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Abstract
A comparative analysis of macromolecule content between processed and unprocessed bovine blood samples was performed to determine the deterioration of the bovine meat. The hypothesis of this study asserted that meat processing practices cause the carbohydrates and proteins in blood, and therein meat, to breakdown. The experimental sample, processed meat blood runoff, was examined alongside the control sample of unprocessed bovine blood in a variety of sugar and protein tests. The lead concentration test assesses the possible presence of lead poisoning within the animal. The amount of lead within the samples gives insight to the chemical aspects of processing, and helps eliminate any source of abnormal deterioration resulting from lead induced illness. The results showed both samples were well within the safe range of lead concentration. The carbohydrate tests and respective results included: Barfoedâs Test showed processed blood having monosaccharides and unprocessed blood having only di- and polysaccharaides, Iodine Test gave a positive result for animal starch, glycogen, in the processed blood and not in the unprocessed blood, Selivanoffâs Test gave a positive result for ketones and a negative result in unprocessed blood, Bradford Assay was predicted to give a higher concentration of protein in unprocessed blood than in processed blood, and the final test, a thin layer chromatography amino acid assay, illustrated that the processed blood had a higher diversity of different amino acids than the unprocessed sample. These combined results yielded a higher level of carbohydrate and protein degradation in the processed sample than in the unprocessed sample. The results of this study supported the original hypothesis, concerning sugars, protein concentration, and lead, yet did not support the amino acid aspect of the hypothesis as expected.
Discussion
The purpose of this experiment was to determine quantitatively and qualitatively how the processing of meat affects the macromolecules such as carbohydrates, proteins, and amino acids present. An unprocessed blood sample was tested as the control and meat runoff blood was used as the experimental sample. Research on meat processing reveals that many chemicals used in meat processing plants and procedures, such as acetic and lactic acid treatments used in packaging, could lead to carbohydrate, sugar, protein, and even amino acid degradation (McCracken and Murphy, 2004). The original hypothesis stated that processed blood sample will show a degraded level of sugars, carbohydrates, proteins, and amino acids due to industrial processing and packaging methods, and that the unprocessed pure blood would show a more complete and unharmed range of these macromolecules. This study was relevant because meat processing effects on essential macromolecule content in bovine meat were compared with macromolecules found in pure unprocessed blood, which contains similar macromolecules to meat (Unknown-2, 2005). By analyzing the degree of degradation of processed meat, harmful denaturizing of beef macromolecules can be assessed. Factors such as whether this lack of macromolecules and amino acids can be used to determine whether human health is being compromised by these processes. This would ultimately lead to meat processing procedures being modified in the future to accommodate a higher human intake of macromolecules and essential amino acids needed from eating meat (McCracken and Murphy, 2004). Basically, this study is the first step in creating a food industry reformation that could result from analyzing the effects of macromolecules by the current packaging system. It was predicted that the processed sample would show higher degradation of carbohydrates, proteins, and amino acids, whereas the unprocessed sample would exhibit fairly intact carbohydrate, protein, and amino acids.
Since the red blood cells in the blood samples gave the experimental samples a very dark coloring (Figure I), both the processed and unprocessed samples were centrifuged and the plasma was removed and used for testing. Since only the red blood cells are removed, the remaining blood plasma contains the same macromolecules and amino acids as the whole blood (Unknown-1, 2005). This renders the plasma acceptable as a substitute for whole blood in all the performed tests in this study (Unknown-1, 2005). The plasma was more translucent than the sample containing red blood cells, and provided a much better reading for the spectrophotometer and sugar tests than the samples with red blood cells.
The lead toxicity test was done for the independent experiment and was achieved by using the Diagnostic Center for Population and Animal Health on the Michigan State University Campus. This test was used as a preliminary examination to determine the level of health and usability of the live and processed blood samples. Results obtained showed that lead content for unprocessed blood was 3.4 ppb, or part per billion, while for processed blood it was 0.4 ppb. These results showed that both samples contained very low levels of lead, and that both were trustworthy to use to continue the investigation and the rest of the tests. The extreme drop in lead content shows that perhaps through processing, lead is removed via health regulations (Llobet, 1990). Although both values found in this test were far from the human toxic level of 100 ppb (parts per billion), meat processing undergoes preventative removal of toxic compounds such as lead (Llobet, 1990).
Barfoedâs test yielded good results upon the first attempted test. In the Barfoedâs test, Barfoedâs reagent was added to the processed and unprocessed plasma samples. Barfoedâs test results exhibited a positive result for monosaccharide content in the processed meat sample, indicated by the red color that was produced by the reduction of copper ions. The unprocessed samples demonstrated a negative result for monosaccharides (Figures I and II). This demonstrated that in the unprocessed blood, more di- and polysaccharides are present, meaning that they remained more intact in the unprocessed blood. In the unprocessed sample, the polysaccharides may have broken down due to processing chemicals and procedures (McCracken and Murphy, 2004). This aspect of the study is relevant to ingest polysaccharides into the human body. The complex structure of polysaccharides provides more energy for the body than ingesting just di- or monosaccharides because the more carbon-carbon bonds that are located in a sugar, the more potential energy is released when these bonds are broken by enzymes in the digestive system (Freeman, 2005). If the sugar is already somewhat broken down, then the body is not receiving as much energy from ingesting the sugar and therefore has to ingest a greater amount to achieve the amount of energy that would have been provided had the polysaccharides not degraded due to processing (Kotz et. al., 2005)
Selivanoffâs test was completed successfully through one attempt. For this test, the processed plasma sampled showed a high content of ketoses, while the unprocessed samples showed negative test results for ketose content, meaning that the sugars present were aldoses (Krha, 2006). In this test, ketoses were distinguishable from the aldoses because the ketoses reacted more rapidly during the test (Krha, 2006). Aldoses contain aldehyde groups which can be easily oxidized to form a carboxylic acid (Royal Society of Chemistry, 2004). Ketoses already contained a carbonyl group, which is a polar functional group (Royal Society of Chemistry, 2004). Because of this, the carbonyl group interacted with water through intermolecular forces, particularly hydrogen bonding (Royal Society of Chemistry, 2004). However, ketoses can bond only to other substances, not to other ketoses, making them more volatile and more prone to quick reactions than aldoses (Royal Society of Chemistry, 2004). The results of the Selivanoffâs test show that only ketoses were found in the processed plasma samples (Figures IV and V). This can be explained by the reaction of an aldoseâs initial aldehyde group being oxidized to form a carboxylic acid group, giving it a similar structure to a ketose, which was present in the processed sample because the aldose had already reacted to form this new structure (Royal Society of Chemistry, 2004).
The Iodine test, like the previous sugar tests, also yielded usable results upon the first attempt. This particular test used iodine to distinguish a starch, with which it reacted and turn the solution a bluish-black color, from other polysaccharides, as well as monosaccharides and disaccharides. Iodine test results exhibit a negative result for starch present in the unprocessed sample, meaning that no starch was detected through the reaction with iodine, and a positive result in the processed samples. In this particular examination, glycogen, or a common animal starch, was the starch being tested for in the processed and unprocessed blood samples. Glycogen provides an intermediary color reaction due to slight differences in structure of this starch (Krha, 2006). No glycogen content was detected in the unprocessed sample, as was predicted, perhaps because the test was not sensitive enough to differentiate between the intermediate and negative result colors. Results may also lost accuracy due to human error such as color misconception or pipetting the wrong volumes into the experimental test tubes.
The Bradford Assay was performed to determine the protein content in our experimental samples by comparing them to a standard curve created with BSA as the standard for comparison. The line obtained from this graph was the basis for calculating the protein concentration from the absorbance readings of the processed and unprocessed samples. This test was repeated four times in order to obtain a standard curve with usable values and that created a valid equation for necessary calculations. A valid equation and standard curve was not achieved repeatedly because although the procedure was completed correctly, the concentration values obtained were always inconsistent and too low to create a good standard curve. On the failed attempts, the values alternated between too high and too low to provide a good standard curve, leading to the conclusion that human error may have caused the fluctuating absorbency readings by not vortexing the solutions sufficiently before removing a portion to create the rest of the dilutions. For the three trials of the processed sample, the calculated protein amount averaged 225.7 micrograms, while the average of the three unprocessed plasma samples averaged 219.6 micrograms. This means that the protein content was lower for the unprocessed sample than the processed sample. Since muscle tissue has higher protein content than blood (Nowak et. al., 2005), it can be extrapolated that through meat processing, the tissue may have been destroyed, causing tissue proteins to leak into the processed blood that was later collected for this experiment. This holds extreme relevancy to health and human consumption of meat because this shows that as meat is processed, proteins are being removed into runoff blood and lacking to serve fundamental benefits through the consumption of meat (Nowak et. al., 2005).
A Thin Layer Chromatography, or TLC, assessment was performed on the two samples to assess and qualify the amino acids present between the two samples. This was done as a comparative analysis between the processed plasma sample and the unprocessed plasma sample. The first attempt failed because undiluted plasma was dotted on the plates and too much protein was present for the n-Butanol mobile phase to pick up and carry down the plate, resulting in large blobs smeared across the plates. After failing to perform this trial of undiluted plasma, 0.1 and 0.01 plasma concentrations were created in order to accommodate the mobile phase with a lower concentration level that it could handle and move more easily across the plate. The results obtained demonstrated that the unprocessed sample showed a greater range of identifiable amino acids than the unprocessed sample. The unprocessed samples exhibited a higher percentage of consistency with proven amino acid Rf values. A chart of known Rf value for each of the 20 amino acids was used as a standard for comparison for the experimentally determined Rf values. The plates run using the processed samples showed fewer identifiable amino acids, exhibiting only eight identifiable amino acids altogether, while the unprocessed samples exhibited ten identifiable amino acids (Figure VI). This is easily due to the denaturation of specific amino acids normally found in blood due to overexposure of blood to the environment. When removed from the body, all the proteins, carbohydrates, sugars, and even amino acids found in blood begin to denature rapidly (Nowak et. al., 2005).
Thin layer chromatography is the method commonly used to separate the amino acids out of a mixture, in this case processed and unprocessed plasma samples, based on the intermolecular forces, charges, and polarity of the different amino acids. (Unknown-1, 2005). The level of separation of the amino acids occurs depending on the affinity of each particular amino acid toward either the mobile phase or the stationary phase of the TLC test. The nonpolar silica plate serves as the stationary phase and attracts amino acids that are nonpolar and large, such as Glycine, Leucine, and Methionine (Figure V). The polar mobile phase, composed of n-Butanol, attracts small, polar amino acids such as Serine, Threonine, and Cysteine (Figure V). The transport mechanism of this separation system can be explained by a ãlike bonds with likeä principle that moves across the system due to surface tension (Kotz et. al., 2006). The non-polar silica gel will have an affinity for the nonpolar amino acids, causing them to move very slowly and only a short distance down the plate (Unknown-1, 2005). The polar mobile n-Butanol solution holds a very strong affinity for polar amino acids, readily carrying them down the plate and depositing them depending on size and degree of affinity (Unknown-1, 2005). Polar and electrically charged molecules will form strong interactions with other polar substances, sometimes due to free radical electrons interacting with a hydrogen atom on a neighboring molecule, called hydrogen bonding (Unknown-1, 2005). Non-polar molecules interact with one another in much weaker intermolecular bonds called London Dispersion Forces, which cause weak, temporary dipoles to be created, in this instance between the amino acid and the silica gel, that hold the two normally neutral molecules together (Kotz et. al., 2005). The result of this TLC test is the ability to identify any particular amino acid present in the sample due to the degree of affinity for the mobile and stationary phases and the distance traveled (Kotz et. al., 2005).
There were several reasons that may have causes the errors seen in this study. In the Bradford Assay, for example, several repetitions were required to obtain a standard curve graph that could be used. Faulty equipment, such as the leaky pipette we kept accidentally using, also caused some miscalculated solution measurement and mistakes in pipetting amounts, leading to major mistakes when making Bradford Assay concentrations. Another source of error was the sensitivity of the equipment used, such as the spectrophotometer. Lack of equipment sensitivity could have caused a slight misreading of the absorbance levels and therefore a skewed calculation of protein level, for example. An additional source of error was the amount of time that the TLC strips were allowed to develop before being sprayed, heated, and analyzed. There simply was not enough time for the strips to run as long as they should have, therefore yielding slightly less accurate results than if the strips had been allowed to run for the full two or two and a half hours. With less stringent time restraints, the results to all the tests could possibly have been repeated for more trials or simply been performed more carefully, yielding a more precise and accurate study.
For future study, it would be very interesting to examine the macromolecules found in blood on different levels. For example, due to complications in obtaining samples, this study focused only on two experimental samples: processed and unprocessed. If redone, more samples would be compared, with a variance of processing level, to answer the original hypothesis more thoroughly. Ideally, experimental samples would range from pure blood, blood from minimally-processed meat such as whole bovine legs, blood from medium processing, such as steak, and high-level processed blood, such as ground beef. This would yield a much greater analytical range of breakdown and denatured amino acids, which would address the hypothesis beyond the scope of this limited scientific study. To improve the present study, more samples from several different bovine blood donors, instead of just one test species, would be used. This would ensure that the results werenât influenced by unknown reasons associated with this one particular donor specimen. Additional tests to consider in future research would include doing different toxicity tests looking for different levels of metals or contaminants. It would be interesting to examine the different grades of meat for different toxicity levels, and to see if there is a direct correlation between meat grade and metal content.
Example Figures
