By: Group Guinness
Jeannette Kelly
Matthew Menna
Eric M. Rosenbaum
Samantha Salvia
LBS 145
Section Th1
Dr. Luckie
February 9, 2005
Abstract
People drink many types of tea, each characterized by the way it is processed (Dashwood, 1999). Green tea undergoes only withering and firing processes, while black tea is fermented before itâs fired (Dashwood, 1999). Our group wondered if fermentation affects black tea composition. We analyzed green and black tea, members of the Camellia sinensis species (Graham, 1992). To compare the components of each tea, the following assays were performed: Benedictâs Test to detect free aldehyde or ketone groups, Barfoedâs Test to distinguish monosaccharides from di- and polysaccharides, Selivanoffâs Test to detect ketoses or aldoses, Bialâs Test to detect furanoses, Iodine Test to detect starch, and Bradfordâs assay to quantify protein. Since each tea represents the same plant species, we predicted equal amounts of protein in both (Graham, 1992). However, our results show that green tea contained more protein than black tea. We predicted that all sugar tests would yield similar results, suggesting that processing doesnât effect which sugars are present; our results agreed. We predicted the greatest variation, due to processing, would be in the absorption spectrum test because processing involves an oxidative fermenting stage which affects the pigments in the leaves, (Ryzner, 2001). These predictions were not supported, since black and green leaves absorbed the same light wavelengths. We predicted the chromatography pigments to be the same and our results agreed. We were surprised to find more caffeine in black tea when we believed there would be equal amounts since fermentation doesnât affect the amount of caffeine in tea (Graham,1992).
Discussion
Our hypothesis was that the processing of tea leaves to make black tea does not have an effect on the components in the final products. To evaluate the components Benedictâs Test, Barfoedâs Test, Selivanoffâs Test, Bialâs Test, Bradfordâs Assay and an Iodine Test were performed on two types of tea, black and green. A caffeine extraction test was also performed to examine a compound that has come under fire for its negative health effects (Lane et al., 2002). At the conclusion of these tests, little difference between green tea and black tea has been found.
Benedictâs test for a free aldehyde or ketone group of a reducing sugar involved adding Benedictâs reagent to a dilute sample of our teas and boiling the mixture for 3 minutes. A positive result would form a red precipitate; however no significant precipitate was formed in all three trials of both teas. We encountered a trace amount in one trial of green tea but this positive result is most easily explained by human error because the other two trials were negative with no trace amounts of precipitate. Benedictâs test therefore yielded all negative results for green and black tea, this result is the first evidence to support our hypothesis that processing does not have an effect on the final product. While these results are definitive both green and black tea contains other reducing sugars, (Anonymous-3, Unknown). Further tests for these sugars could be done to better understand the components in tea leaves
Barfoedâs test distinguishes monosaccharide reducing sugars from more complex di- and polysaccharirides, by adding Barfoedâs reagent to a sample and boiling for two minutes (Krha et al. 2005). A positive result would result in red precipitate like Benedictâs test, but contrary to Benedicts test produced a brown precipitate on every trial. This seems fairly accurate and we can assume that human error can be eliminated, because the positive and negative control tests had the correct result. However, if due to human error, longer heating of the mixture can cause a positive result if polysaccharides are also present. A brown precipitate formed in both green and black tea, in all trials. However, the test formed a great deal more precipitate when performed on green tea than black tea. The test results while not quantitative support there being more monosaccharides in green tea. Although that is the only assumption about quantity of sugar, black tea is processed which could break down polysaccharides into monosaccharides. These results of Barfoedâs test give the first results that refute our hypothesis that teas do not differ after processing. And specifically do not contain reducing sugars with aldehyde or ketone groups, but contain reducing monosaccharides.
A simple iodine test gave differing results for black and green tea also. The test was based on the reaction of IKI reagent added to the tea solutions (Krha et al. 2005). A bluish-black color is the positive result caused by Iodine staining starch. The results were positive for all the trials of both solutions. The amount of color change differed between green and black tea, and by making the same assumption as we did for Barfoedâs test more starch is present in black tea than green tea. These test results are relatively accurate because there are so few steps in the test; however the process to determine if there was a color change was qualitative, relying on the vision of our group members. This is the strangest result insofar as that starch is a storage polysaccharide in plants and for it to be present in a processed tea plant leaf seems to contradict common sense, because the process to make black tea involves simply leaving the leaves to ferment. (cite) And the fermentation process might breakdown starch to do just that.
Selivanoffâs test is based on the dehydration of carbohydrates and differentiates between ketoses and aldoses. Selivanoffâs reagent is added to the tea solutions and placed in boiling water, while observations are taken. For a positive result of ketoses a red color would have to form under one minute while disaccharides and aldoses take several minutes to form the red color. The results for both tea types were the same, yielding no color change after one minute indicating the absence of ketoses. This data further supports our original hypothesis.
Bialâs test used Bialâs reagent added to the tea solutions, this was then placed in boiling water for 5 minutes. For a positive test result of pentose furanoses the tea solution will turn into a green solution while a positive test for hexose furanose will form an olive-brown solution (Krha et al. 2005). All of our tea trials for both tea types yieled no color change indicating the absence of any pentose furanoses and hexose furanoses. Human error in this experiment most likely did not affect the negative test results we had, both the positive and negative trials yielded the appropriate result, and also the consistency of the results rules out human error. This absence of sugars in both tea samples again supports our hypothesis by providing another similarity between the green unprocessed tea and the processed black tea.
The results of Barfoedâs test and the Iodine test are at first confusing. Research indicates that during the processing of black tea, or oxidation, starch is converted into smaller sugars (Graham, 1992). This leads to expectations of opposite findings than our own. It would be expected to find that Barfoedâs test formed more precipitate in black tea if monosaccharides formed during the oxidation of the black tea leaves. To explain these baffling results we looked at the flavanols found in tea. During the oxidation process of black tea, flavanols are combined and converted into oligomers (Yang, 1999). Flavanols are strong reducing agents (Makris, 2004),the flavanols in green tea could then reduce copper ions in Barfoedâs test . We also believe that Black teaâs oligomers could not react fast enough to reduce copper ions in the test which explains why green tea formed more precipitate than black tea in Barfoedâs test. As for the Iodine test, we believe that the oligomers in black tea were stained, this seems possible because the iodine can also stain other molecules like glycogen in animal cells although leading to a lesser degree of color change (Krha et al. 2005). This could explain why more blue specks formed when the test was performed on black tea than green.
Since the above tests only tested for sugar compounds we also performed Bradfordâs protein assay to help discriminate or identify differences in the tea types. The protein assay can only give accurate data after a standardization curve is established. This was done using Bovine Serum Albumin in known concentrations varying from 0 µg to 50 µg. These were then analyzed in the spectrophotometer at 595 nm and the absorbencies were recorded. The standardization curve showed a linear relationship between absorbance and protein concentration. We then analyzed our two teas three times each at 595 nm. The absorbencies obtained were then compared against the standard curve to get the protein concentration. Our data clearly showed similar amounts of protein in both black and green tea lending support to our hypothesis that the processing of black tea does not change the final product (Table 3).
A paper chromatography of the pigments in both green tea and black tea was done to determine the presence of pigments in a qualitative way, to supplement the absorbance spectrum. For the paper chromatography, a crushed sample of our tea leaves was dissolved in an acetone solution to extract the chlorophyll in chloroplasts. A capillary tube was used to deposit small drops onto a baseline on a strip of filter paper. The tip was placed in a tube filled in a petroleum ether chloroform solution to allow the pigments to begin their separation. After this was completed the strips were allowed to dry and the following data was collected.
When we performed the paper chromatography test, the results obtained for green tea and black tea were similar and all very faint. In the green tea, there was the presence of carotenoids (yellow and red pigments) and chlorophylls (green pigment). The carotenoids included α -carotene and β-carotene (both orange-yellow), and the chlorophylls included chlorophyll α (blue-green) and chlorophyll β (light green), (Suzuki, 2003). These would account for the orange-yellow, blue-green, and light green pigments.
With the black tea, we saw the same pigments present; however the intensity of the colors were lower in black tea. The orange bands may be lighter on the black tea chromatography strip, because there are less of α-carotene and β-carotene pigments in the black tea, (Suzuki, 2003). We believe the orange-yellow pigment would travel the farthest because according to other chromatography results, carotenoids travel the farthest, (Bassett, 2000). Next farthest would be the blue-green pigment, which is chlorophyll α followed by the light green pigment, chlorophyll β. These results were obtained when tested previously on ivy leaf extract, that this would be the order of the pigments, (Anonymous-4, 2003).
The processing of black tea clearly had some impact upon the pigments present and their concentrations. (cite) This processing involves crushing the leaves to expose pigments and enzymes to air allowing them to oxidize. (cite)
To supplement the paper chromatography an absorbance spectrum was done to more accurately determine the pigments present. This was done by analyzing samples of the teas at wavelengths from 400 nm to 700 nm. The data collected shows an exponential decay curve across the spectrum measured, with a small hump around 680 nm. This is intriguing data compared to the double humps we expected (cite) however both black and green tea were very close to each other which supports our hypothesis
The caffeine test performed yielded further data to support our hypothesis that the fermentation process has no effect on tea leaves. Our collected data is also supported because the fermentation process that the tea leaves undergo to make black tea does not affect the caffeine content of the tea leaves, (Graham, 1992). During the caffeine test a positive control of caffeinated coffee and negative control of water was used to validate the data to be collected. Using dichloromethane to extract the caffeine from the tea samples and anhydrous sodium sulfate to extract water and isolate caffeine. The results show that the caffeine levels of each trial of tea are consistent, but there is a difference in the amount recovered between black and green (Figure 1).
Overall, the two types of tea have provided very similar results Differing only in the caffeine, Benedictâs Bradford's and Barfoed's test. These tests are the only data that refute our original hypothesis that processing has no effect on the compounds in different tea types.
Figure 1. Results for caffeine test. These pictures show the results of the caffeine test. The most caffeine was extracted from coffee, the positive control, beaker A. Water, the negative control, beaker B had no caffeine. And there was about half as much caffeine in green tea, beakers C and D, as there was in black tea, beakers E and F.