The Effect of

Muscle Supplement

on Yeast Cell Growth

Michael Gemperle Central Catholic High School Grade 9

Problem

• Companies selling muscle supplements claim the supplements will help increase muscle growth.

• In 2014 approximately 32 billion dollars in supplements were sold globally.

• Are cells affected by muscle supplements?

Genuine Muscle Milk®

• Popular brand of muscle supplement

• Consumed in liquid form

• The major active ingredient is whey protein

Nutrition Facts

Major Ingredients: Calcium Sodium Casseinate, Milk Protein Isolate, Whey Protein Concentrate

Grams % Daily Value

Total Fat 7 11

Cholesterol 10 3

Sodium 260 11

Potassium 620 18

Total Carbs 6 2

Protein 20 40

Whey Protein

• Mixture of globular proteins isolated from whey, a byproduct of cheese production

• Contains all the essential amino acids

• Fast digesting protein, 80-90% absorbed by body

• Marketed as a dietary and muscular supplement

Muscle

• Muscle anatomy and physiology are partly maintained by the available amino acid pool.

• Proteins, made of amino acid chains, help build muscle tissue.

• Foods containing high amounts of protein include eggs, meat, fish, and milk.

• Many muscle supplements are enriched with protein (whey) to help repair and grow muscle tissue used during exercise/workouts.

Yeast

• Unicellular eukaryotic Fungi

• Common model organism

• Saccharomyces cerevisiae - species of budding yeast used in research and food industry

Purpose

To determine if Genuine Muscle Milk® has a significant effect on the growth

of Saccharomyces cerevisiae.

Experiment Hypotheses

Hypothesis: Genuine Muscle Milk® will significantly

increase yeast cell growth.

Null Hypothesis: Genuine Muscle Milk ® will have no

significant effect on yeast cell growth.

Materials

• Klett-Summerson Photoelectric Colorimeter

• Saccharomyces cerevisiae

• Genuine Muscle Milk® Protein Nutrition Shake

• Sterile YE broth (1% Yeast Extract)

• Sterile YEP broth (1% Yeast Extract, 2% Peptone)

• Sterile test tubes

• Sterile side-arm flasks

• Sterile pipettes

• Sterile water

Colorimeter

• Klett-Summerson Photoelectric Colorimeter • Scale readings (Absorbance) are directly proportional to the concentration, according to Beer’s Law

Procedure

1. 8.5 ml of YE broth was added to 16 sterile side arm flasks.

2. 8.5 ml of YEP broth was added to a new set of 16 sterile side arm flasks.

Procedure

3. Sterile water and Muscle Milk® were added to 5 flasks of both YE and YEP broth to make a 0.1% dilution.

4. Sterile water and Muscle Milk® were added to 5 flasks of both YE and YEP broth to make a 1% dilution.

5. 0.5 ml of yeast cells were added to 15 of both YE and YEP flasks.

+ 8.5 ml broth + 0.9 ml sterile water + 0.1 ml Muscle Milk ® + 0.5 ml yeast cells

1%

0.1%

+ 8.5 ml broth + 0.99 ml sterile water + 0.01 ml Muscle Milk®

+ 0.5 ml yeast cells

Procedure

6. Klett readings were taken every 30 minutes for 15 hours.

7. The final reading taken at stationary growth phase.

8. Data was recorded and analyzed.

Data Analysis

-20

-10

0

10

20

30

40

50

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0% YEP

0.1% YEP

1% YEP

0% YE

0.1% YE

1% YE

Me

an

Ch

an

ge

in

Ab

sorb

an

ce (

ku

)

Hours

Mean Change in Absorbance verses Time

рYE3 = 0.5334

рYEP3= 0.2889

рYE15 = 6.893E-4

рYEP15= 4.244E-5

рYE12 = 5.581E-4

рYEP12= 7.935E-4

рYE9 = 3.7E-3

рYEP9= 0.5798

рYE6 = 0.5341

рYEP6= 0.6126

Data Analysis

30.8

23.5

7

0

5

10

15

20

25

30

35

40

45

50

0% 0.10% 1%

Mean Total Absorbance Increase; YE

0%

0.10%

1%

Percent Muscle Milk®

Ov

eral

l C

han

ge

in A

bso

rban

ce (

ku

)

34

44

25.8

0

5

10

15

20

25

30

35

40

45

50

0% 0.10% 1%

Mean Total Absorbance Increase; YEP

0%

0.10%

1%

Ov

eral

l C

han

ge

in A

bso

rban

ce (

ku

)

Percent Muscle Milk®

р = 6.893E-4 р = 4.244E-5

Analysis of Variance (ANOVA)

H0: μ0%= μ0.1%= μ1%

H1: not all means are equal α = 0.05

Media F critical F calculated p-value Interpretation

YE 4.1028 16.4434 6.893E-4 Reject H0

YEP 3.8853 26.1258 4.244E-5 Reject H0

Reject Ho; There is a significant difference in the means.

Dunnett’s Test

td =

Mi – Mc

√ 2MSE

nh YE α = 0.05

Percent Muscle Milk® t critical t d

Interpretation

0.1 2.57 -1.738 Not Significant

1.0 2.57 -5.665 Significant

YEP α = 0.05

Percent Muscle Milk® t critical t d

Interpretation

0.1 2.50 3.965 Significant

1.0 2.50 -3.251 Significant

Control sample is 0% Muscle Milk®

Conclusion

YE: Experiment Hypothesis: Not Supported

The yeast cells exposed to Muscle Milk did not show a significant increase in growth.

YEP: Experiment Hypothesis: Supported

The yeast cells exposed to Muscle Milk showed a significant increase in growth.

Interpretation

• At the 1% Muscle Milk concentration the solution was opaque making it difficult to use with colorimeter.

o Cell growth in both YE and YEP broth for the 1% Muscle Milk was significantly less than for 0%.

• A Student-t test was conducted on the mean growth in YE and YEP broth at 0% Muscle Milk.

o There was no significant difference in the growth between the YE and the YEP broth at the 0% exposure.

• Increase in cell growth in YEP broth at 0.1% Muscle Milk is likely the result of the additional nutrition.

Limitations/Extensions

• Limitations:

Time required to reach stationary phase growth

Temperature conditions were not ideal

30 minute reading interval limited replicates

• Extensions

Increase number of replicates

Test additional Muscle Milk ® concentrations

Test growth using whey isolate

Quantify cell growth using hemocytometer or plating

Works Cited

1. Bio Factsheet, Number 114, “Practical Techniques in MicrobiologyII: Measuring the Growth of Microorganisms”, September 2002, <www.curriculumpress.co.uk>.

2. Current Protocols in Molecular Biology 13.0.1-13.0.4 (John Wiley & Sons Inc 2008) 3. David Lariviere, Forbes "Nutritional Supplements Flexing Muscles As Growth Industry“

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

4. Exploreyeast "Yeast, a Cellular Model“ <http://www.exploreyeast.com/article/yeast-cellular-model>.

5. Heidi Skolnik, Andrea Chernus "How our bodies use protein“ <http://www.humankinetics.com/excerpts/excerpts/how-our-bodies-use-protein>.

6. Hemi Weingarten "What's In Muscle Milk?" <http://blog.fooducate.com/2011/12/05/whats-in-muscle-milk>.

7. “How Fast Do You Digest Whey Protein?” <http://www.livestrong.com/article/520142-how-fast-do-you-digest-whey-protein>.

8. Jeff S. Volek, Ph.D., R.D, “Whey vs. casein protein” <http://www.nutritionexpress.com/showarticle.aspx?articleid=787>.

9. Kansas State University, “Yeast Experiments: Baker’s Yeast and It’s Life Cycle”, <http://www.phys.ksu.edu/gene/a1.html>.

10. Megan Nantel "Why Is Yeast Used in Research?" <http://futurescienceleaders.org/researchers2012/2013/03/why-is-yeast-used-in-research>.

11. Operating Instructions Klett Photoelectric Colorimeter, Scienceware,005B-0997-5H-1-IH, REV 5/10.

Thank you

95% Confidence Interval for

Total Mean Absorbance Increase

0

5

10

15

20

25

30

35

40

45

50

0% YE 0.1% YE 1% YE

YE

Ab

sorb

ance

(k

u)

0

5

10

15

20

25

30

35

40

45

50

0% YEP 0.1% YEP 1% YEP

YEP

Ab

sorb

ance

(k

u)

р = 6.893E-4 р = 4.244E-5