By: Joshua McCallum, Tim Karrer, Melissa Southern, Abby DeWildt
LBS 145
October 20, 2003

Abstract
Our group obtained three different samples of energy drinks to use in our investigation: Red Bull, V8 Splash and Gatorade. We performed this investigation to determine which drink was best at replenishing the body. We hypothesized that all three would contain carbohydrates, V8 Splash would possess photosynthetic properties and all would have protein. Also, V8 Splash would be the most efficient. We tested each drink for carbohydrates using Benedict’s, Barfoed’s, Selivanoff’s, Bial’s, and the Iodine test. From Benedict’s and Barfoed’s tests, a red precipitate present in all three drinks showed reducing sugars. From Selivanoff’s test, ketoses present in all three drinks was supported because all reacted in under a minute. Bial’s test showed a presence of furanoses in all three drinks because all reacted as the positive control did. From the Iodine test it was supported that none contained starches unlike the control, starch, which turned a blue-black color. We also performed two photosynthetic tests to determine if our samples possessed photosynthetic properties. The absorption spectrum showed only Red Bull and V8 Splash had any possibilities of pigments. The paper chromatography test showed that only V8 Splash contained one photosynthetic pigment, carotene. The last test performed was the Bradford Protein Assay. Using the spectrophotometer we compared our samples’ absorptions at 595nm with the protein standard assay curve. Our protein concentrations found were V8 Splash=.168mg/ml, redbull=0mg/ml, and Gatorade=.031mg/ml. We determined that V8 Splash is best at replenishing the body due to its carbohydrate content, natural ingredients and the greatest protein concentration.

Figure 2C: Buchner Filter Concentration of Barfoed’s Test. This figure shows the different concentrated of red precipitate from Barfoed’s Test. From left to right, the precipitate from Gatorade was found to be .023g, the precipitate from Red Bull was found to be .014g, and the precipitate from V8 Splash was found to be .021g. This shows that from our three samples found through Barfoed’s Test, Gatorade contained the greatest amount of reducing sugars.

Discussion
This investigation was initiated by a motive to determine which energy drink is the most energizing, revitalizing, and overall most effective. We decided to find our answer by testing Gatorade, Red Bull, and V8 Splash for concentrations and type of carbohydrates, concentrations of proteins, and also by performing photosynthetic pigment tests and absorption spectra. Our initial hypothesis was that all the drinks would contain carbohydrates and proteins and that V8 Splash would be the only one to have photosynthetic properties shown by pigments and the absorption spectra, thus V8 Splash would be the most effective energy drink. V8 splash has ten percent real juice in it yielding the thought since it is more natural, it would be the best energy drink.

We performed experiments and found that while some results supported our hypothesis, others did not. Every experiment was replicated three times and the average results were then analyzed to conclude that our hypothesis was supported or revoked. Controls of a known sample were also done to verify our experimental results. The six sugar tests showed that all three energy drinks contained levels of carbohydrates and our experiment using the Bradford Assay tested for the presence of proteins. In our photosynthesis analysis only the V8 Splash showed that it contained pigments and showed evidence of photosynthesis in the in both the paper chromatography test and absorption spectra.
For our sugar tests we did a variety of tests to determine the presence of reducing sugars. Five different sugar experiments were set up and carried out: Benedicts, Barfoed’s, Selivinoff’s, Bial’s, and Iodine test for Coiled Polysaccharides. In our carbohydrate experiments, we already know that carbohydrates are the main energy source for the human body and by testing for quantity and the different types contained in our test samples, we can conclude which is the most effective energy drink. Fruit drinks are known to have higher concentrations of carbohydrates, proteins and enzymes (Court 1995). We think because the V8 Splash is made from more natural ingredients it contained photosynthetic pigments because it’s the only drink that comes from raw material or natural juice. Proteins are also important to the human body and we predicted all three drinks will have protein content.

In the human body most dietary sugars are converted to glucose. Glucose is the major fuel used by all cells and primary food required by brain tissue for normal function (Anderson 1997). In refueling the body with an energy drink, reducing sugars are an essential component. Monosaccharides are the simplest form of a reducing sugar; Selivanoff’s test concludes that all three energy drinks contained monosaccharide ketose. These refined carbohydrates are converted to glucose, which quickly refilled your intestine with glucose (Anderson 1997). Gatorade, V8 Splash, and Red Bull all reacted in Bial’s test concluding that each of the drinks contain a hexose-furanose ring on their carbohydrates and in the Iodine test none of the solutions reacted leading us to believe that none of the beverages contain starch. So all three energy drinks contained carbohydrates that were monosaccharide ketoses and with hexose-furanose rings and none of them contained starch. Looking at these results the reducing sugars in Gatorade, V8 Splash, and Red Bull all tested positive for the same carbohydrate tests but at different levels of concentration. Taking the precipitates of Benedict’s test and calculating the concentration of reduced copper, Gatorade had the highest concentration, 5.4X10-5 grams of reduced copper per micro liter of solution. The higher concentration of reduced copper leads to the conclusion that there was a higher concentration of reducing sugars because the sugars are what reduced the copper and made the precipitate, so the more sugars there are in a sample the more precipitate there will be. Also in Barfoed’s Test, which precipitates monosaccharides, a form of reducing sugars, Gatorade had the highest concentration of 3.3x10-5 g/ml. Red Bull had the second highest concentration in the Benedict’s test of 2.8x10-5 g/ml and the lowest in the Barfoed’s Test of 2.0x10-5 g/ml. V8 Splash had the lowest concentration in the Benedict’s test of 2.4x10-5 g/ml and the second highest in the Barfoed’s test of 3.0x10-5 g/ml. All three energy drinks have the same types of carbohydrates but in different concentrations, we conclude that Gatorade has the highest level of concentration of reducing sugars, making it the most efficient in replacing burned off sugars such as glucose after exercise. This evidence does not confirm our hypothesis that V8 Splash is the preeminent energy drink, but that in the carbohydrates test Gatorade may be more energizing.
In the photosynthesis portion of our investigation we performed a paper chromatography test to show any photosynthetic pigments in our sample beverages, as well as absorption spectra on the three drinks. Due to the fact that V8 Splash is the only one of our three test samples that contains juice derived from actual plants, we believed that it would be the only beverage to display photosynthetic properties. To test this idea we performed a paper chromatography test and absorption spectra to show any of four possible pigments that may be contained in our samples. The presence of any of these pigments indicates that photosynthesis occurred at one time, which allows us to infer that there are natural, plant ingredients in the beverage. Upon completion of the absorption spectra on each of our samples we plotted the absorbance verses wavelengths and compared the plots to a plot of absorbance verse time of spinach leaves as well as a graph that shows the wavelengths that different pigments peak at. From this we determined that the V8 Splash had its highest peaks in the areas that could show a presence of Chlorophyll a and Carotenoids. The Red Bull had the highest peaks in the area that may indicate Chlorophyll b (Freeman 2002). The absorbencies for the Gatorade were so close to zero for all wave lengths that we could assume that no pigments were present.

To further test these results we performed the paper chromatography test on each of our sample beverages. As expected neither the Red Bull nor the Gatorade drinks showed any evidence of any of the photosynthetic pigments. However, the V8 Splash showed an orange/yellow color about half way up the paper strip which indicates the presence of the pigment carotene. This result also fit with the result we obtained from the absorption spectra. From these tests we were able to determine it is likely that the V8 Splash contains Carotene and the remaining two beverages do not contain any pigments. These results concur with our hypothesis. V8 splash was the only drink that contained pigments and indicated photosynthesis.
The final portion of our investigation into the comparison of three energy drinks consisted of the Bradford Assay to determine protein concentrations in each beverage. We predicted that all three beverages would contain proteins and that each would have a similar concentration. We completed the Bradford Assay on each of our drinks as well as a control BSA sample. From the control BSA we created a Protein Standard Assay curve and used the line of best fit to compare it to our absorbencies of our three samples in order to find the protein concentrations of each. The results of the Bradford Assay show that V8 Splash has a protein concentration of .168 µg of protein per µl of sample. The Gatorade had a concentration of 0.031µg/µl, and Red Bull energy drink had a concentration of zero. These results contest our hypothesis that all three drinks would have proteins and that each would contain similar concentrations of proteins. In fact the V8 Splash had a higher concentration of proteins by quite a large margin.

After analyzing all of our results from the carbohydrate tests, photosynthesis tests, and Bradford Assay we were able to determine that some of the tests confirmed our hypothesis and others did not. The purpose of our investigation was to examine Gatorade, V8 Splash, and Red Bull energy drinks and to determine which was the most beneficial to the human body. We predicted that the V8 Splash would be the best drink and several of the tests confirmed our prediction. The V8 Splash had the highest concentration of proteins, which are important to the human body. From the photosynthesis tests we determined that V8 Splash was the only drink that contained evidence of photosynthesis which indicates that there are ingredients in the drink that are made directly from plants. This leads us to make a preference for the V8 Splash over the other two drinks from the standpoint that it is made from more natural ingredients. However, in the carbohydrate tests it was determined that Gatorade had the highest concentration of reducing sugars, which are important to energize the body. In conclusion we can say that it is likely that the V8 Splash is the most natural of the three beverages, but it is not necessarily the most efficient drink in terms of providing energy for the body.

Although our experiments were performed with precision and accuracy, there are a few weaknesses that we were able to identify in our design. A few problems that we thought we might have during the experimentation with distinguishing the actual color of the results from our tests during the carbohydrates test. The energy drinks already had a distinct color, but didn’t affect the color changes of the reactions so the tests were easy to interpret. Another problem we encountered when doing the carbohydrate tests is that the tests we did only gave us a broad generalization of which types of carbohydrates our drinks contained and not any specific carbohydrates. The distinct colors of the energy drinks could also be a problem in the Bradford’s Assay in testing for proteins but shouldn’t be because the sample size is so small. After completing the Bradford Assay on each of our drinks as well as a control BSA sample we encountered a problem. There were big discrepancies between the three trials done for each drink. This may have been due to dirty test tubes or errors in adding materials according to our procedure. We performed the Assay again from the beginning and achieved much better results. We only used three different energy drinks in our experiment out of hundred of energy drinks on today’s market, and we are well aware that other drinks could be more effective, but due to time and other factors, these are the only three products we tested.