Carbohydrate Structure and Protein, Pigment,
and Vitamin C Concentration Analysis in Organic and Conventional Baby Spinach
By
W. Bush, D. Fasseel, R. Martin, and K. Strehl
Cell and
Molecular Biology
Tuesday Section 3
Dr. Fata-Hartley
February 28, 2006
Abstract
Written by Wesley Bush, Revised by Danielle Fasseel and ReneŽ Martin
ÊÊÊÊÊÊÊÊÊÊÊ A commonly debated topic today is whether organic or conventionally grown vegetables are ãhealthier,ä in respects to concentrations of proteins, pigments, and vitamin C.Ê Organic and conventionally grown baby spinach, Spinacia oleracea, were chosen to investigate this discrepancy.Ê Before the experiments were conducted, it was hypothesized that despite a similar overall basic structure and chemical composition the organic baby spinach would have a lower protein concentration, but higher chlorophyll a and b, and vitamin C concentrations than conventional spinach.Ê Barfoedâs test, Selivanoffâs test, and the Iodine test were used to characterize the carbohydrates of each sample, while a pigmentation test was used to test for the presence of the pigments beta-carotene and xanthophyll.Ê Although these tests were not predicted to provide any qualitative differences, they were used to show that the organic and conventional samples are similar in respects to basic structure and chemical makeup.Ê Each sample was prepared by blending and straining to extract a useable solution.ÊÊ After performingÊÊ a Barfoedâs test and a Selivanoffâs test both spinach samples tested positive for monosaccharides and ketoses.Ê They also tested negative for coiled polysaccharides (starch) using an Iodine test.Ê This supported our predictions that both organic and conventional spinach would contain the same in these aspects.Ê It was concluded after conducting a Bradford Assay that the concentrations of protein within each sample was approximately equivalent (Organic was 1.567mg/ml and the conventional was 1.5276mg.ml).Ê After the photosynthesis test was administered, the presence of chlorophyll a and chlorophyll b was more evident in the organic spinach thus supporting our prediction of organic spinach containing more pigmentation.Ê The mass of vitamin C was greater in the organic baby spinach which was concluded by using a vitamin C titration which supported our prediction that organic spinach would contain more vitamin C.
ÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ ÊÊÊÊÊÊÊÊÊÊÊÊ (a) ÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊÊ ÊÊÊÊÊÊÊÊÊÊ(b)
Figure 6. Results of TLC Photosynthesis Test.Ê This test was performed to determine which pigments each spinach sample contained.Ê After the pigments were identified, and the Rf values were calculated from the solvent front and pigment distances.Ê Sample (a) conventional spinach and (b) organic spinach.Ê Both samples contained a: beta-carotene, b: chlorophyll a, c: chlorophyll b, and d,e, and f: xanthophyll (each sample was replicated three times).ÊÊ
Discussion
Written by Danielle Fasseel, Revised by Kristen Strehl
ÊÊÊÊÊÊÊÊÊÊÊ To answer the commonly asked question of whether organic or conventionally grown vegetables are ãhealthier,ä in terms of protein, pigment, and vitamin C concentration, several tests were conducted. These tests included Barfoedâs Test, Selivanoffâs Test, an Iodine Test, a Bradford Assay, a thin-layer chromatography pigmentation test, and a titration test for vitamin C. Before the testing commenced it was hypothesized that the structural and chemical makeup, pertaining to monosaccharides, carbohydrate ketoses or aldoses, starch, and the pigments b-carotene and xanthophyll of both types of spinach would be the same. On the other hand, it was hypothesized that the organic baby spinach would have a lower protein concentration, but higher pigment concentration, in respects to chlorophyll a and b, and higher vitamin C concentrations than organic spinach.
ÊÊÊÊÊÊÊÊÊÊÊ These predictions were made on the
basis that different growing conditions for organic and conventional baby
spinach could change the rate of production of essential compounds, like
proteins and carbohydrates. When a plant is raised under conventional
conditions it is typically subject to an increase in protein production, this
in turn, decreases the carbohydrate production, which therefore lowers pigment
and vitamin synthesis (
ÊÊÊÊÊÊÊÊÊÊÊ After performing the experiments the carbohydrate tests supported the prediction that both samples of spinach had the same chemical make-up.Ê Barfoedâs Test, Selivanoffâs Test, and the Iodine Test concluded that both spinach samples contained monosaccharides, were ketoses, and that they did not contain starch.Ê It was concluded from the Bradford Assay that the organic spinach samples had more protein, but this amount was so minute that there was not a significant difference between the two protein concentrations.Ê The protein concentration of the organic spinach was 1.5687mg and the protein concentration of the conventional spinach was 1.5276mg.Ê The concentrations of chlorophyll a and chlorophyll b were greater in the organic spinach samples compared to the conventional spinach samples through the use of a thin-layer chromatography test. For chlorophyll a the organic spinach averaged 17.973mg/mL, while the conventional spinach averaged 16.148 and for chlorophyll b the organic spinach averaged 12.924mg/mL, while the conventional spinach averaged 7.0897mg/mL.Ê A titration of vitamin C for the two spinach samples established that there was an increased concentration of vitamin C in organic spinach compared to conventional spinach, .0595g compared to .0467g.
ÊÊÊÊÊÊÊÊÊÊÊ The data obtained in these experiments both supported and refuted the predictions made previous to testing.Ê It was predicted that the organic and conventional spinach samples would contain similar structural and chemical makeup, concerning monosaccharides, carbohydrate ketoses or aldoses, starch, and the pigments b-carotene and xanthophylls, which was confirmed by the results found from the experiments.Ê The protein concentration of the conventional spinach was approximately equal to that of organic spinach, unlike the prediction that the conventional spinach would have more protein.Ê Concentrations of both chlorophyll a and chlorophyll b, were higher in the organic spinach samples compared to the conventional, as predicted. Also as predicted, the concentrations of vitamin C in the organic spinach samples were higher than the conventional spinach samples.Ê
It can be concluded that the application of pesticides and fertilizers do not affect the chemical processes or composition of plants, since the composition of the carbohydrates was the same for both organic and conventional spinach. This is because fertilizers only affect the inner processes of plants by varying the amounts of essential minerals, which only changes the concentrations of compounds produced, not the structures of or mechanisms using these compounds. In other experiments performed only the concentrations of compounds produced in plants or minerals absorbed by plants are tested to find the differences between organically grown plants and conventionally grown plants (Brandt and Molgaard, 2001). The reason for this could be because it is commonly known by experiments on fertilizers that nothing else is affected. The only way to unnaturally change a plants chemical make-up is by genetic engineering, which was not done on the samples tested. ÊIf there was a natural change the plant would potentially become a different species.Ê Ê
Reasons
for the protein concentrations being insignificantly different in the organic
and conventional spinach samples could have been due to environmental factors,
such as soil quality and light availability. Normally with the increase of the
availability of plant nutrients, primarily nitrogen, plants are stimulated to
either increase or decrease the production of compounds that contain nitrogen,
such as protein, and compounds that do not contain nitrogen, such as carbohydrates,
pigments, and vitamins (Brandt and Molgaard, 2001). This
means that the conventional spinach should theoretically contain more protein
than the organic spinach due to the application of nitrogen containing
fertilizers. However, this was proven false by the Bradford Assay conducted.
One reason this may not have been true is because the light and soil conditions
of the samples used were unknown. Plants grown under high levels of light are
usually found to have a decreased level of protein (
On the other hand, it can not be concluded whether the essential amino acid content is the same in both spinach samples.Ê In tests on spinach it was found that when crude protein levels and free amino acid levels are higher due to the increased availability of nitrogen the concentrations of essential amino acids, such as lysine, decrease (Magkos et al., 2003).Ê This increased free amino acid level may possibly be due to the plantâs metabolic focus being on making simple proteins, such as crude proteins, instead of taking the time to manufacture more complex proteins, such as essential amino acids.
ÊÊÊÊÊÊÊÊÊÊÊ Some potential reasons for the differences in concentrations of pigments, primarily in chlorophyll a and b, and vitamin C in organic and conventional spinach could be environmental factors.Ê Oxidative stress due to the lack of essential nutrients, primarily nitrogen, increases the production of pigments.Ê When nitrogen is a limiting factor in plant growth, there is an increase in carbon containing compounds, like carbohydrates, which makeup some pigments, such as chlorophyll a and b, beta-carotene and xanthophyll (Brandt and Molgaard, 2001).Ê Again the factors of light and soil quality can decrease or increase the protein concentrations in either sample.Ê
ÊÊÊÊÊÊÊÊÊÊÊ The belief that organic spinach is ãhealthierä than conventional spinach can only be supported by the data obtained in the vitamin C test and the pigment test, since the concentrations of vitamin C and the pigments chlorophyll a and b were found to be at increased levels compared to the concentrations in conventional spinach.Ê Vitaminsâ antioxidant qualities have been reported to contribute to the general health of a person and decrease the risk of chronic diseases (Magkos et al., 2003). With increased vitamin concentrations in organic spinach compared to conventional, it can be argued that organic spinach is healthier to consume then conventional spinach on the basis that organic spinach has more vitamin C and pigments (chlorophyll a and b). It is significant that the organic spinach contained more pigments because many pigments are the precursors to vitamins, such as vitamin C (Magkos et al., 2003).Ê
ÊÊÊÊÊÊÊÊÊÊÊ The results obtained from the experiments conducted might have possibly been skewed due to slight human and mechanical error.Ê Potential error could have occurred in the Bradford Assay because parts of this test had time restrictions that if were not followed affected the concentrations of protein found. In the first trial of this test the data obtained was incorrect because the spinach samples were not diluted enough prior to adding the BSA solution, so when their absorbance was found it was beyond the absorbencies of the standard curve. Another problem encountered was that the spectrometers used gave inconsistent absorbencies if the cuvettes were not placed in the exact position each time.
The TLC test had to be repeated four times before credible results could be obtained. ÊThe reason for the error in the first trial was that the test would not work if the spinach extract was prepared with distilled water, since pigments are hydrophobic and cannot separate in a water based dilution. Therefore, an acetone dilution was used in the later tests in place of water. Human error was the cause for the failure of the second trial when the cuvettes were knocked over while their absorbencies were being read, which led to the loss of the samples, since there was not enough pigment solution left in the cuvettes to test. From this mishap precaution was learned, which led to the placing of the cuvettes into styrofoam holders. The data from this test could not have been used anyway, since only two repetitions were performed, instead of the standard three tests.Ê In the third trial the TLC strips were cut after applying the spinach solutions, which had an adverse affect on the separation of the pigments, where pigments were blending and appearing more than once. From this test it was learned that it was better to do extra repetitions in case of human error when the applying of the spinach solutions or any other unexpected error.Ê Error may have also occured in the measuring of the pigment bands when calculating the Rf values and when scraping off the pigments to measure their absorbencies, because it was difficult to see where the pigment bands began and ended.Ê Here the spectrometer could have also affected the data obtained, since it continued to give inconsistent absorbencies if the cuvettes were not placed in the exact position each time and did not stay zeroed.
One aspect of difficulty with the vitamin C test was in the volumes because the procedure followed did not state exactly how much starch to use, how much one drop of iodine should equal, nor the concentration of both the iodine and the starch. ÊIt was assumed that the labs iodine, IKI, could be used for the titration, but this was not possible since it reacted with the starch instead of the vitamin C. Another problem was that the only was to get an iodine solution was to make using solid iodine, which was highly insoluble.Ê It was also difficult to tell if there was a color change in the spinach samples because the solutions would change to purple, but after approximately three minutes the samples would turn back to green.Ê
ÊÊÊÊÊÊÊÊÊÊÊ There are many other experiments that could be performed to further investigate the differences between organic and conventional spinach.Ê To further understand protein composition of each sample an essential amino acid test could be performed.Ê Other vitamins, such as vitamin A, which is very abundant in spinach, could be tested and quantified.Ê Similarly, other tests could also be done for chemicals or pesticides such as nitrogen. To fully understand the differences between organic and conventional vegetables a test would need to be conducted under the same growing conditions for several years. Further, it could be tested how manures, crop rotations, and special nitrogen fixing crops changed the compounds organic vegetables produced.