Pigments, Reducing
Sugar Richness & Structure, & Protein Richness Varied Due to Cooking
& Freezing Brassica
oleracea
By: Nathan Fry, Chris Tresnak, Kathleen Riopelle, Stephanie Zafiroff
Table 5.Ê Independent test for glucose and protein.Ê It was found that the uncooked frozen sample (2) had the largest amount of glucose followed by the frozen cooked (4). Both the fresh, cooked and uncooked samples had similar amounts of glucose found.Ê In the protein assay the amount of protein was the most in the fresh samples that had identical amounts in both the cooked and uncooked samples.Ê The frozen samples in the protein test only showed traces of protein in both samples both cooked and uncooked.Ê The glucose and xylose are the positive and negatives, respectively to test the glucose, while the BSA and water are the positive and negative, respectively for the protein test.ÊÊ
|
|
trial 1 |
|
trial 2 |
|
trial 3 |
|
|
|
sample |
glucose
(mg/dl) |
Protein
(mg/dl) |
glucose
(mg/dl) |
Protein
(mg/dl) |
glucose
(mg/dl) |
Protein
(mg/dl) |
|
|
Fresh Uncooked |
250 |
30 |
250 |
30 |
100 |
100 |
|
|
Frozen Uncooked |
500 |
0.1 |
500 |
0.1 |
1000 |
30 |
|
|
Fresh Cooked |
100 |
30 |
250 |
30 |
100 |
100 |
|
|
Frozen Cooked |
250 |
0.1 |
250 |
0.1 |
250 |
0.1 |
|
|
pos
(glucose) |
2000+ |
neg |
2000+ |
neg |
2000+ |
neg |
glucose |
|
xylose (neg) |
neg |
neg |
neg |
neg |
neg |
neg |
glucose |
|
POS (BSA) |
neg |
2000+ |
neg |
2000+ |
neg |
2000+ |
protein |
|
water (neg) |
neg |
neg |
neg |
neg |
neg |
neg |
protein |
Abstract
By: Nathan Fry
Tests were
conducted determining carbohydrate, protein and photosynthetic differences
between frozen and fresh Brassica oleracea,
when cooked and raw. Benedictâs test was preformed to determine if reducing
sugars existed in the samples.Ê Color
changes, indicating precipitate formation, supported the prediction that
reducing sugars were present in all solutions.Ê
For Barfoedâs test it was predicted that
heating or freezing could alter polysaccharides into monosacchrides.Ê Contrary to research and predictions red
precipitate, indicating monosacchrides, formed in
both uncooked samples of broccoli but not in cooked.Ê Contrary to hypothesis,
Paper chromatography was conducted to identify the pigments present.Ê Chlorophyll a only occurred in fresh uncooked with an Rf value of 0.7251cm, all others only had chlorophyll b with an Rf≈0.2cm, indicating freezing and cooking degrade pigments.Ê The Absorption Spectrum found all samples had the greatest absorption around 400nm.Ê We conclude that broccoli then uses mostly lower wavelength light for photosynthesis.Ê The final test was a Uritest which quantified glucose and protein concentrations.Ê For glucose frozen cooked and uncooked samples averaged 250 and 666mg/dl respectively and fresh cooked and uncooked averaged 150 and 200mg/dl respectively.Ê The fresh cooked and uncooked averaged 53.3mg/dl and both frozen samples only had trace amounts.ÊÊ This suggests freezing may preserve glucose but cooking as seen in Barfoedâs may diminish glucose.Ê Also contrary to our hypothesis it suggests proteins break down when frozen.
Discussion:
Written by:Ê Kathleen Riopelle
Broccoli has become a necessity in the American diet due to its dense nutritional content as it has high iron and protein concentration. Broccoli may be obtained and prepared several different ways.Ê Our research team sought out to determine the differences between cooked and raw, as well as frozen and fresh broccoli to find the best way for it to be prepared while maintaining nutritional value. Our hypotheses included:Ê The nutritional content and photosynthetic properties will not be the same for all samples, the nutritional content and photosynthetic properties will be higher in frozen uncooked broccoli.Ê Individual hypotheses and predictions are included for each of the test which support our original hypothesis.Ê The content of the samples, including the quantification of glucose and protein is what was used to determine nutritional content.Ê We predicted that the nutritional content and photosynthetic properties were greater in frozen compared to uncooked fresh and both cooked samples.Ê The cooking of the broccoli denatures both the nutritional and photosynthetic properties compared to uncooked samples (Doliniska 2004).Ê We tested these predictions in the lab to conclude if we can confirm or reject our hypotheses.Ê Through a series of different carbohydrate tests, protein tests, and photosynthetic tests we determined the most nutritional way to obtain and prepare broccoli.
To test whether reducing sugars were present we utilized Benedictâs Test.Ê Results confirmed that there indeed were free or potentially free aldehyde or ketone groups in all frozen, fresh, cooked, and uncooked samples due to glucose being in all samples shown in our independent lab.Ê Therefore we predicted that all samples would contain these reducing sugars.Ê Benedictâs reagent was added to each sample and then heated to see if a reaction took place.Ê All the samples reacted forming a precipitate with the reagent testing positive for the presence of aldehyde or ketone groups.Ê The frozen uncooked reacted the greatest meaning it had the most aldehyde and ketone groups present.Ê The least reactive was the fresh cooked sample.Ê In the next carbohydrate test, the Barfoedâs test, the presence of monosaccharide is tested.Ê Similar to Benedicts, the Barfoed reagent is added to each of the samples and heated.Ê The reduction of copper caused a precipitate to form in the presence of a monosaccharide.ÊÊ We predicted that the cooked samples would contain the monosaccharide and form the precipitate in reaction.ÊÊ The monosaccharides should be formed by the thermal energy of the heat denaturing carbohydrates into their simplest forms, monosaccharides (Link, 2004).ÊÊÊ The results demonstrated only the uncooked samples reduced copper and changed the color of the solution, while the cooked samples did not reduce the copper and thus the color change did not occur and no precipitate formed.ÊÊ The cooked samples tested negative for monosacchrides, but the uncooked samples contained monosacchrides. This refuted our prediction and intellectual isnât correct.Ê According to common sense and previous studies, theses cooked samples should have had polysaccharides reduced into monosaccharides due to heat denaturing the sugars.Ê Very little precipitate did form in the uncooked samples that tested positive demonstrating that they did not contain a large amount of monosaccharides.Ê Also, it is a possibility that the samples may not have been cooked at high enough heat or long enough to break monosacchrides.Ê
Next, to measure protein concentration, the Bradford Assay was utilized to gather the total protein concentration.Ê Different concentrations of our samples and ddH2O were tested in the spectrometer for their absorption at a certain wavelength. We predicted that the fresh uncooked sample would contain the highest concentration of protein.Ê Our prediction was supported by the results.Ê The fresh uncooked sample showed the highest concentration of protein.ÊÊ It was greater than 1 ug/ul concentration of protein.Ê An exact value of concentration could not be determined because the absorption of the sample was too much more than the standard curve.Ê An extension of the linear fit line would have had to be used leaving too much room for error.Ê ÊÊIt was followed by fresh cooked sample at 0.92 ug/ul. The frozen uncooked and cooked were 0.6 ug/ul and 0.32 ug/ul Êrespectively.Ê The cooked samples showed a significantly smaller amount of protein as compared to the uncooked counterpart samples.Ê This phenomenon is due to the denaturing effects of heat on protein (Link, 2004).ÊÊ The fresh broccoli both cooked and uncooked had a higher concentration than the frozen samples.Ê This could have occurred as a result of the freezing denaturing the proteins.Ê The freezing of waters sharp crystals could possibly break the chains in protein.
Paper chromatography and absorption spectrum were the photosynthesis tests that we conducted in our study.Ê The pigment chlorophyll facilitates the photosynthetic process by absorbing light energy.Ê Paper chromatography was necessary to identify the pigments present in each sample.Ê We predicted that the fresh uncooked samples will contain more pigments due to the fact that boiling can leach pigments out and denature them.ÊÊ During this test, the samples were centrifuged to obtain pellets which were used as the solution.Ê Chromatogram strip was made with the pigments, then placed in an chlorophyll acetone solution to separate the pigments.Ê The rate flow of each sample was calculated and analyzed showing that our predictions were confirmed with the results.Ê The fresh uncooked sample contained both chlorophyll a and b.Ê This sample was the darkest green each time it was prepared reflecting these results.Ê This is due to the fact that there was nothing done to this sample to remove or damage the pigments.Ê Both frozen samples and the cooked fresh both contained only chlorophyll b.Ê They were also much lighter green.Ê The chloroplasts and pigments could be leached out during the process of freezing and cooking in the water (Sizer 2003).Ê This explains the lack of cholorphyll a pigments.ÊÊ The next test was to see what wavelengths of light were absorbed by each samples.Ê They all contain different amounts and types of pigments, so theyâre absorbtion of light will vary.Ê In the absorption spectrum assay, it was predicted that the cooked broccoli would have a lower absorbance of wavelength as compared to the uncooked broccoli because some of the pigments will be leeched out in the cooking process (Sizer 2003). The discovery of more pigments in the fresh uncooked would make one think that it would absorb the most light.Ê This in fact did occur.Ê The fresh cooked sample absorbed less, and both frozen samples were the least and similar to one another.Ê Our predictions were correct on the difference between the absorption of cooked and uncooked.ÊÊ However we didnât predict that there would be much of a difference between the fresh and frozen, but the frozen were the least absorbent.ÊÊ The reason fresh samples absorbed more than the frozen could be due to the damage of the chloroplasts during the freezing process again.Ê Cooking also damages pigments at certain heats, inevitably affecting the absorption (Hurst et al, 2004).Ê The greatest absorbance was found at 400 nm and decreased linearly as the wavelength of light increases.Ê This is because chloroplast absorbed red and blue light the best and reflects green giving them their color.
The conclusion of the photosynthesis tests led us to our independent study to further investigate nutritional value.Ê We conducted an iodine test for starch content. We predicted that all samples would contain starch, but at somewhat different concentrations. The process of photosynthesis creates sugars which in turn create starches.Ê One would then conclude that our samples would contain starch.Ê When the iodine starch test was conducted, all of the samples tested negative for the presence of starch.Ê According to the nutrient values in broccoli per serving, it was found that each contains 0 grams of starches, which results in a negative result for starch also (Sizer, 2003). However, from prior knowledge that starch is found in the chloroplasts we assumed that the solution was not of an adequate concentration (Khra 2005). Due to these findings, we diluted the sample with ddH2O and centrifuged it, drained off the supernatent and subjected only the pellet to the Iodine test.Ê Both tests still indicated a negative result for the presence of starch. Since we could not devise a sufficient method to get the starch within the broccoliâs chloroplasts to react with our Iodine sample, we could not complete our experiment designed to quantify the amount of starch based on the absorbancy of the color change due to the reaction with Iodine. ÊThe next test that was conducted was to quantify glucose and protein again.Ê Uristix were dipped into our samples and were observed 30 seconds later.Ê We predicted once again that uncooked samples would contain larger amounts of protein with the fresh containing more than the frozen.Ê This was shown to be true through the results.Ê The protein results generally supported the assay results. Both of the frozen samples found only a trace of protein, while the fresh found to both have an average of 53.3 mg/dl in each sample.Ê This test demonstrates a difference between the frozen and fresh while not showing a difference between uncooked and cooked samples.Ê This could be a result of the fact that this test was not as precise as the Assay because it gave semi-quanitative and not calculated results.Ê It does however demonstrate that the freezing process damages protein concentration.Ê The prediction for the glucose test was that the frozen uncooked would contain the highest concentration of glucose.Ê The freezing of the broccoli preserved the sugars and the cooking will denature and break down the complex forms of glucose decreasing its concentration.Ê The glucose test demonstrated that the frozen uncooked had the highest concentration of glucose, which confirms the prediction.Ê The frozen cooked was found to be next, and the fresh samples had similar amounts just below the frozen cooked sample.Ê The uncooked samples both contained more glucose than their cooked counterparts.Ê The cooking of both samples could have caused the glucose molecules to break decreasing concentration.Ê The freezing of this broccoli when it was obtained could have helped preserve the glucose levels.Ê Something involved in the transport of the fresh samples could have broken some of the glucose molecules down over time.Ê
Conducting an experiment with so many different test and people leaves room for error and flaws in experimental design.Ê Human error could have occurred in measuring solutions, blending the samples, and care taken of the samples.Ê A source of error could have occurred by the company in their process of picking and packaging the product.Ê The transportation of the broccoli also leaves room for time and ways of contamination or loss of nutrient to occur.Ê The destruction of monosacchrides in our samples could have occurred giving us skewed results. The blending and creation of the samples could have been slightly different each time, it was difficult to get exactly equal concentration between samples.Ê This could determine results that donât coincide between different tests.Ê This could also have been a reason that we were unable to obtain starch in our samples.Ê There was also an inadequate number of trials done due to lack of time to perform the experiment to the best of its ability.Ê Inaccurate readings could have also occurred in the photosynthetic test because the chloroplast could be damaged during the preparation.Ê The cooking also draws pigments out of the samples.Ê Even though the errors could have occurred, the results are still conclusive.Ê
ÊÊÊÊÊÊÊÊÊÊÊ Through a series of different sugar tests, protein tests, and photosynthetic tests we determined the most nutritional way to obtain and prepare broccoli.Ê The conclusion was that the fresh uncooked sample was most nutritional.Ê This refuted our hypothesis which was that the frozen uncooked would be the most nutritional.Ê The prediction that the cooked samples would be of less nutritional value than the uncooked samples proved true.Ê The uncooked samples contained more protein, and they performed photosynthesis more effectively by absorbing more light.Ê The sugars, proteins and chloroplasts in the uncooked samples were not damaged providing larger concentrations of each of them, supplying more nutritious value.Ê The frozen samples both contained more glucose, which is needed in the body, but it contained only a trace of protein, making it less nutritious.Ê The freezing process may preserve glucose content, but damages too much of the chloroplasts and protein.Ê The fresh uncooked samples contained more protein, smaller amounts, but still enough glucose, and absorbed the most light with the most pigments, demonstrating efficient photosynthesis. The sugars, higher protein, and most efficient creation of ATP from photosynthesis makes fresh raw broccoli best for you.Ê This supplies a person with the most nutritional value possible.Ê So next time you sit down to dinner, eat the freshly bought broccoli before you cook it.Ê