Chromatography, spectroscopy, and Bradford Assay show broccoli has higher structural complexity than cabbage

Written by Aimee Sutherland, Michael Susner, John Tyler Roasa, and Yasaman Osati

Abstract

This project was designed to examine the relative amounts of various chemicals (i.e. nutrients) – carbohydrates, proteins, and photosynthetic compounds – in species of genus Brassica. Brassica has undergone much genetic manipulation by humans, to expand its utility as a food and a spice (Clairmonte, 2003). Our endeavor was to determine how this manipulation has affected the presence of various nutrients in the plant. To determine this, we subjected samples of two different Brassica species (head cabbage and broccoli), as well as a control vegetable, spinach, which is outside the Brassica family, to several standard chemical tests (Krha, 2003). These tests included methods of determining the presence of different types of carbohydrates (sugars and starches), measuring the absorption spectrum of photosynthetic pigments, and gauging the concentration of proteins in the vegetables. These experiments were designed to test the validity of the hypothesis that further altered species of Brassica would have more complex and more numerous chemical compounds present. We therefore predicted that broccoli would be a more chemically complex plant than cabbage. The results we obtained from the carbohydrate tests were inconclusive, while the photosynthesis tests provided backup for our hypothesis. The protein test showed that broccoli did indeed contain more protein than the either cabbage or spinach. Overall the tests provided evidence to back up our original hypothesis that broccoli is more chemically complex than spinach or cabbage.

Discussion

This experiment was performed to observe whether many years of genetic modifications acting upon the genus Brassica have yielded any significant changes in the types or amounts of such qualities as carbohydrates, photosynthetic compounds, and proteins. Our group first tested the chemical differences between a species outside the genus Brassica, spinach, and two Brassica species- cabbage and broccoli. Our hypothesis was that a higher concentration of nutrients would have developed in a more manipulated species – namely, broccoli. We also expected that broccoli and cabbage would still be reasonably similar in composition, and used spinach as an extra-genus control. To test these ideas, we examined several types of chemicals responsible for nutrition that are present in plants: carbohydrates, photosynthetic compounds, and proteins. We first performed several experiments to examine the carbohydrate content of broccoli, cabbage, and spinach. Our prediction was that broccoli would have a higher concentration of more complex sugars and carbohydrates. After making solutions from the three vegetables by blending each separate vegetable with water (Figure 1), we first performed Benedict’s test, which demonstrates the presence of reducing sugars. A positive test gives reddish orange precipitate (copper that has been reduced). When tested, spinach became slightly cloudier, but had only a small amount of precipitate. Broccoli also became cloudier, to a higher degree than spinach, and turned a dark olive green color (the color the orange precipitate made when mixing with the blue-green broccoli solution). The cabbage, on the other hand, remained clear and blue (Figure 2, Tables 1 and 2). These results show that broccoli and spinach tested positive for reducing sugars, and that cabbage tested negative. Reducing sugars are potentially free aldehyde or ketone groups. More complex sugars have their carbonyl groups involved in the bonds between momomers; a large amount of free aldehydes or ketones indicates a high amount of lower complexity sugars. Benedict’s test failed to support our hypothesis, as cabbage, being the less developed species, should have had the highest concentration of the simple reducing sugars, compared to broccoli and spinach. Next we employed Barfoed’s test to distinguish monosaccharides from di- and polysaccharides. Barfoed’s will turn solutions with monosaccharides an orange red color, due to a copper precipitation reaction (this is similar to Benedict’s test). We had predicted a high monosaccharide content for cabbage. (Clairmonte, 2003). After completing the experiment, the results showed that spinach and broccoli became slightly cloudy and olive-colored, while cabbage stayed clear and blue (Figure 3, Tables 1 and 2). These outcomes contradicted our prediction. They indicate the relatively high concentrations of monosaccharides in broccoli and spinach compared to cabbage. However, this experiment does not tell us definitively which vegetable contains more polysaccharides, since a negative result for monosaccharide content does not necessarily conclude that there are polysaccharides present. Further testing would be needed to determine this for certain. Selivanoff’s test was next used to differentiate between ketoses and aldoses. The rate of color change to a pink or red color determines the nature of the carbohydrates present: a rapid change (less than a minute in duration) to red indicates the presence of monosaccharide ketoses; a change to red that takes about one minute indicates disaccharide ketoses; and a change to red after one minute indicates aldoses. We predicted that the broccoli would yield the shortest time, and have the darkest color change. This would be because broccoli would contain the most varied amount of sugars, some of which would be expected to be monosaccharide ketoses that would react quickly. The dark color change would be due to the fact that broccoli would contain so many sugars – the different forms would continue to react long after the first monosaccharide ketose had turned red. After doing the test we discovered that spinach showed no color change, while cabbage turned a light red by two minutes, and broccoli turned dark red by one minute (Figure 4, Tables 1 and 2). This experiment confirmed our hypothesis: while, due to the length of time it took to change, broccoli apparently does not contain any monosaccharide ketoses, it does have at least disaccharide ketoses for certain. It may be speculated that it contains some amount of aldose, as well, because of its extremely dark red color. Cabbage, on the other hand, can only be said to contain aldoses. Spinach, having no color change, very probably has either an extremely low sugar content, or a high ratio of polysaccharide sugars that take too long to react to show results in the time observed. Bial’s test checks for a furanose ring in the sugars present. If the vegetable contains sugars that have a furanose ring, the solution will turn green. If the sugars are pentose furanoses, the solution will turn green; and hexose or higher furanose will turn the solution a muddy brown. Also, if the sugar is a pyranose there will be no change in color. In our experiment, both spinach and cabbage turned an olive shade of green while broccoli became much darker and greener (Figure 5, Tables 1 and 2). This indicates that spinach and cabbage contain more hexose furanoses (olive being the color change that should happen when a brown color change happens in an already pale green solution). Broccoli, on the other hand, contains more pentose furanoses. This neither supports nor confirms our hypothesis: fructose and xylose, two important sugars for nutrition, are a hexose furanose and a pentose furanose, respectively. We cannot be sure that the results we found positively indicate the presence of those certain sugars, though. The final test for organic compounds was the iodine test for coiled polysaccharides, which distinguishes starch from monosaccharides, disaccharides, and other polysaccharides. If starch is present, the solution will turn a blue-black color. We predicted that broccoli would turn the darkest color because of its presumed higher carbohydrate content. However, none of the vegetables gave a positive result for the iodine test (Figure 6, Tables 1 and 2). This is probably due to the fact that most plants store glucose as cellulose rather than starch. The next battery of tests performed was tailored to the analysis of photosynthetic compounds and light dependent reactions. We used a standard thin layer chromatography analysis to see the differences in these compounds. In a thin layer chromatography, the bands form that indicate the presences of different pigments: since each different type has a different molecular weight and solubility, they all rise to different points on the paper they are tested on. We predicted that the two Brassica species would have similar pigments, but that broccoli would have a larger amount of pigments – both with respect to number and concentration. We also expected that spinach would have a very distinct green band, due to its very dark green color. As it turned out, yellow green was the darkest, and in one trial the only, color band for spinach (Figure 7, Table 4). Broccoli also consistently displayed more color bands than cabbage. This is to be expected; since broccoli has been more developed by humans, part of that development might involve having a wider variation in pigments to permit it to grow more widely with regard to available sunlight and warmth. Another test involved measuring the absorption spectrum of the vegetable chloroplasts. We inserted chlorophyll samples of all three vegetables into separate cuvettes, placed them in a spectrophotometer, and measured each vegetable’s absorption at fifteen nm intervals between 400 and 700 nm. We predicted that spinach would have a higher absorption peaks at 685 and 700 nm, since it is the darkest of the three vegetables, and more pigmentation causes higher absorption. It was also expected that broccoli would have higher peaks than cabbage, due to its darker green coloration. This was in fact very nearly the case (Figure 8): spinach has very distinct absorption peaks at 450 and 675 nm. These correspond to the areas where green pigments and orange / yellow pigments absorb light. Neither broccoli nor cabbage displayed very high absorption peaks, but broccoli consistently showed a higher absorbance than cabbage. This is in line with our prediction, and supports our hypothesis for the same reasons as the pigment identification experiment: a larger amount of pigments maximize the plant’s use of light and enables it to grow more easily in various conditions. The last photosynthesis-related experiment was the action spectrum of chloroplasts to test for the ideal wavelength of light that maximizes photosynthesis for each vegetable. For this we filled cuvettes with vegetable extract, indicator, phosphate buffer, and water. Then a cuvette containing each extract was subjected to red, white, blue, or no light. The absorptions were then read in a spectrophotometer. We predicted that broccoli would have a larger absorbance, indicating a higher rate of photosynthesis, than cabbage in most of the light types. This was not the case: cabbage turned out to have the highest absorbance of the three vegetables in white light and blue light, while broccoli had the highest in red (Table 5). However, cabbage also showed an extremely high absorbance when exposed to no light – the highest of any of its trials – so the cabbage experiment may not have been correctly administered. Assuming that that it is more blue in color. This corresponds to the observation in the pigment identification experiment that broccoli has a more significant blue-green band. However, it is worth noting that we observed highest amount of photosynthesis for cabbage in the zero light trial, so there may have been some error in this experiment. The final experiment performed was a Bradford Assay to determine the quantity of proteins in each vegetable. This experiment called for each vegetable solution to be mixed with NaOH, ddH2O, filtered, and introduced to Bradford reagent. The absorbance of the solutions were then taken at 595 nm and compared to a standard protein concentration curve (Figure 9). We predicted that the broccoli would have a higher quantity of proteins, since it has been genetically altered to be a food source. As it turned out, the average concentration of protein in broccoli was .978 ìg/ìl, compared to .968 ìg/ìl in cabbage (Table 3). This is to be expected: a food designed to be a better food source should have a higher protein concentration. The experiments performed provided some support for our hypothesis, but also raised several questions. The carbohydrate tests generally failed to provide support; however they also cannot be said to definitively refute our hypothesis. Further tests would need to be done to determine the cellulose, polysaccharide, and non-reducing sugar contents of the vegetables, or the exact nature of the furanose rings present in cabbage and broccoli. And while it may be argued that broccoli, containing more reducing sugars than cabbage, is therefore less chemically complex, it should be considered that a high concentration of reducing sugars does not necessarily conclude a low concentration of non-reducing sugars. The photosynthesis experiments provided some better support for our hypothesis. A food modified by man to be a better nutrient source should also be designed to thrive in diverse environments – wherever man may need to take it, grow it, and consume it. The numbers and concentrations of pigments in broccoli seem to show that it is better adapted than cabbage to grow in different types of environments. It is true that there may be other factors – soil types, for example – that cancel out the differences between broccoli and cabbage and equip them equally to grow in different environments. More research would be needed to determine this, but based only on light availability, we conclude that broccoli is the best adapted plant. The protein test was the most supportive of our hypothesis. Broccoli definitely had a higher concentration of proteins, and proteins are a crucial nutritional element, needed for building healthy cells, or recycled for use in the consumer’s own body. A better food would probably include more proteins, and this is what our results showed. It was also helpful to use an extra-genus species (spinach) as a control for our tests. Spinach fairly consistently gave us quite different results compared to broccoli and cabbage, which were often similar. This information helped to verify the validity of our tests; a vegetable from an entirely different genus should tend to have quite a different makeup than those species in the genus. Our results showed this to be true, which helps to prove that the tests were being administered correctly. Overall, our hypothesis gained some helpful support. Further testing, as mentioned above, would help to eliminate any remaining issues and confirm the hypothesis. While our experiments did not give us the perfect results, we conclude that, overall, our hypothesis that broccoli is a more chemically complex and nutritionally useful vegetable is true.