Sugar, action spectra, and protein/phosphate study of variants of Brassica oleracea yields similarity

By: Royce Grewer, Nicholas Lorenz, Ashley Richards, and April Roodbeen

The Green Mile

LBS 145

Section T1

Dr. Doug Luckie

02/28/2005

Abstract

Written by April Roodbeen, First Revision by Nick Lorenz, Final Revision by Royce Grewer

ÊÊÊÊÊÊÊÊÊÊÊ This investigation centered on a possible difference among nutritional values for three varieties of the Brassica oleracea, broccoli, cauliflower, and broccoflower. For the purposes of this study, a holistic approach to nutrition was not used, but instead each nutrient (starch, sugar, protein, and phosphate) was considered individually using Barfoedâs (carbohydrate), Iodine (starch) and Bradfordâs (protein) assays along with a phosphate test. For chlorophyll analysis, paper chromatography, action and absorption spectra were used. After performing Barfoedâs assay, it was determined that none of the samples contained monosaccharides.Ê As predicted, none of the specimens contained starch after performing the Iodine assay.Ê A series of t-tests suggested that there was no significant difference between the amounts of protein in any of the samples. Paper chromatography tests resulted in broccoli containing chlorophyll a and b and carotene pigments, broccoflower containing chlorophyll b, and cauliflower containing no pigments. The absorption spectrum shows that there was a high level of absorption for broccoli and a very low level for cauliflower. Broccoflowerâs absorbance level was in-between that of broccoli and cauliflower. A study of the action spectra of the three vegetables did not support evidence of a difference in the amount of photosynthesis performed by the flower portion of each variety. The phosphate analysis results, while suspect due to lack of robustness, support the conclusion that broccoflower lies in between broccoli and phosphate with respect to phosphate content, and that cooking vegetables tends to decrease their nutrient content.

Figure 11. Results of Bradford assay. The Bradford assay was used to test for total protein concentration by using Commassie brilliant blue G-250 dye (CBBG) which binds to certain proteins and produces different colors based on certain protein residues. The calibration curve has a positive slope, indicating that as protein concentration increases observed absorption also increases. The absorbences measured were fitted on the calibration curve to find the mass of protein in each sample.

Discussion

Written by Nicholas Lorenz, First Revision by Ashley Richards, Second Revision by Ashley Richards and Royce Grewer, Final Revision by April Roodbeen

Genetic combination of broccoli and cauliflower produces a vegetable known as broccoflower which obtains physical characteristics of both vegetable species (Anonymous-3, 2004).Ê Our research team sought out to determine if broccoflower is like its vegetable counterparts by testing nutritional content and photosynthetic properties.Ê We predicted that broccoflower is similar to both broccoli and cauliflower in nutritional content of sugar, starch and protein however; it is most like broccoli in photosynthetic properties.Ê We also predicted that in relevance to nutritional content, cooking the vegetables would decrease nutritional value, meaning less phosphate is present after cooking.Ê Our predictions were rigorously tested in laboratory to see if we could support or negate our hypothesis.

ÊÊÊÊÊÊÊÊÊÊÊ We predicted these results based on the analysis of tests that have been preformed and previous research that was conducted.Ê The first test that was conducted in order to show whether broccoli, broccoflower, and cauliflower have monosaccharides present was Barfoedâs.Ê We conducted Barfoedâs assay doing five repetitions with a positive control of galactose (a monosaccharide), a negative control of double distilled water, cauliflower, broccoflower, and broccoli. When the assay was completed each galactose showed a redish brown precipitate in the solution showing that monosaccharides were indeed present. However, the negative control of double distilled water, and our unknowns broccoli, cauliflower, and broccoflower showed no signs of a color change indicating that there were no monosaccharides present in any of the solutions. This seemed strange because previous research supports that broccoli contains carbohydrates in the form of simple sugars such as glucose and fructose (Esshaki et al., 2002). However, all five repetitions came out to be negative for monosaccharides leading us to conclude that broccoli does not contain monosacchrides like previously showed.

ÊÊÊÊÊÊÊÊÊÊÊ The next test that was conducted was the Iodine test, which tested for the presence of starches in the sampled vegetables.Ê According to the nutrients per serving, the three species of vegetables each contain zero grams of starches (Anonymous-7, Unknown).Ê The Iodine test for all three specimens resulted in no color change for the solutions supporting our prediction that broccoli, cauliflower, and broccoflower do not contain any starch.

ÊÊÊÊÊÊÊÊÊÊÊ In regards to nutrition as a whole thus far, broccoflower appeared to be very similar to both broccoli and cauliflower because none contained monosaccharides or starches. As far as the nutritional value is concerned foods containing monosaccharides tend to be less healthy then foods containing only starches because simple sugars contain unneeded calories, as previously mentioned (Anonymous-8, Unknown). Our results show that these vegetables do not contain either simple sugars or starches so, based on the definition of nutritious that pertains to our experiment; we can conclude that broccoli, cauliflower, and broccoflower are relatively similar in nutritional value of sugars and starches. Bradfordâs assay tells us more about protein content.

ÊÊÊÊÊÊÊÊÊÊÊ Bradfordâs assay was conducted to gather total protein concentration.Ê The assay is a dye-binding assay and it works by the action of Coomassie brilliant blue G-250 dye (Krha et al., 2005).Ê Previous testing found that broccoli, cauliflower and broccoflower show a decrease in total protein concentration respectively (Anonymous-7, Unknown).Ê

ÊÊÊÊÊÊÊÊÊÊÊ To analyze the data of Bradfordâs assay there were two choices: a Chi-square test for goodness of fit and a set of two-sample t-tests.Ê Although the Chi-square test could have assessed all the data at once for any difference among the averages, this test is not as sensitive to differences among just two of the three variables.Ê By inspection it appears that the averages for broccoli and cauliflower are very close together; this would prevent the chi-square test from detecting a difference between either of these two and broccoflower.Ê Thus a battery of t-tests was chosen instead because it allowed for independent consideration of each pair of averages in turn.Ê It should also be noted that a significance level of α = .05 was used.Ê

When broccoli and cauliflower were examined the p-value was p = .456.Ê When broccoli and broccoflower were examined the p-value was p = .519.Ê When broccoflower and cauliflower were examined the p-value was p = .223.Ê All of these p-values are much greater than significance level α=.05.Ê Therefore, none of the t-tests support the conclusion that there is a (statistically) significant difference among these three vegetables with respect to percent protein concentration by weight. This test ended our research on macromolecules and will begin our research on photosynthesis.

We continued our comparison by conducting different photosynthesis tests such as the paper chromatography, absorption spectrum, and the Hill reaction.Ê The pigment chlorophyll acts as the agent, which by absorbing light energy, mediates the photosynthetic process (Krha et al., 2005). The paper chromatography assay was used as a pigment identification test. Previous research showed that broccoli contained both chlorophyll a and b, xanthophyll and carotene pigments, while cauliflower contained carotene and xanthophyll pigments (Esshaki et al., 2002). However, after performing the assay, it was determined that cauliflower contained no pigments, as our strips for cauliflower showed no visible pigments. This may be due to the fact that the leaves of the cauliflower are the part of the plant that undergoes photosynthesis, not the curd (top part of the cauliflower) (Anonymous- 6, 2004). If the actual cauliflower part of the plant did not perform photosynthesis, there would be no chlorophyll pigments found in the curd (Anonymous- 6, 2004). Broccoli was found to contain chlorophyll a and chlorophyll b, and carotene (beta-carotene) pigments (Anonymous 9, Unknown). Broccoflower contained only the chlorophyll b pigment. This is logical because the chlorophyll pigments are common in the green vegetables (Anonymous 9, Unknown).Ê Previous research has shown that the R_f values in our particular solution for carotene, xanthophyll, and chlorophyll a and b should be approximately 1, 0.615, 0.340, 0.231 respectively (Anonymous-10, Unknown). For broccoli the average R_f value for chlorophyll a on the three strips was 0.37 which was fairly close to the known R_f value of 0.340 for chlorophyll a. A t-test was then applied and the p value was 0.112 which is greater than 0.05; this allowed us to support the conclusion that this pigment is chlorophyll a. However, more trials would strengthen support for our conclusion. The average R_f for all three strips of broccoli for chlorophyll b turned out to be 0.196 which is slightly lower than the known R_f for chlorophyll b, 0.231. When applying a t-test to these values the p value was 0.03 which is lower than 0.05, which, allows us to conclude that the pigment found is not chlorophyll b but maybe another pigment. This seems strange because based on othersâ research our prediction was that broccoli would contain chlorophyll b. However, more trials would be needed to make more accurate conclusions. In broccoli, carotene was found in two out of the three strips and both R_f values were 1, matching the known R_f value for carotene. Possible laboratory errors could have resulted in one of the strips not showing carotene while the other two did. Broccoflower just showed the pigment of chlorophyll b, which has a known R_f value of 0.231. When averaging out the three R_f values on these three strips it came out to be 0.308.Ê A t-test was also applied in this situation and the p value turned out to be 1, which is greater than significance level 0.05, allowing us to support the conclusion that the pigment found on the strips for broccoflower is chlorophyll b. Usually a p-value of 1 suggests that the data agree completely with the expected average; however, in this case the data {.287, .387, .250} have such spread that the validity of the t-test is questionable.Ê Thus, it is very important to have more trials.Ê This dataset is a good example of how small sets of data are not always appropriate for testing.Ê For the t-test to be considered robust and reliable, there must be at least 30 trials.Ê In all situations that were considered, more trials or more dotting might have revealed more pigments in the vegetables.

As our group predicted and previous research showed, when performing the absorption spectrum assay, cauliflower had a low absorbance of wavelength, possessing no chlorophyll, and causing its white color (Esshaki et al., 2002).Ê Broccoli is a dark green color and because it contains chlorophyll, previous research showed that it would have a high absorbance of wavelengths of blue and red compared to that of cauliflower (Esshaki et al., 2002). We predicted that broccoflower would have an absorbance of wavelengths somewhere between that of broccoli and cauliflower due to its light green color. Our results supported our predictions, with cauliflower having a very low absorbance level.Ê Broccoli had an overall high absorbance level, with a high peak in the blue absorbance area and a smaller peak in the red absorbance area. Broccoflowerâs absorbance level was similar to the shape of that of broccoli, although a decreased amount of light was absorbed, making absorbance levels in-between that of broccoli and cauliflower for all wavelengths measured.

ÊThe last photosynthesis test was the Hill reaction, which was tested under white light only, to determine the action spectrum of photosynthesis.Ê We predicted that cauliflower would not react in solution because of the absence of chlorophyll.Ê This is because if there is no chlorophyll, photolysis cannot occur and water will not split into its constituents (Krha et al., 2005). We predicted that broccoflower and broccoli would react under the same conditions, causing a color change in solution. Both contain chlorophyll and a hydrogen acceptor such that indophenol is reduced as photosynthesis occurs, converting it to a clear substance that causes the solution to become clearer as photosynthesis progresses.

According to the Hill Reaction, the lower the absorbance level, the less the indophenol that remains, indicating that more photosynthesis has occurred. After performing a series of t-tests, the t-test comparing broccoli and broccoflower yielded p-value=.15. This indicates that there is no statistical difference between the amounts of photosynthesis that has occurred in either of the samples, because the p-value is not less than significance level α=.05. A t-test between cauliflower and broccoflower resulted in a p=.053, and one between broccoli and cauliflower resulted in a p=.12. Both these values indicate that there is no significant statistical difference in the amount of photosynthesis that has occurred.Ê This result has many possible explanations. It could mean that because our samples of broccoli, broccoflower, and cauliflower were all dead, (that is not growing and attached to the plant anymore) no statistical difference in the amount of photosynthesis performed could be observed. It could also mean that the particular part of the vegetable we analyzed (the curd) did not undergo photosynthesis, but instead, another part of the vegetable did, eliminating the need for chlorophyll pigment to be present (Anonymous- 6, 2004). Yet another possibility is that instead of doing a limited amount of trials, as we did, if we had done more trials, our t-tests could have been more accurate and could possibly change our results. The photosynthesis tests aid in the support of our prediction that broccoflower is similar to broccoli and cauliflower in photosynthetic properties.

ÊÊÊÊÊÊÊÊÊÊÊ The phosphate test yielded the weakest support for our hypothesis that broccoflower is similar in nutritional content to broccoli and cauliflower.Ê Although the results indicate that broccoli had the highest concentration of phosphates (with broccoflower coming in second), this result is suspect because the method used to gain it is not robust.Ê Consider the calibration curve used to associate a particular phosphate concentration with each observed absorbance.Ê The calibration curve was constructed to confirm that the concentrations and absorbances measured would conform to Beerâs Law for concentrations, which states that there is a linearly positive relationship between concentration and absorbance.Ê Noting that the R² value is only .8697, we see that the data do not necessarily conform to this straight-line relationship; however, due to time constraints this relationship could not be further explored.Ê Furthermore, although Beerâs Law may apply within the domain of the curve (that section which has actual data to base itself on) any extrapolated data are automatically suspect, because it is not known for certain that the relationship postulated extends beyond the observed domain.Ê Therefore, the results extrapolated from the higher absorbances are not reliable and should be further investigated before being used to support our hypothesis.Ê Finally, only doing one trial of each sample is not nearly enough to constitute any statistical difference between samples.Ê At least 30 trials of each must be done before statistical methods could be considered robust enough to support any conclusion, for the effects of chance are too large below that threshold.Ê However, it should be noted that the phosphate concentrations in the cases of broccoli and broccoflower do decrease markedly after boiling, and this does support our hypothesis that cooking reduces the nutrient content of vegetables.

ÊÊÊÊÊÊÊÊÊÊÊ Other reasonable objections to our results could have been caused from human error in laboratory procedure.Ê Possible solution contamination caused from the extensive use of chemicals in lab from peers could cause an error in findings although our use of a positive control helped to decrease any risks of unknown contamination.Ê While performing the paper chromatography assay, we believe that more dots on the strips might have aided in the ability to see some of the pigments more clearly (like those of cauliflower), but due to time limitations, this was not possible. Another possible source of error could be a result of an insufficient number of trial repetitions, which might weaken support for our findings.

ÊÊÊÊÊÊÊÊÊÊÊ It is important to understand that, although broccoflower shares some of the nutritional qualities of both broccoli and cauliflower, this result only hints at the possible genetic proximity between broccoflower and broccoli and cauliflower.Ê It cannot be directly concluded from these results that broccoflower is genetically in between broccoli and cauliflower.Ê In order to better support that conclusion a new study must be done, one that directly compares the genetic information of the three vegetables: their DNA.

Another intriguing topic for future study could be a comparison or analysis of a new variety of cauliflower, known as orange cauliflower. What started out as an orange mutation of cauliflower was crossbred over generations with normal, white cauliflower to produce the orange cauliflower (Zakour, 2004). This vegetable is high in Vitamin A content (or beta- carotene), with twenty-five times the amount found in normal cauliflower (Zakour, 2004). It would be interesting to see further research conducted in this area in the future.