Carbohydrate and Protein Assays Show Soy and Whey Protein Supplements are Advantageous in Improving Overall Health
By: The Amoebas
Ashlee Aude, Nicole Hedquist, Neeaz Mahmud, Brittany Sheets
Written by: Nicole Hedquist, Ashlee Aude, and Britt Sheets, Revised by: Ashlee Aude and Nicole Hedquist, Second Revision by: Ashlee Aude, Third Revision by: Neeaz Mahmud
Recent controversies have raised debates over which protein source is best for overall health. This nutritional study aimed to show differences between common protein sources, chicken, whey, and soy protein, by using a number of assays. Benedict's, Barfoed's, Selivanoff's, Bial's, and Iodine assays all tested for the presence of different carbohydrates. Benedict's test showed soy has a reducing sugar and Selivanoff's assay indicated both soy and whey have aldoses. Whey and soy have disaccharides or polysaccharides while chicken has neither. None of the protein samples tested, contained starch, but all contained a furanose ring.
Bradford 's assay, used to determine the concentration of protein, showed the highest average protein concentration for whey and the lowest for chicken. A protein analysis using an SDS-PAGE Gel Electrophoresis was used to analyze the protein sizes in order to determine the digestibility of each protein . This assay showed that whey protein was the most digestible of the three proteins tested, and chicken was least digestible.
A paper chromatography assay was used to test for the presence of the amino acids: L-valine, L-lysine, and L-leucine. We separated them using two different amino acid developing solvents. We believed all three would have some type of amino acid present (Cribb, 2004). However, the results of this assay were inconclusive. All of these assays combined attempted to show if there are any significant nutritional reasons for using one of the studied protein sources over another.
Figure: An In Vitro Protein Digestibility assay was used in combination with an SDS-PAGE Gel Electrophoresis. The resulting digested and undigested bands of soy, whey, and chicken protein samples, along with pepsin and pancreatin controls, are shown above. Using a known protein ladder, the chicken samples appeared to have moved to 26,626 Da showing they were still large proteins that did not digest very much compared to the undigested chicken control. The digested soy samples revealed a streak between 26,625 Da and 2,340 Da, showing more digestion than the undigested control soy sample. The digested whey, as well as the undigested whey, moved to 6,517 Da, when compared to the protein ladder. This showed that whey was more quickly digested than soy and chicken since the colored bands moved the furthest in the gel.
Discussion
Written by: Ashlee Aude, Revised by: Nicole Hedquist and Britt Sheets, Second Revision: Britt Sheets, Third Revision by: Nicole Hedquist
We researched and performed a series of carbohydrate and protein assays in order to find differences or similarities among three protein sources, whey, soy, and chicken. We analyzed these protein sources to find why someone might choose one source of protein over another, or add one of the supplemental sources, whey or soy protein, to a diet already containing chicken. Protein is crucial because it aids in all of the body's tasks, making it an important energy source for all cells in the human body (Duffy, 2004). Based on previous research, we concluded that each protein source would be used for different reasons. A woman suffering from pre-menopausal symptoms, for example, would benefit from a diet containing soy protein because it contains a type of estrogen called isoflavones, which relieves symptoms without a high risk of cancer development (Paxton, 1996). The average person, one not trying to gain massive amounts of muscle, or a woman not going through pregnancy or menopause, might not choose whey or soy because chicken protein would supply enough nutrients. Our original hypothesis, based on this research, was that there would be reasons for taking one source of protein, either soy, whey, chicken, or a combination, to meet a variety of nutritional needs. This was supported after performing a series carbohydrate, amino acid, and protein assays including a protein analysis SDS-PAGE gel electrophoresis.
After performing a sequence of carbohydrate assays, we found that soy contained a reducing sugar, ketone or aldehyde group, shown by turning red during the Benedict's assay. Barfoed's test was conducted to show if the proteins contained polysaccharides or monosaccharides. After the completion of Barfoed's assay we found that soy and whey both contain disaccharides or polysaccharides, shown by a slight color change. Selivanoff's assay was used to determine the presence of ketose or aldose in each of the three samples. This assay showed that soy and whey tested positive for aldose when they turned red after one minute of heating. Chicken did not change significantly when heated and, therefore, showed it was negative for ketose and aldose. Bial's assay showed the presence of a furanose ring in each protein sample. The positive control for a hexose-furanose ring, xylose, turned a muddy brown color, which matched the soy and whey samples. In the soy and whey protein, it showed hexose-furanose while in chicken it was pentose-furanose. The Iodine test showed there to be no presence of starch in any of the protein sources because all samples matched the negative control, water, and the samples did not turn a blue-black color.
Most of the carbohydrate conclusions stated above did follow our original predictions. However, we thought that whey would have a reducing sugar and that soy and whey would have tested positive for the presence of a ketose instead of an aldose. We also believed, previous to testing, that soy would have tested positive for starch, since it was derived from a plant, but by using the iodine test, soy was found to be negative for the presence of any starch. Overall, chicken was concluded to have few, if any, carbohydrates. Adding a supplement such as whey or soy to one's diet, would add carbohydrates not found in chicken. From a previous study it was concluded that the uptake of an important amino acid, L-phenylaline, was increased by three times with a protein shake containing carbohydrates (Anonymous -8, unknown). This supports our conclusion that whey protein would aid athletes since it tested positive for carbohydrates.
Protein concentration was analyzed using the Bradford assay, which showed that all three protein sources had high concentrations of protein. The whey protein was found to have a slightly higher concentration than the soy protein and chicken, which followed our original predictions. This might have been significant in understanding the nutritional benefits of soy and whey, however , sources of error may have accounted for the slight difference in the concentration. Error may have included color misinterpretation and inconsistency with time intervals in between additions of solvent. Also, the original protein samples may not have been diluted enough to get an accurate reading based on the BSA curve.
An In vitro Protein Digestibility assay was used in combination with a SDS-PAGE Gel Electrophoresis. By basically recreating the digestive system with some of the enzymes in the body, pepsin and pancreatin, the protein digestibility of soy, whey and chicken protein could be analyzed. The distance that the proteins traveled during the gel electrophoresis led to many possible conclusions when compared to the undigested controls. The proteins traveled down the gel by an electrochemical gradient that was created by a voltage source. The size of the protein determined how far the proteins would travel down the gel, and the exact distance was concluded using a protein ladder that contained known protein sizes. The digested chicken samples mostly traveled to 26,626 Da indicating that they were still very large proteins that did not digest much more when compared to the undigested chicken control. The digested soy samples showed up mostly as a streak between 26,625 Da and 2,340 Da, determined when compared to the standard protein ladder. The bands of digested soy samples illustrated that they were more digested when compared to the undigested soy protein control. Also, by comparing the undigested soy and chicken controls, it was possible to see that the chicken proteins were much larger to begin with. However, this could be due to the physical form. The chicken may not have been grinded up enough to allow for an equal amount of surface area for the enzymes to digest the chicken. Both the soy and whey protein began as a purchased powdered form, which would have enabled pepsin, pancreatin, and HCl to digest the samples more easily.
The whey control showed most of the sample at 6,517 Da and most of the digested sample at the same, 6,517 Da. This showed that even though the whey protein was digested very well, it is necessary to take into consideration the size of the original protein. The beginning size of the whey protein was very similar to the digested protein size, which could explain why whey protein is more easily digested. Our overall results support previous research that soy and whey products are easier to digest than chicken (Cribb, 2004). Therefore, nutritional needs need to be taken into consideration when deciding on the appropriate form of protein consumption. For example, an athlete that needs a lot of protein may consider a whey supplement.
The paper chromatography assay was inconclusive for the presence of amino acids for all three samples. However, from previous research, amino acids are present in soy, whey, and chicken, but from our assay, we were not able to conclude the presence of the specific amino acids, L-Leucine, L-Lysine, and L-Valine. Two different solvents were used with varying concentrations. The first, n-propanol and water with a 1:1 concentration did not show any coloration for the presence of the amino acids. Since it seemed that the samples did not move from the placement at the bottom of the chromatography paper, we concluded the solvent was not polar enough to overcome the polarity of the paper. Therefore, we used a new solvent, 1-butanol and glacial acetic acid at 1:1 and 4:1 concentrations. However, the result of this was also inconclusive.
Possible reasons for our inconclusive results may include defective samples of our amino acid controls since none of the colorless amino acids showed up, even when analyzed under an ultraviolet light. The correct solvent was unfound because so much depends on determining the correct polarity between the paper and the samples. Also, a major source of error could have included the unpurified, physical form and size of our samples. Our samples were not exclusively made up of proteins and even though the solutions were thoroughly blended and mixed, the size of the proteins may have been the reason why the samples did not travel up the chromatography paper.
Nevertheless, conclusions from previous research show that L- valine, L-lysine, and L-leucine are very important to the overall health of everyone, especially athletes. For example, an athlete training for a marathon would best benefit from adding whey protein to their diet because whey contains numerous amino acids, including L-Leucine, which helps in promoting tissue recovery after muscles have been used repeatedly (Cribb, 2004). This study sought to show if there were any significant reasons to consume one protein source, whey, soy, or chicken, over another. It scientifically showed the reasons someone might choose one source over another based on an individual's dietary needs and lifestyle.