A growing number of college students are experiencing testing anxiety now more than ever. According to the American Test Anxieties Association, “about 16 to 20 percent of students experience high test anxiety.” It is one of the greatest learning challenges to encounter and “it affects more than 10 million students in North America alone.” Even if a student is prepared to take an exam, it can cause them to freeze up and receive scores that are not reflective of their knowledge. Test anxiety is a specific type of performance anxiety which invokes a sense of discomfort and worry within the test taker. Common symptoms include: stress, tension, and even physical illness such as stomach pains, headaches, and nausea. As of now there is no reported medication that targets this specific type of anxiety. 

Even though there is no “cure” as of now for testing anxiety, there has been significant progress made on the research of ways to reduce it. One approach is to evaluate the relationship between intestinal microbiota (the set of microorganisms that colonize our digestive tract) and testing anxiety. This is because there is a direct relationship between the microorganisms in our guts and the neurological functions of our brains (the microbiota-gut-brain axis). For that reason it is believed that the abundance or lack of certain microbiota can have significant effects on anxiety within the individual. In order to restore the composition of the gut microbiome, probiotics can be taken because they contain a mixture of live bacteria. Therefore, in February 2021, a group of researchers wanted to know whether regulating intestinal microbiota through probiotic supplements would in fact reduce testing anxiety levels.

Research Methods

 In order to perform this experiment, they first sent a questionnaire out to 300 randomly selected college students. Of the 300, 43 chose not to participate. The survey asked questions about everyday behavior and health history. An additional 137 students were excluded after the survey results were received. This was due to actions such as consuming alcohol, taking antibiotics, and obesity which could interfere with the results. 

They then had the 120 selected students take a survey that evaluated their depression and anxiety. 60 of the students did not have anxiety or depression so they were the control group. The other 60 students of the survey had testing anxiety and were deemed the treatment group. The students were evaluated by a psychiatrist which rated their anxiety and depression on a scale of normal, mild, moderate, and severe depression. 

Next stool samples were collected from the students before and after taking a probiotic The probiotic supplement they chose, probiotic supplement preparation (PSP - Hengxin probiotics), contained a bacterium called Bifidobacterium longum. This bacterium is specialized to live within the gastrointestinal tract of humans so they wanted to see if increasing its levels in the gut would decrease the anxiety levels of the students. The students took two PSP supplements each day for 15 days. The DNA from the fecal samples was extracted and sequenced. Then the team used a statistical software to eliminate the low quality sequences collected. 

Results

In order to evaluate the effectiveness of PSP, the researchers wanted to test if it had improved the mental state of the students with testing anxiety. Therefore, they measured the status of their mental health before and after taking the supplement using the Hamilton depression and anxiety scales. Upon taking PSP, the college students reported significantly lower anxiety and depression levels which were similar to those of the healthy candidates.

PSP effect on microbiome

Next the team wanted to see how PSP affects the intestinal microbiome diversity within the subjects. Overall, they concluded that it had very little effect on the overall composition of gut microbiota. About 87% of the microbiota were the same in the before, after, and control groups, with only 18 unique bacteria in the before group.The researchers suggested that of these 18, some of these bacteria might be contributing to testing anxiety.

Microbiota at the Phylum level

The team further wanted to see if there was any effect on the microbiota at the phylum level. They determined the top 20 microorganism populations for each of the groups. The four most dominant groups observed were Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria.  Of these groups they decided to further analyze the composition of Firmicutes, Bacteroidetes, Actinobacteria. Upon statistical analysis, they discovered that PSP had very little effect on Bacteroidetes, Firmicutes. However, what was significant about the results was that it showed students with testing anxiety had significantly less Actinobacteria than the healthy students. Furthermore, upon taking PSP, their abundance of Actinobacteria increased. This suggested that Actinobacteria might play a role in testing anxiety.

Microbiota at the Genus level

Lastly, the team analyzed the composition at the genus level. To do this they evaluated the top 20 genera of bacteria. Of those, they further evaluated the four most dominant genera which were: Fusobacterium, Prevotella, Clostridium, Akkermansia, and Streptococcus.

According to the results, there was a significant difference in the composition of pathogens of the students before and after taking PSP. Students with testing anxiety  had increased Fusobacterium and Clostridium and a limited number of Prevotella and Streptococcus. Furthermore, after the students had taken PSP, there proved to be a reduction in the number of Fusobacterium and Clostridium, as well as an increase in Streptococcus and Akkermansia. 

Main Takeaway and Future Directions

Overall from this study, their results prove that PSP could significantly reduce the number of pathogens through increasing the probiotics. This in turn can help reduce the testing anxiety that college students experience. The researchers acknowledge that their study would have benefited from monitoring the neurotransmitters of the students as well. Therefore, they propose future research which would include this in the study. 

In a world with science and technology evolving every day there are major advancements being made in the improvement of human health. With new studies being done evaluating the relationship between microbiota and probiotics, hopefully studies such as this one will help us find a way to reduce anxiety. For instance, a similar study that was conducted at Shanghai Mental Health Center also evaluated gut microbiota and anxiety. They evaluated intestinal flora and also found that regulation of the intestinal microbiome can reduce anxiety. Therefore, while there is still so much we do not know, there are several studies being done that have published promising results on this matter. So if you are someone who suffers from testing anxiety, next time you have an exam it might be worth a chance to take probiotics and see if there is a change in your anxiety and test performance.

Meet the Research Team

Tingtao Chen: Nanchang University, Professor at Institute of Translational Medicine, published over 107 peer reviewed publications. Main focus is micro microbial ecology. 

Hui Lui - PhD candidate,Jiangnan University, Nanchang University 

Qi Quin: National Engineering Research Center for Bioengineering Drugs and the Technologies, Institute of Translational Medicine, Nanchang University, Nanchang, Jiangxi Province, China.

Puyuan Tian: Senior Researcher at Nanchang University

Shengjie Li: Research Assistant (Institute of Translational Medicine), Nanchang University: Nanchang, Jiangxi, CN, Zhejiang University: Hangzhou, Zhejiang, CN

Yulu Yang: Lanzhou University | LZU · School of Public Health, Nanchang University

Chaofei Xia: National Engineering Research Center for Bioengineering Drugs and the Technologies, Nanchang University

Main Reference

Qin, Qi, Hui Liu, Yulu Yang, Yifei Wang, Chaofei Xia, Puyuan Tian, Jing Wei, Shengjie Li, and Tingtao Chen. “Probiotic Supplement Preparation Relieves Test Anxiety by Regulating Intestinal Microbiota in College Students.” Disease Markers 2021 (April 20, 2021): 5597401. https://doi.org/10.1155/2021/5597401.

Other Sources used

O’Callaghan, Amy, and Douwe van Sinderen. “Bifidobacteria and Their Role as Members of the Human Gut Microbiota.” Frontiers in Microbiology 7 (June 15, 2016): 925. https://doi.org/10.3389/fmicb.2016.00925.

BMJ. "Anxiety might be alleviated by regulating gut bacteria: Review of studies suggests a potentially useful link between gut bacteria and mental disorders." ScienceDaily. ScienceDaily, 20 May 2019. <www.sciencedaily.com/releases/2019/05/190520190110.htm>

Sokol H. Définition et rôles du microbiote intestinal [Definition and roles of the gut microbiota]. Rev Prat. 2019 Sep;69(7):776-782. French. PMID: 32233323.

Emily Ahearn (‘23) is a rising Senior at Bucknell University working towards a B.S. in Biology and minor in Sociology, while on the Pre-Med track.

Outside of the lab, Emily is involved in a variety of activities at Bucknell University such as: Pre-health Society, Women in STEM, Biology Club, Relay for Life, and Environmental Club. She is also on the executive board of the Doctors without Borders Club and a member of Epsilon Pi chapter of Kappa Alpha Theta.

Interested in the medical field, Emily is a research assistant at Hackensack Meridian Health Center. Here, she hopes to expand her knowledge of the healthcare system and improve patient care. She plans to pursue a MD-PhD in order to continue research in a clinical setting.