Grade 11Central Catholic High School

Effects of Whey Protein on Microbial Survivorship

William McCarthy

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

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.

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

Purpose:

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

Hypothesis:

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

Materialso Labeling tapeo Ethanolo Latex gloveso E. coli DH5 Alphao Saccharomyces cerevisiae

(Common Yeast)o Micropipetteso Micro racko Micro tubeso 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 Turntableo Luria Broth agar plateso Bunsen burnero Vortexo Sidearm flasko Spreader barso Klett spectrophotometero Incubator

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 107 cells/mL for yeast and 108 cells/mL for E. coli.

4. The culture was diluted in a sterile dilution fluid to a concentration of approximately 105 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:

Liquid Pulse Concentration Chart

Yeast/ E.coli

Whey/Water Solution

SDF Total Volume

0% 1 ml 0ml 1ml 2ml

1% 1 ml 0.02 ml 0.98 ml 2ml

10% 1 ml 0.2 ml 0.8 ml 2ml

20% 1ml 0.4 ml 0.6 ml 2ml

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 hours9. The resulting colonies were counted visually. Each

colony is assumed to have risen from one cell

Effects of Whey Protein on E.coli Survivorship

control 1% 10% 20%0

20406080

100120140160180

Whey Protein Concentration

Aver

gae

Surv

ivin

g Co

loni

es 164.6170

144.3125.5

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

E.coli -ANOVA

• The P-Value suggests that there is a significant effect

Alpha: 0.05

Anova: Single Factor

SUMMARYGroups Count Sum Average Variance

control 6 1020 170 302.41% 6 988 164.6667 129.8667

10% 6 866 144.3333 187.466720% 6 753 125.5 70.7

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 7454.458 3 2484.819 14.39571 3.15E-05 3.098391Within Groups 3452.167 20 172.6083

Total 10906.63 23

Dunnett’s Test: E.coli

Variable Concentrations

T-Value Interpretation

T-Crit=2.447 Whey Protein

1% 0.634 not significant

10% 3.170 SIGNIFICANT

20% 5.289 SIGNIFICANT

Effects of Whey Protein on Saccharomyces cerevisiae Survivorship

control 1% 10% 20%0

20

40

60

80

100

120

140

160

180

200

Whey Protein concentration

Aver

age

Surv

ivin

g Co

loni

es

173.8160

138 119

Saccharomyces cerevisiae -ANOVA

• The P-Value suggests that there is a significant effect.

Alpha: 0.05

Anova: Single Factor

SUMMARYGroups Count Sum Average Variance

control 6 1043 173.8333 251.76671% 6 960 160 341.2

10% 6 828 138 35.220% 6 714 119 221.2

ANOVASource of Variation SS df MS F P-value F crit

Between Groups 10512.13 3 3504.042 16.5019 1.24E-05 3.098391Within Groups 4246.833 20 212.3417

Total 14758.96 23

Dunnett’s Test: Saccharomyces cerevisiae

Variable Concentrations

T-Value Interpretation

T-Crit=2.447 Whey Powder

1% 1.664 not significant

10% 4.259 SIGNIFICANT

20% 6.518 SIGNIFICANT

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%

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

Extensions

• More trials • Different microbes• Varying exposures and exposure

times• Synergistic effects• Wider variety of variables

References

• www.medicalnewstoday.com/articles/68511• http://www.cdc.gov/ecoli/• http://onlinelibrary.wiley.com/journal/10.111

1/%28ISSN%291567-1364

• http://www.forbes.com/sites/davidlariviere/2013/04/18/nutritional-supplements-flexing-their-muscles-as-growth-industry/

• http://www.bbc.co.uk/news/business-12277808