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
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

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) Sterile pipette tips Staph
Sterile Dilution Fluid ([SDF] 10mM KH2PO4, 10 mM K2HPO4, 1mM MgSO4, 0.1 mM CaCL2, 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

11. 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.

12. 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

13. 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

14. P-value=2.22 E-12

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

16. P-value= 4.37 E-18

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

18. 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%

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/
consumption/
sports-nutrition-market
Mark Krotec, PTEI

22. Single Factor Anova for E. coli
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Control 8
779
97.375
0.05%
824
103
0.50%
909 5%
1190
148.75
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 3
E-12
Within Groups 28
Total 31

23. Single Factor Anova for Staph
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Control 8
1179
0.05%
1296
162
0.50%
1496
187 44 5%
1757
87.125
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups 3
E-18
Within Groups 28
Total
25344 31