagent of resorcinol in hydrochloric acid (HCl) is used to distinguish between aldoses and ketoses in carbohydrate chains.  When heated, the acid and any sugar in the broccoli will turn a red color.  A quick change in color indicates aldoses, and a slow change indicates ketoses.  The uncooked and the cooked broccoli both turned quickly indicating that both types contained a significant amount of aldoses. This does not indicate directly that there are no ketoses present but that they were undetected by our studies (Figure 3).

            The next test used was to distinguish the presence of starches from the presence of mono-, di-, and other polysaccharides.  This experiment is done using the iodine test.  Iodine was placed in separate test tubes with our solution of uncooked and cooked broccoli.  Neither broccoli types displayed any color change indicating that there was no detectable starch within these solutions (Figure 4).

            Subsequently to testing different carbohydrates, we tested for the photosynthetic pigments of the cooked and uncooked broccoli.  First, we did paper chromatography on the samples of both cooked and uncooked broccoli. We prepared chromatography strips and then dipped them into an acetone solution for 15 minutes.  The uncooked broccoli displayed the colors pale green and blue-green in that order on the paper chromatography strips. The cooked broccoli had no display of colors and therefore an unobservable rate of flow. These results were contained in all three trials of the plant. The displayed colors of pale green and blue-green had similar R_f values compared to our controls of the pigments of a spinach chloroplast (Table 3). This suggests that there is a presence of the pigments chlorophyll a and chlorophyll b which were the known pigments in the spinach chloroplast. The rate of flow for the three different trials was very similar in value in trials number two and three but the first trial was slower. This could be a factor of differing concentrations.  The lack of a rate of flow may indicate a smaller concentration of pigments in cooked versus uncooked broccoli.  

            Following that, we tested the absorption spectrum of both broccoli types by inserting a sample of broccoli solution (two tubes for each type separately) into the spectrophotometer. The solutions were tested from wavelengths 400- 700 nm at intervals of 15 nm, with the highest absorption occurring at 400 nm (Table 2).  The absorption steadily decreased as the wavelength increased.  The different absorptions were placed in a graph to show the comparison of the cooked and uncooked (Figure 5).

In the last experiments we conducted, we examined the presence of active enzymes in the broccoli types.  In exploring the presence of PPO we discovered that there was no indication of the presence of PPO in the uncooked or the cooked broccoli.  We tested this using two different methods both coming out negative. After this we were still inquiring if PPO was present in our broccoli. So we conferred with another lab group testing broccoli to determine if they had gotten the same results, and they also found no detectable PPO.

In accordance with enzymes, an enzyme contained within uncooked broccoli stops broccoli from treating people with BPH (prostate enlargement), urinary tract infections, or prostatitis. This enzyme must be deactivated in order for broccoli to treat these ailments (Saracoglu, 2000). This study was done by The Prostate Foundation. In this study they suggest that eating broccoli that has been boiled for just five minutes is more nutritional than that of the uncooked broccoli (Saracoglu, 2000). These findings demonstrate that there are advantages to eating cooked broccoli as well as fresh, but for the typical human being, the nutritional value of fresh broccoli seems to be greater.  But maybe a later study that could be done is the greater nutritional value of carbohydrates in uncooked broccoli versus the greater nutritional value in enzyme activity of cooked broccoli (Saracoglu, 2000).

            Looking at the three categories of tests conducted (carbohydrate distinction, photosynthetic pigment recognition, and enzyme action); our results indicated that there were higher amounts of photosynthetic pigments and the tested carbohydrates in the uncooked broccoli.  This indicates to us that on a basis of the results of our test uncooked broccoli is of greater nutritional value than cooked broccoli. But our lab experiments were not definite and did have room for error. There were the obvious error mentioned previously that could be measurements and though our broccoli was purchased at the same store the same distance apart they are not directly from the same stock and this could cause error as well.  Further investigations must be done to find the extent to which cooking causes carbohydrate and pigment loss.  Tests must also be done to determine the effect of cooking on other enzymes

Collective data comparison of the carbohydrate tests performed on both the raw and cooked broccoli solution samples.