Introduction

Written by Barbie Balasko

Revised by Corrinne Thomas

Revised by Scott Pontious

            In order to pursue optimal health, humans demand a diverse and balanced diet of both macronutrients and micronutrients (DellaPenna, 1999). The umbrella term macronutrients encompasses proteins or amino acids, lipids and carbohydrates (DellaPenna, 1999). It is this group of macronutrients that harness the majority of foods net energy (DellaPenna, 1999). Without a well balanced diet numerous degenerative diseases including dementia, scurvy and diabetes can develop (Blackman, 2006). Kwashiorkor, a disease that originates out of insufficient protein and overall calorie intake is associated in its early stages with fatigue, overall edema, and eventually leads to immunity failure or even coma in its severe stages

(Blackman, 2006). This disease, along with all of the diseases listed above, can be corrected by altering the diet of the affected individual to compensate for the missing elements. The debate lies in discovering the best source of these elements. Meat has traditionally been regarded as a superior source of protein, essential amino acids and some vitamins such as zinc, iron and B12 (Lion, 2006). Meat is not only difficult to attain in some regions of the world but it is also much more expensive than its plant competitors, such as soy. Along with its expense, red meat such as beef has been criticized in the past due to its association with obesity and cancer because of its high fat content (Biesalski, 2005). Soy, on the other hand, has been accredited with extending the length and raising the quality of life for those who consume it on a regular basis. Soy protein products can substitute for animal based protein products because soy offers a comprehensive protein report (Henkel, 2000). Soy protein which is low in saturated fat and cholesterol may reduce the risk of certain heart diseases (Henkel, 2000). The people of Japan and Greece have the longest life expectancies in the world; their dietary source of protein derived mainly from soy products has been thought to be the reason for such long lives (Savige, 2002).

            In order to better assess the nutritional content of soy and beef a comparative study using various assays and test has been preformed. Several studies have supported the health benefits of soy over that of red meat such as beef. It was initially hypothesized that gram for gram, fresh soy beans would be overall more nutritious than raw Meijer ^© beef. We defined nutritious as a high protein content in addition to the presence of complex carbohydrates, starch and essential amino acids. The following five assays were used to analyze the macronutrients. For all tests a stock solution was prepared of beef and soy for consistency purposes in results. Barfoed’s test was used to distinguish between simple and complex sugars or monosaccharides, disaccharides and polysaccharides by way of an oxidation and reduction reaction. It was predicted that the soy beans would have a higher presence of complex carbohydrates than beef because prior testing has shown that carbohydrates are present at higher levels in soy than in beef (Karr-Lilienthal et al., 2005). A lack of precipitate in Barfoed’s test indicated that soy beans and beef contain disaccharides and polysaccharides. Although one sample of the soy was found to have a white precipitate is was concluded that it resulted from the product falling out of solution. Selivanoff’s test was used to detect ketoses and aldoses. This reaction was made possible by placing resorcinol in concentrated HCl. In the presence of ketoses or aldoses and enough heat, the resulting solution turned red in color. It is known that ketoses react faster than aldoses; roughly one and two minutes respectively. It was predicted the soy beans would have both aldoses and ketoses while the beef would have neither. This was speculated after an extensive literary review beef had been found to have a zero percent of the recommended daily allowance or RDA for carbohydrates (Lion, 2006). Both soy beans and beef were found to have aldose sugars. The Iodine test for coiled polysaccharides was used to test for the presence of starch, a complex carbohydrate. In the presence of iodine-potassium iodide (I₂KI) compounds containing starch turn a bluish black. It was predicted that starch would be present in the soy solution and absent from the beef solution. This is because it is known that plants store extra energy as starch and because animals store complex sugars and use glycogen, not starch, for structural support of their tissues. Starch was present in soy and absent in beef. The Bradford assay was used to test for the total protein concentration in the beef and soy solutions. Our dilutions were compared to a pre-set standard curve and their concentrations were disclosed. It was predicted that the soy solution would have a higher protein concentration, which was not supported by our experimental data. It was found that beef contained more protein per serving than soybeans. TLC was used to detect the presence of the essential amino acids Arginine, Tryptopan, Histidine, and Phenylalanine. Different amino acids were detected based on the idea that amino acids with heavier molar masses migrate more slowly up the TLC plate than amino acids with lower molecular weights. Intermolecular forces also help determine which amino acids migrate faster and slower. Differential movement up the TLC plate grants RF values and allows various amino acids to be distinguished from each other. It was predicted that the soy solution would have a more amino acids than beef because past research has shown that soy has all of the essential amino acids (Henkel, 2000). This was not supported in our research; it was found that beef contained more essential amino acids than soy. Soy contained Valine, Methionine and either Cysteine, Lysine, or Histidine . Beef contained Phenylalanine, Proline, Tyrosine and either Cysteine, Lysine, or Histidine. An independent lipid assay was performed using the lysochrome Oil Red O. Known dilutions of canola oil and hexenes were compared to unknown dilutions from the pellet extract of both soy and beef in hexenes. This was used to approximate the amount of lipids in beef and soy solution. It was predicted that beef would have a higher lipid content than soy (Anonymous- beef, Anonymous-soy). This was predicted partially because the beef that was used in the tests contained 20% fat initially. This test was found to be inconclusive. Bials test was used to test for pentose or hexose sugars. This test was preformed due to time constraints after the lipid test failed to produce usable data. It was found that both beef and soy contain hexose sugars.

            After performing the above tests it was found that beef had a net higher nutrition value due to its more complete set of essential amino acids, its complex carbohydrate content and its high concentration of protein per serving, negating our hypothesis.

Methods

Written by Scott Pontious

Revised by Barbie Balasko

Revised by Jennifer Johnson

Stock Solution Preparation

            An eight ounce bag of Meijer brand dried soy beans (Figure 1) were obtained and soaked in distilled water for seven days, blended with 25g to 300mL of water , and strained through 5 layers of cheese cloth.  The resulting stock solution had a concentration of 0.08g/ml of soy to distilled water. The mixture was then centrifuged for five minutes, the supernatant was isolated for use and the pellet discarded.  Nine ounces of fresh uncooked Meijer^© brand ground chuck (Figure 2) was obtained from Meijer^© on Grand River in Okemos. The beef solution was obtained by grinding the raw meat in a blender, 25g to 300mL of water, and then sifting it through 3 layers of cheese cloth.  The resulting stock solution had a concentration of 0.08g/ml of beef to distilled water. This mixture was then centrifuged for five minutes, the supernatant was isolated for use and the pellet discarded. 

Barfoed’s Test

            First, 3ml of Barfoed’s solution were placed into two standard test tubes labeled ‘soy’ and ‘beef’; 500μl of stock soy bean solution were placed in the soy test tube and 500μl of beef stock solution were placed in the other labeled beef. All test tubes were placed in a boiling water bath (100 degrees centigrade), observed and heated for 2 minutes. Three trials were performed to account for anomalies, n=3. Positive, negative and mock trials were preformed using glucose, sucrose and water respectively (Krha et al., 2006).

Selivanoff’s Test

                        For this test, 350 μl of stock beef solution and 350 μl of stock soy bean solution were placed into labeled test tubes to identify treatments and replicates (to rule out error). Next, 3 ml of the Selivanoff’s reagent were added to each tube. The individual test tubes were then heated in boiling water (100 degrees C), and the observations were recorded. Three trials were performed to account for anomalies, n=3. Positive, negative and mock trials were performed using fructose, glucose and water respectively (Krha et al., 2006).

Iodine test for Coiled Polysaccharides

            First, 350 μl of stock beef solution were placed into a test tube and 350 μl of stock soy bean solution were placed into a separate test tube labeled beef and soy respectively. Each solution was vortexed vigorously before testing. Thirty-Five μl of I₂KI were then placed into each test tube. Three trials were performed to account for anomalies, n=3. Positive, negative and mock trials were preformed using starch, glucose and water respectively (Krha et al., 2006).

Bradford Assay

            A standard curve was produced using the absorbance values of a dilution series created from bovine serum albumin or BSA. In order to make a standard curve, concentrations of 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 μg/μl of BSA to ddH₂0 were prepared. These concentrations were read at 495 nm in a spectrophotometer and a graph of their absorbencies was created. Next, 50 μl of each concentration were placed test tubes with 50 μl of 0.1 molar NaOH. The test tubes were vortexed. After 5 minutes 3 ml of Bradford’s reagent was added to each test tube. The test tubes were vortexed and left to stand for 5 minutes. BSA, NaOH and ddH₂0 were used as a blank for the spectroscope. The absorbance of each dilution was recorded at 595 nm and a standard curve was prepared. The standard curve itself was created by using the protein concentration (μg/μl) as the x-axis and the absorbance as the y-axis. The second step included repeating the above procedure for a standard curve only replacing the BSA with beef and soy solution. The protein content of the unknowns could not be found without first finding the protein amount (μg) of the BSA. This procedure was done by multiplying the concentration by 50 μl. Once that was accomplished, by using the line on the standard curve, the protein content of the soy and beef were found. Three trials were preformed to account for anomalies, n=3 (Krha et al., 2006).

Thin Layered Chromatography (TLC) Test

            Thin-layered chromatography was used to test for the presence of essential amino acids in beef and soy. The beef and soy solution used in this experiment were prepared as they were in the previous experiments, only the concentrations differed. Five grams of sample to 50mL of n-Butanol-acetic acid-water were mixed in an 80:20:20 mixture. A pre-set stock amino acid solution, from the Sigma Chemical Company which contained known amino acids was obtained and used as a standard. Large glass test tubes were used for developing chambers. The solvent was an 80% acetone mixture. The chambers had 2ml of solvent placed in them and were allowed to reach equilibrium, approximately 10 minutes (Fried, 1999). Two TLC strips measuring 2cm x 20cm from Whatman Company, had an origin line drawn 2 cm from their base. The first TLC plate had a 5 μl solution of the mixed amino acid standard painted in a thin line on its line of origin. The second TLC plate had a 5 μl solution of the beef solution painted in a thin line on its line origin. The two TLC plates were placed into the developing chamber and allowed to develop for 1.5 hours. This process was simultaneously run for soy. Each beef and soy had three trials preformed as to account for anomalies, n=3 (Krha et al., 2006). After 1.5 hours the TLC plates were removed, placed in a fume hood, and sprayed with 0.5% ninhydrin, and allowed to dry. The strips were placed in a 60 degree centigrade oven for 15 minutes. The Rf values were calculated.

Independent Lipid Assay

            First, stock solution preparation was initiated in the manner described above for both beef and soy bean. After the solution was centrifuged for 10 minutes the supernant from each centrifuge tube was extracted using a pipette and set aside. A minimal amount of hexenes (3-6 μl) was then added to the pellet and this mixture was centrifuged again for 10 minutes. Next 500 μl of pellet-hexene mixture was placed in a test tube. Oil Red O was added in drops until a color change was noticed. Three trials of the above procedure were preformed for both beef and soy, n=3. Each trial had a different concentration of pellet-hexene mixture. The first trial of both beef and soy used the mixture created above. The second trial of both used a 50:50 mixture of the above solution to hexenes. The third trial used a 40:60 mixture of the above mixture to hexenes. The number of Oil Red O droplets required to produce a significant color change was recorded. This data was compared to the data received from the olive oil dilutions.

            Meijer^© canola oil dilutions were preformed by running three trials of different concentrations of canola oil to hexenes. The first trial was pure canola oil. The second trial was a 50:50 mixture of canola oil to hexenes. The third trial was a 40:60 mixture of canola oil to hexenes. A graph of amount of oil against number of drops of Red O required to produce noticeable color change was produced.

 Bials Test

            The Bials solution was made by combining 0.05g of Orcinol, 0.05g Ferric Chloride, 50mL water, and 50mL of 12 molar HCl. The resulting solution was mixed well. Next, four test tubes were labeled beef, soy, xylose and glucose. These were then filled with 500μl of stock beef solution, stock soy bean solution, xylose and glucose respectively. Xylose was the positive control for pentoses, and the negative for hexoses while glucose as the negative control for pentoses and the positive control for hexoses. Water was also present and labeled in its own tube as the mock control. Once each sample was in its own test tube, 3mL of the Bials solution was added, and the test tubes were placed in 100^o(C) water for 10 minutes. After 10 minutes, the tubes were removed from the water, and the results were recorded. This test was repeated for a total of three times, n=3.

Results

Written By Barbie Balasko

Revised By Jennifer Johnson

            Barfoed’s carbohydrate test, Selivanoff’s test, the Iodine test for coiled polysaccharides and the Bradford assay were all performed on January 30^th 2006 in the LBS 145 laboratory. Barfoed’s test was negative for the presence of monosaccharides in all three trials of both beef and soy. It was positive for disaccharides or polysaccharides in all three trials for both beef and soy (Figure 3). A white precipitate was found in one trial of the soy solution (Figure 4). Selivanoff’s test showed that in 3 of 3 trials beef was positive for aldoses. In 3 of 3 trials soy was positive for aldoses (Figure 5). Neither beef nor soy was positive for ketoses. The iodine test for coiled polysaccharides showed 3 of 3 trials that soy was positive for starch. It showed for 3 of 3 trials that beef was negative for starch (Figure 6). An average of three trials of the Bradford assay showed that an average 3 ounce serving of beef contained approximately 0.017g of protein. An average 2.66 ounce serving of soy was found to contain approximately 0.0093 g of protein.

            On February 20^th 2006 the Amino Acid TLC, Independent Lipid Assay, and Bials Test were also performed in the LBS 145 laboratory. The Amino Acid TLC yielded Arginine, Proline, Tyrosine, Phenylalanine, and either Lysine, Histidine or Cysteine for beef. Soy tested positive for Valine, Methionine and either Lysine, Histidine or Cysteine (Figure X₁). The Independent Lipid Assay was run without success. There was no apparent color change to create the standard curve needed to obtain the desired results (Figure X₂). Hexanes were then tried in place of Hexenes, but to no avail. Bials Test concluded that both beef and soy tested positive for hexoses. Both beef and soy tested negative for pentose.

Discussion

Written By Corrinne Thomas

Revised By Jennifer Johnson

Revised by Barbie Balasko

A well balanced diet is essential for healthy living. In the past beef has been known for its high protein content and health benefits when consumed in small portions (Biesalski, 2005). Yet with the rise of vegetarianism in the United States, soy now has the potential to become a more nutritious substitute. This rise in popularity has raised the question of which of the two really are more nutritious. Nutrition in this case is considered to be a higher amount of protein, more complex sugars, and a higher concentration of essential amino acids. Essential amino acids are those that the body can not produce however they are important to proper development and growth. Essential amino acids must be obtained through diet (Biesalski, 2005). It was predicted that soy would have a more complete compliment of protein, have more complex carbohydrates, and have more essential amino acids. Because of these predictions soy was also hypothesized to be overall more nutritious. A comparison of both beef and soy was made by testing the two in the fields of proteins, amino acids, carbohydrates, and lipids.

            In order to make these evaluations the first series of tests compared the carbohydrates in soybeans and beef. The first carbohydrate test, Barfoed’s test, was used to distinguish between the simple and complex sugars monosaccharides, disaccharides and polysaccharides. Both soy and beef were shown to contain disaccharide or polysaccharide sugars. A limitation of Barfoed’s test is that a distinction between disaccharides and polysaccharides can not be made. The second carbohydrate test used to analyze the nutritional content of beef and soy was Selivanoff’s Test. In Selivanoff’s test, if ketoses or aldoses are present a color change will occur and the solution will turn a pink-red in color. Because it is known that ketoses react faster than aldoses, roughly one and two minutes respectively, the two sugars can be differentiated from each other. This test showed the presence of aldose sugars and not ketose sugars in both beef and soy. A third carbohydrate test, the Iodine test for coiled polysaccharides revealed that the soybean sample contained starch and the beef did not. This was expected due to the fact that plants store complex carbohydrates as starch while animals store their complex carbohydrates as glycogen. Animals do not produce starch. The final carbohydrate test was an independent assay. Our original independent assay was a lipid assay that will be discussed in detail later in the paper. Because the lipid test failed at the last minute time constraints left no choice but to find an alternative test. The alternative independent assay was Bials test which differentiates between pentose and hexose sugars by way of a color change. When placed in boiling water with Bials solution, the stock beef or soy solution will turn yellow-green for a hexose and blue-purple for a pentose sugar. Bials test revealed that beef and soy both have hexose sugars. After Barfoed’s, Selivanoff’s, the Iodine test, and Bials test were preformed it was concluded that soy had a slightly higher nutritional value due to the presence of more complex carbohydrates than beef (Henkel, 2000). Complex carbohydrates are healthier than simple carbohydrates because the body breaks them down more slowly preventing a spike and sharp drop in blood glucose levels. The gradual digestion of complex carbohydrates promotes elevated moods and steady energy levels. This is due in part to a consistent release of serotonin from the breakdown and digestion of the carbohydrates. (Anonymous-4, 2005). With a qualitative investigation of the sugar content of both beef and soy accomplished, further investigations on other macromolecules were then explored.

            In order to further compare and contrast soybeans and beef an independent Lipid test was attempted. The Lipid test was performed using the lysochrome Oil Red O. A standard curve was formulated by plotting the number of drops of Oil Red O it took to induce a color change against varying concentrations of canola oil and hexenes. A graph of unknown dilutions of pellet extract in hexanes was then compared to the standard curve to approximate the amount of lipids in each beef and soy solution. The results of this test were inconclusive as the amount of Oil Red O it took to induce a color change in any concentration of either canola oil to hexenes or stock solution to hexenes was 1 drop. In fact, the color change of all concentrations was the same hue. The use of hexane as a substitute for hexenes was also attempted with the same result. This made the standard curve and the comparison line both perfectly horizontal lines with a slope of 0. It is speculated that our stock solutions were not different enough in their lipid concentrations to show visible color difference. After the Lipid test was inconclusive and with only experimental information on the carbohydrate content of beef and soy, the macromolecule of protein was considered.

            Both the Bradford assay and TLC tests were used to broaden the scope of our investigation of the nutritional content of beef and soybeans. The Bradford assay was used to test for the total protein concentration in the beef and soy solutions. Beef was found to have 0.0017 grams of protein per serving and soy was found to have 0.00093 grams of protein per serving. A standard serving of beef is 3 ounces while a standard serving of soy is 1/3 cup. For comparison purposes, three ounces is equivalent to a deck of cards and 1/3 cup is about 2.66 ounces (Anonymous-1, unknown). When compared to the commonly ascertained value of 11 grams of protein per serving for soybeans and 17.5 grams of protein per serving for beef, it is strongly indicated that our experiment has wide reaching error (United States Department of Agriculture, 2006). With our three trials for soy having a standard deviation of +0.134 and +0.452 for beef our experimental data clearly represents the impreciseness of this test. While the Bradford assay quantified protein content the TLC test was used to qualify the essential amino acids in both beef and soy. It was found that soy contained Valine, Methionine and either Cysteine, Lysine, or Histidine. Beef contained Phenylalanine, Proline, Tyrosine and either Cysteine, Lysine, or Histidine. Our protein tests supported the conclusion that beef has a higher protein content per serving and contains more essential amino acids than soy (Lion, 2006).

            Through carbohydrate tests and protein assays the hypothesis that soy has overall more nutrition per serving has not been supported in our studies. Our studies have upheld that beef is more nutritious per serving than soy when comparing various complex carbohydrates and overall protein complements. While beef and soy had about the same content of complex carbohydrates beef was shown in both the Bradford assay and the TLC test to have more protein and essential amino acids than soy.

            While most tests preformed in our investigation of soy and beef went smoothly, some aspects of each of the experiments could be improved in future studies. All of the sugar tests were simple in their execution and accurate; it is recommended that the procedures for these tests be kept the same in future. As mentioned earlier, one drawback to Barfoed’s test is that it can not distinguish between disaccharides and polysaccharides. Being able to distinguish between these sugars would allow a better determination of the level of complex carbohydrates in both beef and soy. Future studies could account for this and utilize alternative tests that can distinguish between the two sugars. The protein tests did not run as smoothly as the carbohydrate tests. The Bradford test was neither precise nor accurate as the machine that is vital to its execution was not functioning properly. The spectrophotometers available in the LBS 145 laboratory are in very poor repair. After zeroing the spectrophotometer it would read different absorbencies for a stock solution after the solution was removed and replaced. The readout wavelength in nanometers was not changed during this time period. Re-zeroing the machine a second time with the same blank yielded yet another different absorbency for the same stock solution at the same wavelength. A second weakness in the Bradford assay is that interpretations of protein concentrations by way of comparing samples to a standard curve are close approximations at best. Also, the end quantity of protein per serving for both beef and soy was much lower than commonly reported values. Possibly because of the limitations of the solvent or the spectrophotometer some protein in the sample was not detected. While preformed with fewer errors than the Bradford, the TLC test did have some problems of its own. The TLC test is a qualitative test that is time limited test that is dependent on constancy. An amino acid TLC can take upwards so 5 ½ hours to run to completion while the lab allotment for time is only 3 hours. While additional lab periods were made available, other laboratory groups were in the lab at this same time creating a shortage of equipment and general tension between parties. This undoubtedly lead to hurried actions and error. Because of the tense and hurried conditions under which the TLC test was preformed the consistency of applying the sample to the TLC plates varied greatly. This affected the results with even more magnitude in that thin, even additions of sample to the TLC plate are needed for clear band patterns to develop in the end. Uneven additions of sample to the plate create wavy or even vertical bands and therefore the RF values which identify the amino acids are altered. In the future more time should be allotted for tests and an alternative, more accurate and precise protein tests should be used. Aside from specific testing procedures being enhanced there are some general aspects of this laboratory that should be addressed in impending investigations on beef and soy.

            More trials and newer equipment could be used in the future to avoid inaccurate and imprecise data. Altering testing sites could reduce random error, and switching experimenters could reduce human bias. By addressing these issues in the future it is speculated that tests such as the Bradford would yield much more accurate results. While improving upon current tests could improve this investigation in the future, looking into new areas will also broaden the depth of future results.

            Future research could include comparing of isoflavones in soy and beef. Isoflavones are a secondary vegetable substrate which have been found to have protective functions (Anonymous-1, unknown). Isoflavones are known to have health benefits such as reducing the risk of cancer, easing menopausal symptoms, reducing the risk of heart disease, protecting against problems regarding the prostate and improving bone health (Anonymous-3, unknown). For these reasons isoflavones would have an impact on soy’s overall benefit or nutritional value. Future studies comparing beef and soy nutritional value should include isoflavones.

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Written By Corrinne Thomas

Revised By Jennifer Johnson

Revised by Barbie Balasko

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Figure 5. Results from Selivanoff’s test. This test is used to differentiate between ketoses and aldoses. 350μl of sample and 3ml of Selivanoff’s reagent were placed into separate test tube. Each test tube was placed in boiling water, observations were recorded immediately.. From left to right: beef (A), soybean (B), glucose (negative control) (C), fructose (positive control) (D), and de-ionized water (mock trial) (E).