A Cup of Tannins: The Link Between Tea Fermentation and Antioxidants

My-Lien Nguyen

Westmoore High School Oklahoma City, Oklahoma March 30, 2006

Abstract The purpose of the project was to find how the amount of fermentation of tea affects the amount of tannins in tea, and therefore, the antioxidants in teas. The project consisted of preparing plates, grinding tea samples, and then placing tea samples in the plates for testing. The project would determine if the amount of fermentation in teas directly affects the tannin amounts in teas. In all, nine variations of teas were used: Green tea, Decaf Green tea, White tea, Jasmine tea, Oolong tea, Ti Kuan Yin tea, Black tea, Pu-erh Tea, and dried tea leaves. A control of methanol and another control of tannic acid were also used for comparison among the tea types. There were few patterns in the results of the experiment. Several tea types including Pu-erh tea and Black tea samples yielded no visible results and no data could be taken. The amount of tannins in tannic acid increased steadily with greater amounts. Overall, the Decaffeinated Green Tea had the most amounts of tannins, with White and Green teas as second most in tannins. The lesser fermented teas had more tannins than the teas that were semi-fermented or fully fermented except for Jasmine tea and the dried tea leaves. If this was the only data taken into consideration, then fermentation in teas could have a direct connection with the amount of tannins since the tannin amounts increased with less fermentation. Overall, the hypothesis was supported, although further testing is necessary. Introduction The topic was chosen in order to find if the amount of fermentation in different kinds of teas from the same plant species affect the concentration of tannins, a bitter-tasting plant polyphenol that can act as an antioxidant or as a factor that interferes in digestibility3. If fermentation was found to affect the amount of tannins in teas, then the amount of antioxidants in teas can indirectly be predicted. The research, experimentation, and results can benefit botanists researching in the field involving plant compounds and their benefits to humans. The experiment can also benefit people by proving how beneficial tea can be for the human health. The experimentation involves separating tannins from different types of tea leaves and isolating them, and then measuring the tannins by using a method called radial diffusion assay2. Radial diffusion assay method has been used by Ann Hagerman in her research with tannins in fresh tea leaves1. Her research with radial diffusion assay has proved effective and successful. Thus, by determining the amount of tannins in teas with various

fermentation levels, the tannin amount can be related to the fermentation level in tea leaves. The question that is being asked is: How does the amount of fermentation of tea affect the concentration of tannins in its pigments? The hypothesis for the experiment is: If a tea has a lower level of fermentation when processed, then the concentration of tannins will increase. The data will be collected and analyzed so that conclusions about the relationship of tannins and tea fermentation can be drawn.

Materials and Methods To execute the project, first place 1.425 milliliters of acetate into a 500 milliliter beaker to make the buffer. Then, add distilled water until the volume of the buffer reaches 400 milliliters. Add 10.6 milligrams of ascorbic acid to the solution. Then add 2N Sodium Hydroxide to bring the pH level of the solution to five. Pour the solution into a 500 milliliter graduated cylinder. Then, to bring the volume of the buffer to 500 milliliters, add more distilled water. Pour 100 milliliters of the prepared buffer into a 250 milliliter beaker. Add 1.0 grams of agarose to the 100 milliliters of buffer in the beaker. Place a stirring bar into the beaker and place the beaker onto a hot plate/ stirring plate until the agarose powder in the buffer has completely dissolved. Place the solution into a water bath set for forty-five degrees Celsius. When the solution reaches forty-five degrees Celsius, remove the solution immediately and place .01 grams of Bovine Serum Albinum into the beaker. Making sure that the temperature of the solution stays a constant forty-five degrees Celsius, stir the contents by placing the beaker on the stirring plate. Using a 10 milliliter serological pipette, measure out 9.5 milliliters of the solution and place in a Petri Dish. Fill ten Petri Dishes, making sure that all of the surface of the plate is covered by the solution. Seal each dish with Parafilm, and place the plates in a four degrees Celsius refrigerator. Set the Petri Dishes in the refrigerator for a week. Next, measure out 50 milliliters of methanol using a 50 milliliter graduated cylinder. Pour the methanol into a 150 milliliter Erlenmeyer flask. Using a 50 milliliter graduate cylinder, measure out 50 milliliters of distilled water. Pour the distilled water into the Erlenmeyer flask and set aside. Measure out 2 grams of green tea using an automatic balance. Place the 2 grams of tea into a small mortar and pestle. Measure out 5 milliliters of the 50% methanol solution placed in the Erlenmeyer flask. Pour the methanol solution into the pestle along with the tea leaves. Grind the leaves in the methanol solution. Pour the green tea sample into a centrifuge tube and set

aside. Grind the other teas in the same fashion with 5 milliliters of methanol solution and place into a centrifuge tube. Balance the tea samples in a centrifuge machine and centrifuge the samples. Next, remove the Petri Dishes from the refrigerator. Label each Petri Dish as Tea Leaves, White tea, Decaf Green tea, Green tea, Jasmine tea, Ti Kuan Yin tea, Oolong tea, Black tea, and Pu-Erh tea respectively. Divide each Petri Dish into four sections by labeling it with a Sharpie pen. Label each section 8 microliters, 16 microliters, 24 miroliters, and thirty-two microliters. Using the upper part of a pipette and a 5 millimeter straw, punch four holes into each quarter of each Petri Dish where it was labeled. Using a micropipette, set for 8 microliters, measure out the green tea sample and place one aliquot of the sample into the first punched hole labeled 8 microliters. Place two aliquots of the sample in the hole labeled 16 microliters, three aliquots in 24 microliters, and four aliquots in 32 microliters. Using a different pipette tip for each tea, place the tea sample in its corresponding plate. After all the tea samples have been placed in the well punch holes, seal up the Petri Dishes with Parafilm once again. Then, label another Petri dish as Control and divide the bottom of the plate using a Sharpie into four sections labeled as 8 microliters, 16 microliters, 24 microliters, and 32 microliters respectively. Punch holes using the straw into each quarter. Then, using a micropipette set for 8 microliters, measure out one aliquot of the methanol solution and place it in the hole labeled 8 microliters. Do the same with each hole with the different amounts of methanol labeled. After the four holes are filled, seal up the plate with Parafilm and set aside along with the other plates containing the tea samples. Then, measure out 50 milligrams of tannic acid using the automatic balance. Place the tannic acid in a 150 milliliter. Measure out 10 milliliters of water using a twenty milliliter graduated cylinder. Pour the distilled water into the Erlenmeyer with the tannic acid. Measure out another 100 milligrams of tannic acid and place in a 150 milliliter Erlenmeyer flask. Measure out 10 milliliters of water and place in the Erlenmeyer flask. Taking two Petri Dishes, punch four holes using the straw and pipette in each quarter of each plate. Divide the bottom of each dish into four quarters using a Sharpie and label on both, 8 microliters, 16 microliters, 24 microliters, and 32 microliters respectively. Cover the plates with Parafilm once again. Take the plates and place them in a 32o Celsius incubator. Remove the plates two days afterwards and measure the rings using a plastic ruler formed by each sample in each well hole in each plate. Collect the data and draw conclusions. During the experimentation, several

mistakes were made. Wrong amounts were sometimes measured out and measurements were forced to be redone. Also, time was an essential factor in the experiment; many times time forced the experimentation to delay.

Results The control dish with the methanol solution yielded no band formation after being placed in the incubator for two days. The 50 milligrams of tannic acid in 10 milliliters of water yielded a band of 8 millimeters in the 8 microliter aliquot, 10 millimeters in the 16 microliter aliquot, 12 millimeters in the 24 microliter aliquot, and 13 millimeters in the 32 microliter aliquot. In the 100 milligram of tannic acid with 10 milliliter dish, the diameter of the band for the 8 microliter aliquot was 9 millimeters, 12 millimeters for the 16 microliter aliquot, 14.5 millimeters for the 24 microliter aliquot, and 17 millimeters for the 32 microliter aliquot. The Black tea and Puerh tea samples yielded no detectable or measurable bands in the samples, so no data was recorded for the two teas. Green tea had a band of 10 millimeters for the 16 microliter aliquot, 11 millimeters for the 24 microliter aliquot, and 12 millimeters for the 32 microliter aliquot. In the 8 microliter aliquot, Green tea had no visible results; therefore there was no data on the 8 uL aliquot. Oolong tea had a 7.5 millimeters diameter band in the 24 microliter aliquot and 10 millimeters in the 32 microliter aliquot. In the 8 microliter and 16 microliter aliquots, there were no visible bands. Jasmine tea samples had only one visible band measuring 10 millimeters in the 32 microliter aliquot. The 8 microliter, 16 microliter, and 24 microliter aliquots had no measurable rings formed around the samples. Ti Kuan Yin tea had moderate bands formed for all of the well holes; in the 8 microliter aliquot, a diameter of 7.5 millimeters was formed; a diameter of 9 millimeters formed for the 16 microliter aliquot, 10 millimeters for the 24 microliter aliquot, and 11 for the 32 microliter aliquot. White tea formed strong bands with an 8 millimeter diameter for the 8 microliter aliquot, a 10 millimeter diameter for the 16 microliter aliquot, a 12 millimeter diameter for the 24 microliter aliquot, and a 24 millimeter diameter for the 32 microliter aliquot. Decaffeinated Green tea had similar results to those of White tea; an 8 millimeter diameter for the 8 microliter aliquot formed, a 12 millimeter diameter for the 16 microliter aliquot formed, a 23 millimeter diameter for the 24 microliter aliquot formed, and a 24 millimeter diameter for the 32 microliter aliquot formed. Dried tea leaves grown in the University of Oklahomas greenhouses had no results for the 8 microliter, 16 microliter, and 24 microliter aliquots. In the 32 microliter aliquot, a weak 8 millimeter diameter formed.

Protein Precipitation by Tannins on Agarose Gels

Measurement of Protein Precipitation Bands

Diameter (mm) Diameter (mm) 16 ul aliquot

Diameter (mm) 24 ul aliquot Diameter (mm) 32 ul aliquot Band Intensity

Treatment / Tea Control (solvent only)

8 ul aliquot

Tannins 50 mg/10 ml water Tannins 100 mg/10 ml water 9 12 14.5 17 strong 8 10 12 13 strong

Dried Tea Leaves from plant Green Tea Decaffeinated Green Tea White Tea (greenish) Ti Kuan Yin (greenish) Oolong Tea (greenish) Jasmine Tea (greenish) Black Tea Pu-Erh Tea ND ND 8 8 7.5 ND ND ND ND ND 10 12 10 9 ND ND ND ND ND 11 23 12 10 7.5 ND ND ND 8 12 24 24 11 10 10 ND ND weak strong strong strong moderate weak weak ND ND

Notes: ND = not detectable

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Discussion of Results The problem of the experiment was, How does the amount of fermentation of tea affect the concentration of tannins in its pigments? The hypothesis in the experiment was, If a tea has a lower level of fermentation when processed, then the concentration of tannins will increase. The procedures were to isolate the tannins in several teas and measure the approximate amounts of tannins in the tea sample. The teas that were used were: Green tea (unfermented tea), Decaffeinated Green tea (unfermented tea), White tea (unfermented tea), Jasmine tea (lightly fermented tea), Oolong tea (semi-fermented tea), Ti Kuan Yin tea (semi-fermented tea), Black tea (fully fermented tea), and Pu-erh tea (twice feremented tea). A control of methanol, a solution of tannic acid, and a sample of dried tea leaves grown in a greenhouse were also measured. The tannin amounts from 8 microliters, 16 microliters, 24 microliters, and 32 microliters of each sample were measured. There was an apparent trend in the data. The teas that were least fermented during the tea making process contained more tannins. Overall, Decaffeinated Green tea had the highest amount of tannins. White tea followed with the second most amount of tannins. However, when White tea was tested, 3 more milliliters of the methanol was added to the grinded White tea in order for there to be enough liquid to test the presence of tannins in White tea. Green tea had the third most amount of tannins in its grinded leaves. Ti Kuan Yin, a variation of Oolong tea, had the next highest amount of tannins; Oolong tea had the most amount of tannins after Ti Kuan Yin tea. Yet, since Jasmine tea, lightly fermented tea, had the sixth highest amount of tannins, it does not follow the trend. Jasmine tea is less fermented than both Ti Kuan Yin tea and Oolong tea, and according to the hypothesis, should have more tannins than both the teas. Therefore, the data for Jasmine tea does not justify the hypothesis. The least amount of tannins was found in the Green tea leaves from a greenhouse. The data of the leaves also does not justify the hypothesis; since, the leaves were not fermented, they should contain the most amount of tannins according to the hypothesis. Yet, because the tea leaves were grown in a greenhouse, several nutrients could have not been absorbed by the plant; therefore, the lack of nutrients could have caused the lack of tannins in the dried tea leaves from the greenhouse. Tannic acid was tested in order to compare the amounts of tannins in tannic acid and the teas. Several teas had less tannins than the tannic acid, but Decaffeinated Green tea and White tea had more tannins in the 32

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microliter aliquot than the tannic acid. However, since the amount of tannic acid in the solution varied, and was not identical to that of the teas, the data is inaccurate. More testing is necessary to draw conclusions about the effects of fermentation on tannins. Since Pu-erh and Black tea did not have measurable data, the conclusions may be inaccurate. Also, Jasmine tea, the dried Green tea leaves, Green tea, and Oolong tea did not have measurable data for one or more aliquots. Therefore, data may be inaccurate and further experimentation or repetition of the experiment is needed to support the hypothesis. There were several errors in the experiment. Measurements were wrong and had to be made again. Instead of measuring 8 microliters, .8 microliters was measured out once, and two Petri Dishes had to be discarded due to this mistake. The method of using a straw as a hole punch was also difficult; perfect holes were not made, which may have affected the data and outcome of the tannin rings. Bubbles that formed while pouring the solution into the Petri Dishes may have affected the results of the experimentation as well. Teas were also from different processing companies; since there was no way of knowing how each of the teas was processed, the data may be inaccurate because each company may process their teas differently. During the experimentation, new information was learned. It was learned that fermentation may affect pigments in leaves. Processing leaves, therefore, may destroy important pigments in a plant. Also, the importance of plant pigments to both plants and humans was learned before and during the experimentation. The experiment provided opportunities in which research had to be done relating to the benefits and the toxicity of tannins in plants. New questions concerning the experiment were asked after the experimentation. These questions included: How does the actual tea processing affect the pigments in plants? and How do plants react in exposure to oxygen? Acknowledgements I would like to thank my mentor, Dr. Wayne Elisens, a curator at the University of Oklahoma in the field of Botany, who helped me execute the experiment, making my project successful in every aspect. Both his advice and supervision were main keys that contributed to my overall project. Without his help, I would never have had such a successful project or such a great learning experience that will continually affect me. I would like to thank him again for giving me his time and efforts in my project. I would also like to thank my Science Seminar teacher, Mr. Jeffrey Baughman, for being there for me and helping me throughout my project. His and the classs support

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were factors that motivated me throughout the extent of my conduction of my project. Finally, I would like to thank my parents, Tuyet Mai and Nam Nguyen, for supporting me throughout my research and experimentation, and providing me with all the necessities for my project. Their support was a major factor that allowed my experiment to be what it is. . Bibliography 1 Hagerman, Ann E. Extraction of Tannins From Fresh and Preserved Leaves. Journal of Chemical Ecology 1988; 14: 453-461. 2 Hagerman, Ann E. Radial Diffusion Assay For Tannins. Tannin Handbook [serial on the Internet]. 2002 [Cited 2005 Oct 19]. Available from: http://www.users.muohio.edu/hagermae/tannin.pdf. 3 Tannins: Chemical Analysis. [serial on the internet]. [Cited 2005 Oct 13]. Available from: http://anci.cornell.edu/plants /toxicagents/tannin.

Fermentation of Teas. [serial on the internet]. [Cited 2005 Oct 19]. Available from:
http://ww.tenren.com/fermentation.html.

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