By Gabrielle Lamontagne, Contributing Writer

Gabrielle Lamontagne/CONTRIBUTOR Laura Van Beaver working in the Lab.

Gabrielle Lamontagne/CONTRIBUTOR
Laura Van Beaver working in the lab.

Laura Van Beaver is working to naturally change the caffeine-producing gene — but not in coffee. Instead, Van Beaver is working under Professor Subhash Minocha in order to change the genes in tea to naturally decaffeinate it.

Project

Rather than working directly with caffeine, Van Beaver, a University of New Hampshire junior, is working on changing a caffeine-producing gene in tea. Her work should stop or hinder the plant from producing caffeine so that it is naturally decaffeinated.

Caffeine is a natural insecticide, so completely stopping the gene from producing it naturally is difficult at best.

“You can’t take out just one chemical from something that has so many chemicals in it naturally,” Minocha said. That’s why chemical caffeine extraction also affects the flavor, aroma and health benefits. If the genetic production of naturally un-caffeinated or decaffeinated plants works — and it might not — it should only affect caffeine. Theophylline and theobromine are two other chemicals produced by tea plants. They can also be found in cocoa plants. They have the opposite effect of caffeine in that they help to lower blood pressure.

“That is why dark chocolate can be considered healthy for people with higher blood pressure,” Minocha said. It also explains why people love to eat chocolate when they are anxious or stressed, because it helps to calm them down.

One theory of this project is that once the caffeine-producing gene has been silenced, theophylline might accumulate more than it does otherwise, so that the tea will retain its calming effects as well as the health benefits and smell. This effect is not something that is likely to occur after a chemical decaffeination process.

According to Teas Etc, Inc’s “Tea and Decaffeination Guidelines,” the FDA follows the guidelines set for best practices by the Tea Association Technical Committee for what qualifies as “decaffeinated” tea, which is currently up to 0.4 percent caffeine in dry weight of tea leaves.

​Van Beaver’s research consists of confirming that the caffeine-producing gene pairs are lined up the way that she expects them to be — as they are in the wild.

“There’s a lot of different steps to the project, and I’ve made it through a few,” Van Beaver said. “I’ve learned a lot of the techniques we use, so now that I have that information under my belt it’s easier.”

Van Beaver uses an enzyme, called restriction enzyme, to cut apart the gene pairs in a few places so that they can be re-aligned in the opposite order. This process is called antisense orientation. ​When the mRNA pairs are re-aligned in the wrong order, they shouldn’t match up with the original ordered mRNA, so pairs won›t be formed. This prevents the gene, once replaced in the plant, from producing caffeine. 

Van Beaver said she wants to help produce decaffeinated tea that retains the natural flavor and health benefits, which are usually processed out of the tea along with the caffeine. She also wants to help people avoid drinking tea that has been processed with ethyl acetate, a chemical that — in large quantities — can cause health issues.

Black and green teas are the most commonly decaffeinated teas. Decaffeinated tea is never truly caffeine-free, but it is legally below 2.5 percent of the original caffeine level (or less than two milligrams per cup), according to Arbor Teas. There are currently four recognized tea decaffeination processes: the methylene chloride method, the ethyl acetate method, the carbon dioxide method and the Swiss water method.

The ethyl acetate method is the one most commonly used in the U.S. It involves filtering tea with the chemical ethyl acetate to remove caffeine. The carbon dioxide method seems to be healthier, since humans are carbon-based organisms, and also retains more of the original benefits of tea.

Green tea has more decreased risk of cancer and heart disease than other types of tea. The catechins in tea, a type of polyphenol, can help prevent bad cell growth and aid programmed cell death; both of which help to control the spread of cancer.

Other polyphenols include antioxidants. Antioxidants prevent damage to healthy cells and oxidation of LDL cholesterol. These tend to stop plaque buildup in arteries. The decaffeination process affects the amount of polyphenols in tea.

Ethyl acetate leaves only about 30 percent of the original catechins, whereas carbon dioxide can leave up to 95 percent, according to Teas Etc. However, carbon dioxide processed tea has an unusual molecular composition, so scientists are not sure how much healthier the process is than the chemical methods.

Antioxidant compounds are called flavonoids. The content of flavonoids varies greatly in tea, so the levels depend on the type of tea and how it is processed. According to Berkeley Wellness, there is research being done to test whether it is possible to breed decaffeinated coffee plants.

According to The Health Wyze Report, decaffeinated tea is not necessarily healthier. Caffeine is a natural ingredient in tea and coffee. It can be healthy for the human body as well. Problems occur due to large doses, personal tastes or the separate health issues of a particular person.

There are both environmentally friendly and chemical ways of processing tea to decaffeinate it, but this specific project does not seem to have been attempted before. 

The CO2 Method

At a high temperature, carbon dioxide becomes solvent with small, non-polar molecules. This attracts the small caffeine molecules but not larger flavor molecules. The caffeine is then filtered from the carbon dioxide used, and the carbon dioxide is re-used. This method retains more flavor and antioxidants than the ethyl acetate method. Antioxidants are the “health benefits” of tea, in that they help to prevent cancer and heart disease.

In the Lab

Van Beaver’s work began with confirming that the gene she currently has is in the proper orientation. The genes are held in a petri dish by a plasmid in bacteria. Using restriction enzymes, she is cutting the gene in specific places in order to reverse the gene’s make-up.

According to Minocha, “Research is a very independent activity,” and Van Beaver has become quite well-adapted to lab work. Many students come in with a limited idea of how to start research, and Van Beaver was no different. Minocha gave Van Beaver examples of past projects worked on and she expressed interest in the tea decaffeination project.

“She’s done a very good job working independently,” Minocha said, adding, “Of course, she was guided at first by a graduate student who knew all the lab equipment and techniques.”

Although Van Beaver has brought the project to an advanced stage, Minocha would be surprised if she gets to the point of an entire new tea plant. He expects that she will end with cell tissues for the new tea plants.

During the project, Van Beaver created posters that explain details of her work, which were hung in various scientific conferences. She was also involved with Project SMART, for younger students interested in science, over the summer as a TA.

Van Beaver plans to work three hours per week because she’s receiving one research credit for her work. She thinks that she will probably spend more than that in the lab, though. “If everything goes perfectly, I could probably finish in six months,” Van Beaver said of her project. She’s realistic about it though, as she said, “Science never really works out the way you want it to.”

How Van Beaver got Started

Van Beaver took an Honors Biotechnology and Society class taught by Minocha last fall semester, and he offered his students an opportunity to work in his lab.

“So I decided to go for it,” she said.

The SURF (Summer Undergraduate Research Fellowship) is a program run through the Hamel Center for Undergraduate Research. Minocha, who teaches courses in biology and genetics, is the faculty adviser and supervisor of the tealeaf genetics project. Van Beaver applied and received funding to work with him as part of the SURF program.

Looking Forward

Once the gene has been reversed and put back together, the two non-matching strips should cancel each other out so that caffeine is not produced. The next step will be to insert the gene back into tea cells and begin to grow plants with the new gene. When the plants have grown enough, they can be tested to check whether or not the caffeine-silencing process worked or not. Minocha asserts that Van Beaver has finished the antisense orientation process and is ready to insert the gene into tea plant cell tissue.

After the current project, Van Beaver and Minocha hope to do more research with the caffeine-producing gene in tea. This time, instead of decreasing the amount of caffeine, they would like to increase it for its natural insecticide properties. They’d like to see if it possible to adapt this gene for other plants and crops, such as tomatoes.

As for Van Beaver, she is currently applying for an IROP, a grant for research abroad, through the Hamel Center. She has a connection with someone doing biomedical research in Italy, and she hopes to be able to join that project during the summer of 2015. If Van Beaver studies abroad next summer, she will work on a different project, but Minocha implied that she will practice the same techniques that she learned in his lab.

Executive Editor