Discussion:
Macromolecules affecting the longevity and practicality of organic paint made from non-pasteurized milk and blueberries were tested through five sugar presence tests, a photosynthetic macromolecules test called TLC, protein test, glucose concentration monitoring test, and longevity observations.
Benedict’s Test: The hypothesis formed was the macromolecule carbohydrates would cause the longevity of the organic paint to be compromised. The predicted result was that there would be reducing sugars present in the organic paints composed of non-pasteurized milk and blueberries. This was due to the knowledge that pectin, an element found in the cell wall of plants like blueberries, consists of neutral sugar polysaccharide molecules, for instance galactose, which consists of chains of monosaccharides, like the sugar glucose (What is Pectin?, 2001). Therefore, reducing sugars were expected to be present in the milk and blueberry paint solutions due to the pectin present in the blueberries. After performing Benedict’s test, the conclusion was that the only samples with reducing sugars present were the positive control of glucose and the control containing blueberries and distilled, deionized water. This was concluded based on the observation that the glucose positive and blueberry control changed colors from blue to a rusty red (Figure 1A, 1E).
An explanation for the negative results in the two paint solutions is that the reducing sugars in the blueberries reacted with another molecule in the milk. Furthermore, the ability of heat to break chemical bonds during the Benedict’s procedure must also be taken into consideration. Some research that supports the explanation was the Maillard reaction that causes amino acids and proteins to react with the reducing sugars, ketoses and aldoses (Brands 2001). This was a suitable explanation because the structures of ketoses and aldoses contain a carboxyl functional group. The double bonded oxygen was somewhat reactive by itself because of the lone pairs of electrons and the addition of heat increased the kinetic energy causing the oxygen to be even more reactive. Possible troubleshooting is that once the tubes were placed in the boiling water for the procedure, the water stopped boiling for a little while. This was found to be a common problem with many of the sugar tests. The lack of positive results for the reducing sugars, ketoses and aldoses, does not support the prediction that the ketoses and aldoses would be present. Therefore, because the ketoses and aldoses were not present in the different dilutions of the organic paints, the hypothesis that carbohydrates would compromise the longevity of the milk and blueberry solutions was not supported due to lack of evidence.
Barfoed’s Test: The presence of pectin in blueberries containing neutral sugars with monosaccharides, such as glucose and fructose, is recognizable with not only with the taste of blueberries, but also the very content of pectin (“What is Pectin?” 2001). The predicted results for this test were that the Barfoed’s test would test positive for monosaccharides. Due to this prediction, the hypothesis states that the positive test for this macromolecule would be a factor in the spoiling and curdling of the milk and blueberry organic paint. The presence of red precipitate occurred only in the glucose positive and blueberry control (Figure 3-1B, 2B; 1E, 2E). The glucose was present in the blueberry control because the pectin in blueberries contains coiled polymers of monosaccharides, which can potentially be broken down to monosaccharides, like glucose (“What is Pectin?” 2001).
The dilutions of the organic paint and the milk control had no change, which did not support the presence monosaccharides. No monosaccharides were present because the heating in the procedure for Barfoed’s Test caused the ketoses and aldoses to react with the proteins in the milk (Brands 2001). Sizes of the carbohydrates seemed to have influenced the possibilities of reacting, so the presence of monosaccharides was not detectable. Monosaccharides were more apt to react with proteins after the addition of heat, especially sucrose (Lee 2006). A possible reason for the negative results for monosaccharides is the Maillard reaction. Because the oxygen was more reactive with the addition of heat, the structure of monosaccharides was altered and then not detected by the Barfoed’s Test (Brands 2001). Heating was not consistent and when the test tubes were placed in the hot water bath, the boiling ceased for a minute. The hypothesis that the presence of monosaccharides would discredit the longevity of the organic paint was not supported by the collected data.
Selivanoff’s Test: This test was performed to determine whether ketoses and aldoses exist in the organic paints. The anticipated results were that the different dilutions of organic paint would test positive for the existence of the ketoses with the prior knowledge that pectin present in the blueberries consists of neutral sugars (“What is Pectin?” 2001 ). Because the ketoses were expected to be present, the hypothesis was that the longevity of the organic paint would be reduced with the presence of more carbohydrate macromolecules. Results discovered were that the positive control, fructose, tested positive for ketoses (Figure 4-1A, 2A). However, the negative control, the two dilutions of the organic paint, and the milk control tested negative for ketoses, therefore positive for aldoses (Figure 4-1B, 2B; 1C, 2C; 1D, 2D; 1F, 2F). The blueberry dilution was already a blue-red color and determining the color change was difficult with the expected color change in the dilutions for Selivanoff’s to be a shade of red, as well (Figure 4-1E, 2E). It was assumed that ketoses reacted with the protein present in the milk, thus denaturing the ketoses, which follows the Maillard equation (Brands 2001). Also, the paint solutions sat overnight and had to be mixed with a magnetic stir bar, which left for more room for error especially with the spoiling rate of milk. Because the tests determined that the organic paints did not contain ketoses, the hypothesis was neither fully supported nor rejected.
Bial’s Test: The hypothesis was that the presence of furanoses was expected to discredit the viability of the organic paint. The predicted result was the organic paints would contain furanoses, also known as five-membered rings, due to the presence of pectin in the blueberries used in the organic paints (“What is Pectin?” 2001). The positive control, fructose, turned an olive green meaning that the control was positive for pentose furanose (Figure 2-1A, 2A). The blueberry control also turned the dark green concluding that furanose rings were present in the blueberry (Figure 2-1E, 2E). The negative control galactose, the milk control, and the two dilutions of the organic paint did not show color change (Figure 2-1B, 2B; 1C, 2C; 1D, 2D; 1F, 2F). No color change meant that the sugars that were present were most likely a pyranose. Because the pentose furanose contains an oxygen molecule in the ring structure, a possibility is that the heating caused a reaction with other molecules in the milk (Brands 2001).
Trouble shooting included the fact that the solutions were a couple days old by the time this test was completed so spoiling may have affecting the results, and the boiling of the test tubes may not have been at the same temperature for all test tubes. The hypothesis was not completely supported or rejected by the data. Research was not found that would help explain why the mixtures of organic paint only contained pyranoses.
Iodine Test for Coiled Polysaccharides: The hypothesis was that the iodine test for coiled polysaccharides would confirm the notion that the degradation of the organic paint would be influenced by the presence of a polysaccharide macromolecule. The cell walls of plants, specifically blueberries, consist of pectin with complex polysaccharides (Silva 2005). This presence of polysaccharides in pectin of blueberries lead to the expectation that the solutions of milk and blueberries will also contain coiled polysaccharides (“What is Pectin?” 2001). In results, however, the only solutions that contained coiled polysaccharides were the starch positive control, and the blueberry control (Figure 5-1A, 2A; 1E, 2E). These two samples turned a bluish black color when reacting with the iodine. The other tests did not change into the distinctive bluish black color (Figure 5-1B, 2B; 1C, 2C; 1D, 2D; 1F, 2F).
Since the procedure did not call for heating, either, the negative result for coiled polysaccharides in the milk and blueberry solutions can only be reasoned with the knowledge that another factor in the reaction caused the breakdown of the coiled polysaccharides other than denaturing of heat as explained in Maillard equation (Brands 2001). Reason for error includes the possibility of spoiling of the solutions by the time this test was completed. The hypothesis was not supported, because polysaccharides were not detected by the iodine test.
Thin-Layer Chromatography (TLC) of Plant Pigments: The presence of chlorophyll a, chlorophyll b, xanthophylls, and carotene expected to be retrieved through the TLC experiment will support the hypothesis that photosynthetic macromolecules present in the organic paint compromises the longevity of the paint. The control of blueberry dilution contained pigment results (Figure 6- 1A, 2A). However, the TLC experiment with the two solutions of paint yielded very light pigment results (Figure 6-1C, 2C; 1D, 2D). Despite the fact that blueberries have antioxidant power and contain beta-carotene as shown in Figure 6-1A, 2A, their presence in the milk and blueberry paint solution was not recognizable (Dunlap 2006).
Proteins are said to react with beta-carotene with the addition of heat (Marx 2003). Therefore the proteins in the milk could potentially react with the beta-carotene. This would then influence the longevity of the organic paint. Milk does not contain any photosynthetic pigments since cows are not autotrophs. Therefore, the acetone in the TLC was not expected to extract any pigment from the milk control (Figure 6-1B, 2B). The lack of photosynthetic pigments could possibly have deterred the photosynthetic pigments in the blueberries. Another explanation is that the solvent acetone used to remove the pigments was not ideal for extracting the red pigments that were unique to the blueberries, yet was good for extracting green pigments, like those of leaves. This would be one cause for error, as well as the age of the solutions, which could have been spoiled, the paint strokes may not have been saturated enough, or the centrifuging of the dilutions may not have been complete. The light pigment extracted was not concentrated enough to achieve a reading for the amount of photosynthetic macromolecules present in the blueberry and milk solutions. The hypothesis was not able to be supported or refuted based on the lack of results in the TLC experiment on the organic paint.
Longevity Independent: The hypothesis was that the more macromolecules present in the solution of milk and blueberry paint, the more the longevity of the paint would be compromised through curdling and spoiling of the ingredients. The results of the longevity test observing the curdling were of the two milk and blueberry controls, as well as the two different mixtures of organic paint. Through the observations, it was found that the milk control, C2 separated and curdled the most (Figure 9-1B, 2B). The two mixtures of blueberry and milk organic paint curdled the same in the beginning, but by the later dates, it was observed that M1 had spoiled faster than M2 (Figure 9-1C, 2C; 1D, 2D). The blueberry control C1 had very little observed curdling or spoiling (Figure 9-1A, 2A). A thin film did form on top, however this was not a lot compared to the other three samples.
Since curdling was most apparent in the milk controls, the protein and lactose in the milk has a profound affect on the longevity of the organic paint (Anonymous-1, 2005). The carbohydrates present in the blueberries, specifically polysaccharides, did not spoil in a way that would change or separate the solution, therefore explaining the slight differences in observation results for M2 and M1 in the longevity independent test. The spoiling and curdling of milk is due to bacteria present within the milk that changes the carbohydrate lactose into lactic acid (“Milk Products” 2006). Therefore, the milk control was observed to contain the most curdling, followed by the two solutions of blueberries and milk, due to the amount of lactose that was present in the milk compared to the milk mixture. It is possible that the polysaccharides present in the pectin of blueberries slightly deterred the curdling of the milk and making it less susceptible to separation and bacterial infestation, however this amount would be very little (Silva et al., 2005; “What is Pectin?” 2001).
Glucose Independent: Since sugar tests were already completed confirming or rejecting the presence of specific carbohydrates in the solutions, a glucose test was necessary in order to find out the amount of glucose that was present in each concentration and in each of the two organic paint mixtures. The expectations that the solutions with the most blueberries in them would have higher amounts of sugar, therefore it was hypothesized that the higher the amount of glucose in the sample, the shorter the longevity of the paint with the affects of curdling. The results found from completing the glucose test with a CVS Complete Blood Glucose Monitoring System by Prestige Smart System, it was supported that the control of blueberries, C1, had the highest concentration of glucose by far (Figure 8). In addition, M2 with more blueberries originally added to it had a higher amount of glucose than M1 with the smaller amount of blueberries originally added (Figure 8). The non-pasteurized milk control, C2, did not yield any results from the diabetes glucose test most likely because carbohydrates are not one of the macromolecules present in milk. Lactose, a carbohydrate, very abundant in the milk samplings, would not be measured by a glucose test, and neither would the high concentration of protein in the milk (Anonymous-1, 2005). Therefore, the fact that the milk control did not yield a concentration of glucose further supports the expectation that milk does not contain glucose as a carbohydrate.
Bradford Assay: This experiment questioned whether or not the presence of increased protein macromolecules from the organic materials shortened the longevity of the organic paint. Research done provided the information that because the paint contained milk, the Bradford Assay will show a positive result for the presence of protein macromolecules (Anonymous-1 2005). The results yielded the expectations that the milk control contained more protein macromolecules than the blueberry control (Figure 10). This is due to the knowledge that there is a high protein content in milk (Milk Products 2006). Regarding the organic paint solutions, M1 contained the least amount of blueberries yielding more protein per volume of solution. M2 contained more blueberries and would therefore contain a lesser amount of proteins in solution (Figure 10). Logically, the reasoning behind the different amounts of protein in the two paint solutions is that with more blueberries present in solution there are more sugars present to react with the protein in the milk in accordance to Maillard’s reaction (Brands 2001). Ultimately, there is an inverse relationship between the sugars present from the amount of blueberries to the protein present in the milk.
Possible reasons for error include the fact that bubbles were present in the blank and some samples, the pipette may not have measured exactly 1mL, and during vortexing a small amount of the solution comes out of the tube. A very important reason for error in Bradford Assay is the fact that a new Bradford reagent had to be used halfway through the experiment because the original ran out. The expectations of protein present in the blueberry and milk solutions were supported by the collected data, and the hypothesis regarding the longevity of the organic paint was also supported.
Room for error includes the incomplete testing of the milk control due to error on the glucose monitor, as well as new concentrations of mixtures and controls because the originals had spoiled. The glucose monitor was also designed for use with blood and its use with blueberry and milk solutions was not ideal for retrieving glucose levels of that composition. The machine may have been out of date compared to newer, more expensive models and the glucose strips may have been fresh enough for the exact results that were expected.
The concentrations received from the diabetes glucose test did support the expectation that those solutions with more blueberries would have higher glucose concentrations. However, Barfoed’s Test confirmed the presence of monosaccharides in the blueberry control only, not in the milk and blueberry solutions. This is most likely due to error in Barfoed’s Test. The boiling may have denatured the monosaccharides making them more reactant than before, which was previously addressed in the trouble-shooting and in the Maillard reaction (Brands 2001).
The importance of the longevity of the organic paint concerns the milk itself because research has shown that Pasteurized milk held at proper refrigeration temperature has an average life span of 12-16 days (Anonymous-3 2000). However, early colonists had no refrigeration, which may have been a factor of the shorter longevity and practicality. The overall hypothesis is that the organic paint sample will have a shorter longevity because the greater the presence of macromolecules, the faster the sample spoils and curdles. This would not have as much influence as not being able to keep the paint stored at the proper refrigeration temperature due to lack of technology at the time these paint solutions were originally used.
The four solutions of organic paint provided blue-black color results, which will support the presence of polysaccharides in the iodine test for coiled polysaccharides and support the original hypothesis that their presence compromises the longevity of the solutions (Figure 3). The other sugar tests did not support the presence of reducing sugars, monosaccharides, ketoses, or furanoses in the solutions. This is most likely due to the heating factor in the procedure of three of the sugar tests. The Maillard reaction explains that the carbohydrates, specifically the reducing sugars and ketoses, react with the amino acids in proteins are triggered with heat and ultimately change the makeup of the carbohydrates normally present (Brands 2001). Since milk contains amino acids in the form of proteins, then the heating of the paint for the original mixture of the paint solutions, as well as the sugar tests, would denature the carbohydrates and provide results different from what were expected (Anonymous-1, 2005). The absorbance protein amounts from the Bradford Assay, when compared to the standard protein curve (Figure ?), also support the hypothesis for the presence of protein macromolecules. The TLC test for photosynthetic macromolecules yielded inconclusive results.
The results do not support whether the longevity of the solutions is directly influenced by the concentrations of macromolecules. The unexpected reactions between the sugars and the proteins with the knowledge that heating denatures macromolecules merely confirmed the presence or absence of specific macromolecules, with the result that polysaccharides were the only positive test for carbohydrates. The knowledge that lactose is present in the milk could not be directly tested by any of the sugar tests. The presence and concentration of protein in the blueberry and milk solution confirmed the high quantity of protein. In addition, proteins seem to be influenced by varying environments and the reactions with the sugars may even help stabilize the sugars (Lee et al. 2006). Since the curdling of milk is due to the lactose and lipids present, the concentration of those macromolecules specifically were not determined with the tests that were performed for the experiment (Anonymous-5 2006). After discovering that only specific macromolecules were present in the milk and blueberry organic paint, our results do not fully support the hypothesis that longevity is compromised with an abundance of macromolecules, so it is rejected.
Future Discussions: Throughout the stream of experiments, there were some problems. For the sugar tests, it seemed that constant boiling of the test tubes in order to retrieve a color change result was very hard to achieve. Instead of placing many test tubes in the water to boil, possibly boiling one at a time may be more accurate, however not practical on time. Furthermore, test tubes were being reused throughout the lab and may not have been cleaned properly. The diabetes glucose testing machine belonged to the lab and may not have been as technologically advanced as would have been ideal for the experiment, however this was not practical with the budget. A large possibility of error in the experiment is the fact that all of the tests were not able to be completed in one day, or in a short series of days. Over the weeks that the tests were completed, the solutions began to spoil, which could have severely changed the results that were retrieved. To amend for this, halfway through the series of tests in the stream a new set of controls (C1, C2) and mixtures (M1, M2) had to be made, however, they were with slightly different ratios of blueberries to milk in the mixtures compared to the original solutions. The new blueberries were bought from the same store, however possibly not from the same patch of blueberries, and the milk was retrieved from the same dairy farm, however not the same cow. This could have slightly changed the results of some of the tests, however these changes would have been very minimal. For instance, it was discovered that different breeds of cows produce different amounts of fat and other macromolecules in their milk compared to other breeds, and that all breeds produce milk with more fat in the winter months (Anonymous-5 2006).
ΚΚΚΚΚΚΚΚΚΚΚ With the realizations of the errors made during the experiment, possibilities of future experiments would include the following changes. For carbohydrates testing, a specific test for disaccharides would detect the lactose that is present in the milk (Anonymous-5 2006). The sugar tests that were completed for the experiment did not specifically test for disaccharides. Instead they detected monosaccharides and polysaccharides, both carbohydrates present in blueberries alone, not taking into account the heating of the milk with proteins in the mixtures of the paint. Furthermore, it was the milk that was influential in the longevity of the organic paint solutions. The TLC extraction of photosynthetic pigments in the blueberry and milk solutions was not successful due to the fact that the solvent used to extract pigments, acetone, was not ideal for the red colored pigments of the blueberries present in solution. Instead, the high performace liquid chromatography (HPLC) would extract the correct photosynthetic macromolecules present in the blueberries, providing results of higher carotene level than the chlorophyll that is normally present in green colored pigments (Minguez-Mosquera, 1994; May 2006). Another possibility is to complete a TLC test with amino acids, since protein is a large and very influential component of milk especially with spoiling (Anonymous-5 2006). This could specifically be used with the organic paint solutions to recognize the amount of amino acid concentration in relation to curdling in the blueberries and milk solutions (Tomek 1992). With more time to address these errors and made the changes to the experiment, a more conclusive verdict could be reached.
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