By comparing the presence of carbohydrates, proteins, pigments, and vitamin C in cooked and uncooked fresh and frozen green beans, Phaseolus vulgaris, a better understanding of the cooking process can be obtained. This experiment was performed to better understand the impact different methods of preparation on the nutritional content of the foods we eat. It was predicted that fresh green beans would lose the most macromolecules when comparing cooked to raw, because they possessed the most nutrients from the start (Feldman et al, 2006). Preparing 15g samples of fresh and frozen green beans and comparing them to their cooked counterparts allows for five tests to be used in the macromolecule analysis. Carbohydrates were tested using Barfoedâs and Iodine tests because of their ability to test for mono- and polysaccharides, respectively, showing that present in each bean sample. The other tests mentioned in lab 1 were not performed since this study focused on cooking and the possibility of complex sugars breaking into simpler sugars. Barfoedâs test indicated monosaccharides in all samples but water and the Iodine test indicated coiled polysaccharides for cooked beans only. The Bradford assay tests for protein by comparing unknown contents in green beans with a standard curve of known absorbencies, concluding proteins were lost by both beans during cooking. Thin layer chromatography separates pigments, allowing for their identification, which did not reveal any sound results. The vitamin C titration quantifies vitamin C based on an oxidation reaction, indicating the presence of vitamin C in boiled water, evidence that vitamin C was lost. These tests created a comprehensive understanding of macromolecule characteristics through the processes of preparation and cooking.
Understanding the macromolecules ingested in the body allows for better decisions in the management of diet and health. When food is cooked, irreversible chemical changes occur that alter how the food is dealt with in the body. Green beans, Phaseolus vulgaris, were the representative vegetable that was chosen for this experiment. Fresh and frozen green beans were used as varying treatments to determine which beans will be altered more, if at all, upon cooking. Our hypothesis was that the fresh green beans would be altered more and would have lost more nutrients after boiling than the frozen green beans because previous research stated that numerous vitamins and proteins were denatured and lowered in concentration after boiling (Pastuzewska, 2004). Each of the tests performed, the carbohydrate tests, Bradford Assay, TLC test, and Vitamin C titration, were predicted to support our original hypothesis that fresh green beans would lose the most macromolecules when cooked.
For the carbohydrate tests, it was predicted that the polysaccharides in the frozen and fresh carbohydrates would be broken down into monosaccharides when they were cooked (Khatoon, 2004). This would make them easier to digest because it is only one carbon ring compared to multiple. In the carbohydrate tests, we found that the frozen beans were altered slightly more than the fresh beans when cooked. When looking at the solutions made from the beans, it was hard to tell the difference between the fresh and frozen, cooked and uncooked. The Barfoedâs test showed us that the presence of monosaccharides in the uncooked green beans became weaker in the cooked beans, though it is difficult to draw quantitative results from a qualitative test. In the Iodine test, polysaccharides were found in the water the frozen green beans were cooked in whereas there was no reaction for the water the fresh beans were cooked in. The Iodine test showed a stronger color change in the cooked beans, possibly indicating that the presence of polysaccharides was stronger in the cooked beans than the uncooked beans. If true, this may contradict our prediction that cooking the beans would break the polysaccharides into monosaccharides. It is possible this increase in starch may have occurred because the beans were heated and energy was added which may have caused the monosaccharide molecules to bond and form polysaccharides (Claeys, 2005).
It was predicted for the Bradford Assay that the concentration of proteins in the green beans would decrease more after cooking in the fresh beans than frozen. When beans are heated, some of the proteins may be altered so greatly that they might not be detected as proteins by the Bradford reagent. Using the absorbance and the Bradford standard curve, it was possible to determine the amount (in micrograms) of protein present in the beans. We found that the frozen green beans lost more proteins than the fresh green beans after being cooked (Above Figure). This refutes our hypothesis. Prior to being cooked, the frozen beans had a higher absorbance than the fresh beans and therefore contained a higher concentration of proteins. However, after being cooked, both the frozen and the fresh bean solutions had approximately the same absorbance and, as a result, had around the same concentration of proteins. Looking at the water the green beans were cooked in reinforces the evidence that the frozen beans lost more proteins than the fresh beans during the cooking process. The concentration of proteins in the water that the fresh beans were cooked in was lower than the concentration in the water the frozen beans were cooked in. Evidence indicates that frozen green beans lose more proteins than fresh because during the freezing process, it is possible that the cytoplasm pierced the various membranes in the cell during the freezing process (Nursal, 2000). This would have created gaps for the proteins and other nutrients to escape through during the cooking process.
For the TLC test, it was predicted that the amounts of the pigments in the frozen green beans would decrease more than the amounts of pigments in the fresh after the cooking process. However, after several trials with two different mobile phases of 7:3 hexane: acetone and 7:3 anhydrous ether: acetone, no usable separation of pigments could be achieved. This may have been for several reasons. Since both mobile phases included acetone, it may be possible that acetone plays a role in inhibiting the proper development of separation by interacting with a substance within the green beans. It is also possible that the coarse hairs of the brushes used in the lab may have damage the silica gel on the TLC strip, also inhibiting the separation. This is a distinct possibility because small scratches on the strip could be observed by the naked eye. There also may have been some possible contamination of the mobile phase components and TLC strips due to the heavy volume of use and handling by LBS 145 students.
It was predicted that the Vitamin C titration would indicate a greater change in the levels of vitamin C in the frozen green beans than in the fresh green beans after they were cooked. For each trial in our replications, an endpoint was reached during the titration. However, different trials produced ambiguous results and displayed either blue or orange with no correlation between color and presence of vitamin C. The positive control of pure ascorbic acid as well as a crushed vitamin C tablet both displayed an orange endpoint, while the majority of the experimental bean trials displayed a blue endpoint. Theoretically, the iodine ions were expected to oxidize ascorbic acid until reaching endpoint, at which point iodine is then free to indicate the starch added by means similar to the iodine carbohydrate test. Since each sample had a varying amount of starch due to the presence of starch in green beans, shown by our own experiment, color changes may simply been caused by that variation. A negative control was performed containing starch, water, and no ascorbic acid: contrary to expectations this did not turn blue. It is worth noting that fresh uncooked and fresh water, both reaching orange endpoints, also tested negative for starch in the iodine tests. From this experimental data alone, it is difficult to draw conclusions of the cause of these color inconsistencies and further experimentation would be required to reach these conclusions.
Future Directions
During the Bradford Assay, absorbance values were obtained using a spectrophotometer. The values measured varied considerably, which is possibly due to the method used to gather these values. It is possible that the spectrophotometer used may be out of calibration and it also must be considered that the sample tubes inserted into the spectrophotometer may not have been completely cleaned or have had the ability to fully transmit all light wavelengths. This conclusion is reached because the Bradford reagents used in the tubes prior to the use of this experiment visibly tinted the cuvettes blue. To solve this problem, a more advanced machine could be used, and/or fresh cuvettes could be used in an accurately calibrated device.
The experimental chromatographies were replicated three times, once using an alternative mobile phase. Even though these replications occurred, no useable data was obtained. After brushing the pigment onto the TLC strip, small scratches could be seen due to the coarse hairs of the paintbrush. A softer, finer brush could be used to apply the pigment to the silica gel without damage. Additionally, rather than brushing on the pigment, contact could be avoided altogether by dripping or using a controlled spray to apply the pigment to the strip. Since both mobile phases contained acetone, another mobile phase, such as pure ethanol, could be tried that does not include acetone as an ingredient. This would rule out the possibility of an interactions occurring between the chemicals of the green beans and the mobile phase. Also, to eliminate the use of acetone, a different extraction agent would have to be used in the preparation of the pigment solutions. It would also be helpful to eliminate magnesium sulfate from the process to remove yet another variable. There are other types of chromatography available that can be performed. Several of these could be tried until a successful result was obtained.
The method used for vitamin C titration involves the reaction of iodine and starch. Using an alternative food known to contain vitamin C, such as lemons, a titration could be performed under the same conditions of this experiment to determine whether or not there was a component in green beans, specifically, that altered the results and changed the color. If the lemons did not show differences in colors as green beans did, a case could be made supporting the hypothesis that a substance exists in green beans that interferes with the titration. An alternative process, avoiding titration entirely, is High Performance Liquid Chromatography (HPLC), which involves ultraviolet and fluorescent light and/or electrochemical detection with photodiode array which will detect the presence and concentration of vitamin C (Lopez, 2005). This HPLC test could be performed, theoretically providing more usable and accurate results.
DePew, Single, Spencer, and Uzarski. 2006.