Protein Supplement Influence on Microbial Survivorship

1. Protein Supplement Influence on Microbial Survivorship Ryan Murray Grade 11 Central Catholic High School

2. Protein Powder Background Information • Annual sales total $4.7 billion; expected to increase to $7.5 billion by 2020 • Not monitored for safety by the FDA • Potential dangers: harming kidney functions, excess calcium loss, excess protein, and heavy metal contamination • Results can be harming

3. Microbial Flora • Refers to microorganisms living in or on the body • Little is known about the association between humans and their flora • Effects are mutualistic, parasitic, pathogenic, and commensal • Perform functions beneficial to the host, including the manufacture of essential vitamins, and the prevention of colonization by undesirable microbes • Human foods and supplements may have unintended effects on the flora populations and their functions

4. Gram Positive vs Gram Negative Bacteria • Gram Negative (-) • Cell membrane of peptidoglycan and lipid membrane • Outer membrane is a thin extra layer of lipopolysaccharide • Outer membrane protects the bacteria from several antimicrobials • Gram Positive (+) • Simple, thick membrane • Most human pathogens • Antibiotics such as penicillin prevent linking of peptidoglycan and formation of cell wall

5. Staphylococcus epidermidis • Gram positive bacteria • Most are harmless skin-dwelling micro-organisms • Most strains considered non-pathogenic • The ones that are pathogenic can be life threatening • Commonly used in microbio experiments because of the variety of strain types

6. Escherichia coli (c600) • Gram negative, unicellular bacteria • Commonly found in the digestive tract of animals • Major cause of food-borne illnesses • Model prokaryote cell

7. Whey Protein • Gold Standard 100% Whey Protein • Mixture of proteins isolated from whey • Contains many amino acids that get absorbed by the bloodstream quickly • Often ingested following a workout • Marketed to increase strength, gain muscle, and lose body fat

8. Purpose • To determine the effects of whey protein supplement on Staph and E. coli survivorship.

9. Hypothesis • Null Hypothesis- The protein powder will not significantly effect the survivorship of E. coli and Staph. • Alternate Hypothesis- The protein powder will have a significant effect on the survivorship of E. coli and Staph.

10. Materials • E.coli (c600) • Staph • Gold Standard 100% Whey Protein • LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride) • Agar plates • Spreader bars • Bunsen Burner • Vortex • Incubator • Micropipettes • Sterile pipette tips • Sterile Dilution Fluid ([SDF] 10mM KH2PO4, 10 mM K2HPO4, 1mM MgSO4, 0.1 mM CaCL2, 100 mM NaCl) • Turntable • Sterile test tubes • Test tube rack • Ethanol • Matches • Sidearm flask • Labeling tape

11. Procedure • 1. Bacteria (E.coli and Staph.) 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 an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^8 cells/mL. • 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL. • 5. Solutions were made up in sterile test tubes using sterile fluid and variable. • 6. 0.1mLof the cell cultures were added to the solutions which brought them to a total volume of 10mL. • 7. Tubes were allowed to sit at room temperature for 10 minutes. • 8. Solutions were pipetted onto LB plates using a micro-pipette. • 9. Plates were incubated at 37 degrees Celsius for 48 hours. • 10. Colonies were counted, data was recorded, results were analyzed.

12. Concentration in Tubes 8 replicates for each group

13. Statistical Analysis Anova: Single Factor 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 • Statistical test that compares the means of multiple groups • P-value from ANOVA testing identifies significance if P-value is < 0.05

14. P-value=2.22 E-12

15. Dunnett's Test: E. coli

16. P-value= 4.37 E-18

17. Dunnett's Test: Staph

18. Conclusion • The Null Hypothesis that the protein powder will not significantly effect the survivorship of E. coli and Staphcan be accepted for: • E. coli: 0.05% • The Alternate Hypothesis that the protein powder will have a significant effect on the survivorship of E. coli and Staph can be supported for: • E. coli.: 0.5% and 5% • Staph: .05%, 0.5%, and 5%

19. Limitations • Plating was not perfectly synchronized • Inability to count the exact amount of cells on each plate • Limited number of low concentrations • Only one exposure time • Only survivorship was tested • Media contain amino acids (excess amino acids?)

20. Extensions • More types of protein powders should be tested, to allow for more variance in the data • Protein powder could be tested on additional microbial models • Varying exposure and exposure times could be tested

21. Works Cited • https://authoritynutrition.com/whey-protein-101/ • http://www.livestrong.com/article/540079-what-are-the-dangers-of-protein-powder-consumption/ • http://www.nutraingredients-usa.com/Markets/Protein-powders-The-heavyweight-in-the-16bn-sports-nutrition-market • Mark Krotec, PTEI

22. Single Factor Anova for E. coli

23. Single Factor Anova for Staph