Investigating Probiotics

By: Mei Rosa Ng

 

With cheerful personalities and encouraging messages about digestive health, advertisements on TV and internet often urge us to “eat yogurt!” Similarly, doctors often advise us to ingest probiotics – beneficial microorganisms found in yogurt –  when we’re under antibiotics treatments in order to help maintain the “good” bacteria, and combat the “bad” bacteria, in our intestines. Probiotics are even available as over-the-counter pills, to be taken daily just as we would a multi-vitamin. But does eating probiotics through yogurt or pills really help us? Two seniors from Quincy High School in Massachusetts, Peter Giunta and Eoin Moriarty, were skeptical, and decided to investigate the question.

Using the scientific method, Peter and Eoin began their investigation by focusing their broad question into more specific ones that are testable by experiments. To confer benefits to our digestive health, the probiotics we eat must be able to pass through our stomach to reach the intestines; they must also be able to keep harmful bacteria at bay. The two young investigators therefore asked: Can the types of bacteria we commonly ingest as probiotics survive conditions similar to the human stomach? If so, can these bacteria then inhibit the growth of harmful bacteria? Peter and Eoin hypothesized that the probiotic bacteria likely do not survive the stomach environment, which is highly acidic, presents vigorous physical agitations, and could therefore be hostile to microorganisms.

To test their hypothesis, Peter and Eoin first selected two common types, or strains, of probiotic bacteria as their “test subjects”: Lactobacillus acidophilus and Lactobacillus casei. They acquired these two strains of probiotic organisms in various forms, including from pills, capsules, and yogurt, and also as pure living cultures from a science supply store. This allowed them to study all the possible ways the probiotics can arrive in our stomach. The investigator pair also selected a strain of “bad” bacteria whose growth the probiotics are supposed to stop, Escherichia coli. The high school seniors were careful to handle disease-causing bacteria in a protected environment, so the version of E. coli they chose, called K-12, was actually not harmful to humans.

Finally, to mimic the hostile conditions of the stomach that the probiotic bacteria have to survive, the duo built a model “stomach.” To do so, Peter and Eoin used a beaker filled with water mixed with hydrochloric acid, an ingredient that makes our stomachs acidic, and pepsin, an enzyme in our stomach that breaks down proteins. They heated the mixture to 37 degrees Celsius, the normal temperature of the human body, and stirred the mixture with a magnetic stirrer that simulated the mechanical breakdown that takes place in the stomach. With these components in place, Peter and Eoin’s experimental design was beautifully straightforward: digest the two probiotic bacteria strains, in their various forms, in the model “stomach” for a few hours, then incubate the resulting product with E. coli to see if the growth of E. coli can be inhibited by the digested probiotics.

In less than a year, Peter and Eoin had made significant progress in their research project. The pair started experimentations in November 2010 after conceiving the project idea during the summer, and by March, they had preliminary results showing that L. acidophilus in pill and capsule forms, but not in yogurt and pure strain forms, can survive the stomach conditions. They submitted their results to the school science fair, and successfully advanced to first the regional, then the state-wise competition. They went on to win 2^nd place in the Team Division of the 2011 Massachusetts State & Engineering Fair. By conducting hands-on experiments, Peter and Eoin learned quite a lot about how certain probiotic bacteria may function in our digestive tract. More importantly, they got a taste for the demands of being microbiology researchers, and gained great appreciation for the joys and challenges of scientific research.

Peter and Eoin’s scientific journey was not without obstacles. For example, the pair knew early on that in order for their experiments to be conclusive, they had to perform proper scientific “controls,” a set of independent experiments that verify and validate their approach. These scientific controls would have to show that there were no causes other than the specific condition being tested that could explain the observed results. One such control experiment was to demonstrate that all their test subjects – the probiotics L. acidophilus and L. casei in the various forms – could inhibit the growth of E. coli K-12 under normal incubation conditions. This is an important control, because if the probiotics could not inhibit E. coli growth under normal conditions, then there would be no reason to expect that they could inhibit E. coli growth after stomach treatment. On the other hand, if the probiotics could inhibit E. coli growth under normal conditions, but not after stomach treatment, then the young scientists could attribute the incapacitation of the probiotics to the stomach treatment. Unfortunately, the team had a difficult time completing this control, because the optimal incubation conditions for L. acidophilus, L. casei and E. coli are all slightly different. In other words, if the team used incubation conditions that were ideal for growing E. coli, but not the probiotics, they might find that the probiotics were unable to prevent the E. coli growth not because the probiotics were ineffective after the stomach treatment, but because the E. coli simply outgrew them. To work around this problem, Peter and Eoin are actively looking into published reports to see how other scientists typically grow these bacteria. They are also consulting with microbiologists at Massachusetts General Hospital and Harvard Medical School for advices on alternative ways to test their hypothesis.

In addition to scientific complications, Peter and Eoin also had to overcome the challenge of limited resources during their project. The team conducted all their research in their high school, which means their experiments had to be designed with the limited space, equipment, and reagents of the school, as well as keeping the safety of all teachers and students in mind. Fortunately, their high school was recently renovated with a new science teaching facility, which provided a majority of the crucial equipment. The young scientists proudly recalled using the autoclave in one classroom to sterilize the petri dishes on which they would grow the bacteria, borrowing the magnetic stirrer from the chemistry classroom for their model “stomach,” and traveling to yet another classroom to measure the acidity of their model “stomach” with the pH meter.

As scholar-athletes, Peter and Eoin also had to balance their experiment schedule with classes, homework, sports, and other extracurricular activities. The high school scientists quickly learned the power of collaboration and teamwork. The two coordinated their respective schedules to complete the experiments: when Peter was at hockey practice, Eoin would often “cover” for him, and Peter would return the favor when Eoin was busy with soccer practice. Still, the pair often found themselves getting up early to start their experiments before their first-period classes, and staying after school – sometimes as late as 9 p.m. – to finish their lab works. The two became expert time managers and multi-taskers, completing homework assignments while they waited for certain steps of their experimental protocol to finish.

Fortunately, Peter and Eoin were blessed with supportive parents and teachers who helped and guided them along their scientific exploration. The pair especially credited their science teacher, Mr. Lawrence Johnson, for helping them translate their idea into doable experiments within the school environment. In Peter’s words, “He really… taught us everything we need to know about doing a legitimate experiment.” The pair also credited Peter’s mother, Mrs. Leah Giunta, for encouraging them to pursue their skepticism about probiotics and yogurt through active scientific investigation. Without her initial encouragement, and later tireless efforts in helping the team acquire the necessary reagents for their experiments, the project would not have progressed.

Was all their hard work worth the experience? Peter and Eoin both replied “yes” without hesitation. They might have had a self-professed interest in science prior to initiating their research, but it was only after they began actively pursing their curiosity through careful experiments that they realized what science actually entails. Unlike typical classroom laboratory lessons, science research does not necessarily have results that are clear-cut and easily predictable. This makes research challenging, but also makes the work exciting and worthwhile. In conducting their experiment and remaining committed to the challenges of the research process, Peter and Eoin learned first-hand the power that lies in a set of well-documented results generated from careful experiments. They also experienced the incredible reward of personally discovering the answer to their own question.

The high school seniors appear to be “hooked” by this experience. Currently applying to colleges, both Peter and Eoin want to major in biology or biochemistry, and continue doing science research in the future. Peter hopes to become a leading microbiologist one day, conducting cutting-edge research in a laboratory of his own. Eoin plans to apply his enthusiasm about science toward helping others through both research and medicine. In the short term, Peter and Eoin are looking forward to writing up their scientific report for publication in JEI. It is clear that no matter what the future holds, Peter and Eoin will both treasure their high school research experience, which provided them with unique insights into the life of a scientist and the power of discovery through scientific experimentations.

Mei Rosa Ng

Graduate Student

Harvard University