Antibiotic Interplay of the Keto Diet, High-Protein Diet, and the Green Mediterranean Diet on Gut Microbiota

Student: Akshaj Sama
Table: MED10
Experimentation location: Independent Lab
Regulated Research (Form 1c): No
Project continuation (Form 7): No

Display board image not available



Jie Cai, Zhongxu Chen, Wei Wu, Qinlu Lin & Ying Liang (2022) High animal protein diet and gut microbiota in human health, Critical Reviews in Food Science and Nutrition, 62:22, 6225-6237, DOI: 10.1080/10408398.2021.1898336


Murphy, E. Angela; Velazquez, Kandy T.; Herbert, Kyle M.. Influence of high-fat diet on gut microbiota: a driving force for chronic disease risk. Current Opinion in Clinical Nutrition and Metabolic Care 18(5):p 515-520, September 2015. | DOI: 10.1097/MCO.0000000000000209


Wu S, Bhat ZF, Gounder RS, Mohamed Ahmed IA, Al-Juhaimi FY, Ding Y, Bekhit AEA. Effect of Dietary Protein and Processing on Gut Microbiota-A Systematic Review. Nutrients. 2022 Jan 20;14(3):453. doi: 10.3390/nu14030453. PMID: 35276812; PMCID: PMC8840478.


Bartlett, Alexandria, and Manuel Kleiner. “Dietary Protein and the Intestinal Microbiota: An Understudied Relationship.” IScience, vol. 25, no. 11, Nov. 2022, p. 105313, Accessed 21 Jan. 2023.


Rodríguez-Romero, José de Jesús, et al. “What We Know about Protein Gut Metabolites: Implications and Insights for Human Health and Diseases.” Food Chemistry: X, vol. 13, 30 Mar. 2022, p. 100195,,


FAQ: E. Coli: Good, Bad, & Deadly: “What is true for E. coli is true for the elephant”. Washington (DC): American Society for Microbiology; 2011. Available from: doi: 10.1128/AAMCol.1-2011


Merra, Giuseppe, Annalisa Noce, Giulia Marrone, Marco Cintoni, Maria Grazia Tarsitano, Annunziata Capacci, and Antonino De Lorenzo. 2021. "Influence of Mediterranean Diet on Human Gut Microbiota" Nutrients 13, no. 1: 7. 

Additional Project Information

Project website: -- No project website --
Research paper:
Additional Resources: -- No resources provided --
Project files:
Project files

Research Plan:

The cultures of Escherichia coli (E. coli), Salmonella enteritidis (S. enteritidis), Enterobacter aerogenes (E. aerogenes), and Enterobacter cloacae (E. cloacae) will be obtained from Carolina Biologicals. Initially, each of the bacteria slants and/or cultures will be grown in nutrient agar broth and will be propagated and used for studying the testing products.


These bacteria will have a 3mL culture that will be grown in the lab in a 32º bacterial shaker for 14-17 hours in order to achieve optimal growth. The optical densities of these cultures will be monitored at 600nm on a spectrophotometer, and the absorbance (abs) value will be taken. Gentamicin will be used as the main control antibiotic throughout the experiment.


To test the effects of the keto diet, high-protein diet, and green mediterranean diet, we plan to use MCT oil and keto pills (keto diet), liquid collagen protein and protein powder (high-protein diet), and green tea (green mediterranean diet) to test the variables’ positive or inhibitive effects on growth of bacteria.


For each product, we will make 15mL solutions dissolved with distilled water, except for MCT oil, which will be used in its base form. For each of the products, we will run triplicate trials with 1µL, 3µL, 10µL, 30µL, 50µL, 100µL, 200µL, and 300µL of each product added to 3mL cultures of each bacteria, and will monitor the growth using a spectrophotometer. All products will be stored according to manufacturer instructions.


If any of the products show to have major inhibition or especially increase the growth of bacteria compared to the control settings, with no product, we will reconfirm these results by doing experiments on nutrient agar bacterial culture plates. We will measure the zone of inhibition for these products in triplicates, and confirm the validity of our experiments.


This experiment will be taking place in an independent lab, where all potentially hazardous materials will be placed into red medical waste bags after being used. The lab will have Choice MedWaste take all of the potentially hazardous waste that may be a part of this experiment, and dispose of them appropriately. 

Questions and Answers

  1. What was the major objective of your project and what was your plan to achieve it? 

The major objective of my project was to find the direct effect that different diets would have on several species of gut bacteria. I decided to test the effect of 3 popular diets: the keto diet, the high-protein diet, and the green mediterranean dietTo test the effects of these diets, we grew these bacteria with a 15µL solution of keto pills and MCT oil for the keto diet, a 15µL solution of protein powder and liquid collagen for the high-protein diet, and green tea, on of the most crucial parts of the green mediterranean diet, to see what effects these diets would have on bacteria’s growth. 

Initially, For each product, I made 15mL solutions dissolved with distilled water, except for MCT oil, which was used in its base form. Each product had a triplicate trial with 1µL, 3µL, 10µL, 30µL, 50µL, 100µL, 200µL, and 300µL of each product added to 3mL cultures of each bacteria. The growth was analyzed through a spectrophotometer set at 900nm.

After the initial trial, I narrowed down my research to three main products which showed the best results during the initial testing: Protein Powder, Keto Pills, and Green Tea, one for each diet I was testing. Each of these products was tested by creating a new batch of 15µL solution of the product for each test, except for the Green Tea, which we tested the effect of for different times after being brewed. Each of these products was grown on plates, and the growth was plotted.


  1. Was that goal the result of any specific situation, experience, or problem you encountered?  

This product came to be as a result of my questioning on the direct positive or negative effects that different diets would have. Over the summer, I volunteered at a hospital, and through my volunteering, I was able to be in the operating room throughout the course of procedures with one of the gastroenterologists that I was shadowing. This experience brought about my questioning of different factors that affect the gut, and its bacteria. The most direct way that the gut is affected is by what you eat, and with the increase in popularity of different diets and increasing intakes of specific products as a part of different diets, I was interested in seeing what direct effects these diets would have.  

  1. Were you trying to solve a problem, answer a question, or test a hypothesis?

With this experiment, I was trying to answer if the high-protein, keto, and green mediterranean diet would directly show an effect on gut bacteria in the human body. 


  1. What were the major tasks you had to perform in order to complete your project?

In order to complete my project, I had to go through a couple of main trials. 

  1. First, I grew the bacteria in tubes, and measured their growth using a spectrophotometer for an initial test to see if it would be beneficial for me to continue the testing of these products. 
    1. Through this initial test, I was able to narrow down my research to only a few products, being Protein Powder, Liquid Collagen, Keto Pills, MCT oil, and Green Tea, as other products being tested weren’t showing significant results
  2. After the initial tests, I was able to start actively testing these products on bacterial plates in order to gain a much better visual understanding of the bacteria. I plated each of these plates with the product, and the data was collected. On plates, only Protein Powder, Keto Pills, and Green Tea were able to show significant results, which is why most of my data was collected from tests with those three products. 


3.  What is new or novel about your project?

       a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before?

       b. Is your project's objective, or the way you implemented it, different from anything you have seen?

       c. If you believe your work to be unique in some way, what research have you done to confirm that it is?


My project is unique, as it’s one of the only studies that has taken place on multiple different types of bacteria alongside accounting for multiple diets in order to visualize the direct effect that these diets would have. In order to confirm the novelty of my research, I conducted an extensive literature review into studies that relate to the effects of different diets on gut bacteria. Additionally, most other studies that I’ve gone through in my literature review didn’t account for multiple diets and the effects they’d have on multiple species of gut bacteria. One paper I found that accounted for a wider variety of gut bacteria didn’t account for multiple specific diets, further proving how my experiment is unique. My research is the only one I’ve seen that’s accounted for the effects a direct exposure of different diets would have on singular types of bacteria in the gut. 


4. What was the most challenging part of completing your project?

      a. What problems did you encounter, and how did you overcome them?

      b. What did you learn from overcoming these problems?

One of the major problems I had was me not being able to run complete trials on all of my products. To overcome this, I took only the products that had given the best results and advanced them to being plated on bacteria for further analysis of the growth of bacteria while being directly exposed to the products. By doing this, I was able to put most of my effort into three main products, allowing me to get higher quality, more accurate results that would allow for the best research outcomes. 

5. If you were going to do this project again, are there any things you would do differently the next time?

I’d implement the use of other products related to the diets, such as different protein powders or other protein products, other keto specific products, and other parts of the green mediterranean diet in order to give a much broader scope of what effects the diets could have. Additionally, I’d like to test more bacteria in the gut in order to give a broader scope of the effects that these diets would have on a broader scope of gut bacteria. 


6. Did working on this project give you any ideas for other projects? 

After working on this project, I’d like to expand the scope of this project by branching out to different diets and having more products to simulate the effects that these diets would have on bacterial growth. I’d also like to simulate a wider scope of gut bacteria by implementing more types of bacteria into my experiment from different parts of the gut in order to explore the effects through the gut. This will allow me to gain a broader scope of the overall effect each diet has on each different part of the gut’s microbiota.


7. How did COVID-19 affect the completion of your project?

COVID-19 did not play a significant role in the completion of my project. The entirety of my experiment was done at an independent lab, which didn’t have a major effect from COVID-19.