1. The Effect of Muscle Supplement on Yeast Cell Growth Michael Gemperle Central Catholic High School Grade 9

2. Problem Companies selling muscle supplements claim the supplements will help increase muscle growth. In 2014 approximately 32 billion dollars in supplements were sold globally. Are cells affected by muscle supplements?

3. Genuine Muscle Milk® Popular brand of muscle supplement Consumed in liquid form The major active ingredient is whey protein Nutrition Facts Major Ingredients: Calcium Sodium Casseinate, Milk Protein Isolate, Whey Protein Concentrate % Daily Value Grams% Daily Value Total Fat711 Cholesterol103 Sodium26011 Potassium62018 Total Carbs62 Protein2040

4. Whey Protein Mixture of globular proteins isolated from whey, a byproduct of cheese production Contains all the essential amino acids Fast digesting protein, 80-90% absorbed by body Marketed as a dietary and muscular supplement

5. Muscle Muscle anatomy and physiology are partly maintained by the available amino acid pool. Proteins, made of amino acid chains, help build muscle tissue. Foods containing high amounts of protein include eggs, meat, fish, and milk. Many muscle supplements are enriched with protein (whey) to help repair and grow muscle tissue used during exercise/workouts.

6. Yeast Unicellular eukaryotic Fungi Common model organism Saccharomyces cerevisiae - species of budding yeast used in research and food industry

7. Purpose To determine if Genuine Muscle Milk® has a significant effect on the growth of Saccharomyces cerevisiae.

8. Experiment Hypotheses Hypothesis: Genuine Muscle Milk ® will significantly increase yeast cell growth. Null Hypothesis: Genuine Muscle Milk ® will have no significant effect on yeast cell growth.

9. Materials Klett-Summerson Photoelectric Colorimeter Saccharomyces cerevisiae Genuine Muscle Milk ® Protein Nutrition Shake Sterile YE broth (1% Yeast Extract) Sterile YEP broth (1% Yeast Extract, 2% Peptone) Sterile test tubes Sterile side-arm flasks Sterile pipettes Sterile water

10. Colorimeter Klett-Summerson Photoelectric Colorimeter Scale readings (Absorbance) are directly proportional to the concentration, according to Beer’s Law

11. Procedure 1.8.5 ml of YE broth was added to 16 sterile side arm flasks. 2.8.5 ml of YEP broth was added to a new set of 16 sterile side arm flasks.

12. Procedure 3.Sterile water and Muscle Milk ® were added to 5 flasks of both YE and YEP broth to make a 0.1% dilution. 4.Sterile water and Muscle Milk ® were added to 5 flasks of both YE and YEP broth to make a 1% dilution. 5.0.5 ml of yeast cells were added to 15 of both YE and YEP flasks. + 8.5 ml broth + 0.9 ml sterile water + 0.1 ml Muscle Milk ® + 0.5 ml yeast cells 1% 0.1% + 8.5 ml broth + 0.99 ml sterile water + 0.01 ml Muscle Milk ® + 0.5 ml yeast cells

13. Procedure 6.Klett readings were taken every 30 minutes for 15 hours. 7.The final reading taken at stationary growth phase. 8. Data was recorded and analyzed.

14. Data Analysis Mean Change in Absorbance (ku) Hours Mean Change in Absorbance verses Time р YE 3 = 0.5334 р YEP 3 = 0.2889 р YE 15 = 6.893E-4 р YEP 15 = 4.244E-5 р YE 12 = 5.581E-4 р YEP 12 = 7.935E-4 р YE 9 = 3.7E-3 р YEP 9 = 0.5798 р YE 6 = 0.5341 р YEP 6 = 0.6126

15. Data Analysis Percent Muscle Milk ® Overall Change in Absorbance (ku) Percent Muscle Milk ® р = 6.893E-4 р = 4.244E-5

16. Analysis of Variance (ANOVA) H 0 : μ 0% = μ 0.1% = μ 1% H 1 : not all means are equal α = 0.05 MediaF critical F calculated p-valueInterpretation YE4.102816.44346.893E-4Reject H 0 YEP3.885326.12584.244E-5Reject H 0 Reject Ho; There is a significant difference in the means.

17. Dunnett’s Test t d = M i – M c √ 2MSE nhnh YE α = 0.05 Percent Muscle Milk ® t critical t dt d Interpretation 0.1 2.57-1.738 Not Significant 1.0 2.57-5.665 Significant YEP α = 0.05 Percent Muscle Milk ® t critical t dt d Interpretation 0.12.503.965 Significant 1.02.50-3.251 Significant Control sample is 0% Muscle Milk ®

18. Conclusion YE: Experiment Hypothesis: Not Supported The yeast cells exposed to Muscle Milk did not show a significant increase in growth. YEP: Experiment Hypothesis: Supported The yeast cells exposed to Muscle Milk showed a significant increase in growth.

19. Interpretation At the 1% Muscle Milk concentration the solution was opaque making it difficult to use with colorimeter. o Cell growth in both YE and YEP broth for the 1% Muscle Milk was significantly less than for 0%. A Student-t test was conducted on the mean growth in YE and YEP broth at 0% Muscle Milk. o There was no significant difference in the growth between the YE and the YEP broth at the 0% exposure. Increase in cell growth in YEP broth at 0.1% Muscle Milk is likely the result of the additional nutrition.

20. Limitations/Extensions Limitations:  Time required to reach stationary phase growth  Temperature conditions were not ideal  30 minute reading interval limited replicates Extensions  Increase number of replicates  Test additional Muscle Milk ® concentrations  Test growth using whey isolate  Quantify cell growth using hemocytometer or plating

21. Works Cited 1.Bio Factsheet, Number 114, “Practical Techniques in MicrobiologyII: Measuring the Growth of Microorganisms”, September 2002,. 2.Current Protocols in Molecular Biology 13.0.1-13.0.4 (John Wiley & Sons Inc 2008) 3.David Lariviere, Forbes "Nutritional Supplements Flexing Muscles As Growth Industry“. 4.Exploreyeast "Yeast, a Cellular Model“. 5.Heidi Skolnik, Andrea Chernus "How our bodies use protein“. 6.Hemi Weingarten "What's In Muscle Milk?". 7.“How Fast Do You Digest Whey Protein?”. 8.Jeff S. Volek, Ph.D., R.D, “Whey vs. casein protein”. 9.Kansas State University, “Yeast Experiments: Baker’s Yeast and It’s Life Cycle”,. 10.Megan Nantel "Why Is Yeast Used in Research?". 11.Operating Instructions Klett Photoelectric Colorimeter, Scienceware,005B-0997-5H-1-IH, REV 5/10.

22. Thank you

23. 95% Confidence Interval for Total Mean Absorbance Increase YE Absorbance (ku) YEP Absorbance (ku) р = 6.893E-4 р = 4.244E-5