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

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

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

Gram Positive vs Gram Negative Bacteria Simple, thick membrane Most human pathogens Antibiotics such as penicillin prevent linking of peptidoglycan and formation of cell wall 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

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

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

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

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

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.

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

Procedure 1. Bacteria (E.coli and Staph.) were grown overnight in sterile LB Media. 6. 0.1mL of the cell cultures were added to the solutions which brought them to a total volume of 10mL. 2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 7. Tubes were allowed to sit at room temperature for 10 minutes. 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. 8. Solutions were pipetted onto LB plates using a micro-pipette. 9. Plates were incubated at 37 degrees Celsius for 48 hours. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL. 10. Colonies were counted, data was recorded, results were analyzed. 5. Solutions were made up in sterile test tubes using sterile fluid and variable.

Concentration in Tubes   Control (0%) 0.05% 0.5 % 5% Sterile Fluid 9.9 mL 9.895 mL 9.85 mL 9.4 mL Whey Protein 0.0 mL 0.005 mL 0.05 mL 0.5 mL Bacteria 0.1 mL Total Volume 10 mL 8 replicates for each group

Statistical Analysis Anova: Single Factor Dunnett's Test Statistical test that compares the means of multiple groups P-value from ANOVA testing identifies significance if P-value is < 0.05 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

P-value=2.22 E-12

Dunnett's Test: E. coli Concentration T-Value Significance 0.05% 1.346 Not Significant 0.5% 3.887 Significant 5% 12.290

P-value= 4.37 E-18

Dunnett's Test: Staph Concentration T-Value Significance 0.05% 4.267 Significant 0.5% 11.561 5% 21.079

Conclusion The Null Hypothesis that the protein powder will not significantly effect the survivorship of E. coli and Staph can 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%

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?)

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

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

Single Factor Anova for E. coli Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control 8 779 97.375 36.26785714 0.05% 824 103 41.14285714 0.50% 909 113.625 29.41071429 5% 1190 148.75 172.7857143 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 12749.625 3 4249.875 60.79780304 2.21738E-12 2.946685269 Within Groups 1957.25 28 69.90178571 Total 14706.875 31  

Single Factor Anova for Staph Anova: Single Factor SUMMARY Groups Count Sum Average Variance Control 8 1179 147.375 27.69642857 0.05% 1296 162 29.14285714 0.50% 1496 187 44 5% 1757 219.625 87.125 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 24028.25 3 8009.416667 170.4455 4.36789E-18 2.946685269 Within Groups 1315.75 28 46.99107143 Total 25344 31