1. Grade 11 Central Catholic High School Effects of Whey Protein on Microbial Survivorship William McCarthy

2. Background Information Annual sales of muscle mass products in the US is over $2.7 billion 50,000 health problems a year due to muscle mass products How these products affect microbial flora and the mechanism of the health problems has not been well researched The supplement industry is largely unregulated

3. Saccharomyces cerevisiae (Yeast) Saccharomyces cerevisiae Easy to manipulate in the laboratory. Most commonly studied cell. Very similar in structure and biochemistry to more complex eukaryotes, including human cells. Buds in single colonies, can be easily counted.

4. Escherichia coli Large and diverse group of gram (-) bacteria  Surrounded by an extra cell wall composed of lipopolysaccharides Free living symbiont, or pathogen Common prokaryotic cell model

5. Purpose: To determine the effects of whey protein muscle supplements on yeast and E. coli survivorship.

6. Hypothesis: Null hypothesis: The Whey protein powder will not have a significant effect on the survivorship of E.coli or Saccharomyces cerevisiae.

7. Materials o Labeling tape o Ethanol o Latex gloves o E. coli DH5 Alpha o Saccharomyces cerevisiae (Common Yeast) o Micropipettes o Micro rack o Micro tubes o LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride). o CVS brand whey protein powder o SDF (sterile dilution fluid) (10 mM KH2PO4, 10 mM K2HPO4, 1 mM MgSO4, 0.1 mM CaCl2, 100 mM NaCl) o Turntable o Luria Broth agar plates o Bunsen burner o Vortex o Sidearm flask o Spreader bars o Klett spectrophotometer o Incubator

8. Liquid Pulse Procedure 1.E. Coli and Saccharomyces cerevisiae were grown overnight in sterile LB media. 2.Samples of the overnight cultures were added to fresh media in a sterile sidearm flask 3.The cultures were placed in incubators (30°C and 37°C) until a density of 50 Klett spectrophotometer units were reached. This represents cell densities of approximately 10 7 cells/mL for yeast and 10 8 cells/mL for E. coli. 4.The culture was diluted in a sterile dilution fluid to a concentration of approximately 10 5 cells/mL 5.10 grams of whey powder was mixed with 16 ounces of water to create the recommended concentration 6.The resulting whey/water solution was mixed with SDF to create the following experimental concentrations:

9. Liquid Pulse Concentration Chart Yeast/ E.coli Whey/Wat er Solution SDFTotal Volume 0%1 ml0ml1ml2ml 1%1 ml0.02 ml0.98 ml2ml 10%1 ml0.2 ml0.8 ml2ml 20%1ml0.4 ml0.6 ml2ml

10. Liquid Procedure cont. 6.The solutions were then mixed by vortexing and allowed to sit at room temperature for 15 minutes 7.After vortexing to evenly suspend cells, 100 µL aliquots were removed from the tubes and spread on LB agar plates 8.The plates were incubated at 37°C for 24 hours 9.The resulting colonies were counted visually. Each colony is assumed to have risen from one cell

11. Effects of Whey Protein on E.coli Survivorship 164.6 170 144.3 125.5

12. Statistical Analyses ANOVA: Single Factor – Analysis of Variation – Statistical test that compares the means of multiple groups – P-value from ANOVA testing identifies significance if P-value is < 0.05 Dunnett’s Test – Statistical test that compares an experimental group directly to the control group – Identifies that the experimental group is significantly different than the control if t- value > t-crit

13. E.coli -ANOVA The P-Value suggests that there is a significant effect Alpha: 0.05 Anova: Single Factor SUMMARY GroupsCountSumAverageVariance control61020170302.4 1%6988164.6667129.8667 10%6866144.3333187.4667 20%6753125.570.7 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups7454.45832484.81914.39571 3.15E-05 3.098391 Within Groups3452.16720172.6083 Total10906.6323

14. Dunnett’s Test: E.coli Variable Concentrations T-ValueInterpretation T-Crit=2.447 Whey Protein 1% 0.634 not significant 10% 3.170 SIGNIFICANT 20% 5.289 SIGNIFICANT

15. Effects of Whey Protein on Saccharomyces cerevisiae Survivorship 173.8 160 138 119

16. Saccharomyces cerevisiae -ANOVA The P-Value suggests that there is a significant effect. Alpha: 0.05 Anova: Single Factor SUMMARY GroupsCountSumAverageVariance control61043173.8333251.7667 1%6960160341.2 10%682813835.2 20%6714119221.2 ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups10512.1333504.04216.5019 1.24E-05 3.098391 Within Groups4246.83320212.3417 Total14758.9623

17. Dunnett’s Test: Saccharomyces cerevisiae Variable Concentrations T-ValueInterpretation T-Crit=2.447 Whey Powder 1% 1.664 not significant 10% 4.259 SIGNIFICANT 20% 6.518 SIGNIFICANT

18. Conclusions The null hypothesis may be rejected, for both E.coli and Saccharomyces cerevisiae, the Dunnett’s Tests show a significant effect in the 10% and 20% of the Whey protein It may be accepted for 1%

19. Limitations Slight lag in synchronization of plating Inability to count the exact amount of cells on each plate Limited number of low concentrations Only one exposure time Only survivorship assessed

20. Extensions More trials Different microbes Varying exposures and exposure times Synergistic effects Wider variety of variables

21. References www.medicalnewstoday.com/articles/68511 http://www.cdc.gov/ecoli/ http://onlinelibrary.wiley.com/journal/10.111 1/%28ISSN%291567-1364 http://onlinelibrary.wiley.com/journal/10.111 1/%28ISSN%291567-1364 http://www.forbes.com/sites/davidlariviere/2 013/04/18/nutritional-supplements-flexing- their-muscles-as-growth-industry/ http://www.forbes.com/sites/davidlariviere/2 013/04/18/nutritional-supplements-flexing- their-muscles-as-growth-industry/ http://www.bbc.co.uk/news/business- 12277808 http://www.bbc.co.uk/news/business- 12277808