By: Christian Wawrzonek

The Metabolic Enhancer Effect on Microbes

Supplements taken to increase exercise duration and reduce recovery time.

GNC is a common distributor of “metabolic enhancers.” Some for diet, vitamins, fitness, body care, and sports

nutrition. How many of them really work? One recommended drug is L-Glutamine Powder.

Metabolic Enhancers

Glutamine Claims Decreases muscle deterioration during a

workout.

Used as a fuel for cells.

May increase protein metabolism.

Can increase hormone production.

One of the 11 nonessential amino acids.

The body produces glutamine from glutamic acid and ammonia.

Found in many protein rich food sources.

Glutamine

Nitrogen carrier in the body. Precursor for immune cell division. Important for proper immune system functioning. When damaged, the body uses glutamine for

repair. Studies show an increase in recovery

speed and fewer infections in correlation to glutamine dosage.

Glutamine Uses

Saccharomyces cerevisiae Two strands of Saccharomyces cerevisiae

were used. Bakers Yeast: best known for alcohol

fermentation and bread baking. Laboratory strain: used in many

cell/biochemical investigations (Jones, CMU).

Saccharomyces cerevisiae as a Model The most investigated cell in the world.

Used in microbiology due to its ease of manipulation and rapid growth.

Eukaryotic, shares similar biochemistry, cell cycle, and genetics with more advanced organisms, including humans.

Purpose Do Metabolic Enhancers really work?

To determine if the metabolic enhancer L-Glutamine Powder has any effect on the population growth and cellular respiration of Saccharomyces cerevisiae.

Hypothesis and Null Hypothesis Null Hypothesis: There will be no

significant difference between the population growth and cellular respiration of Saccharomyces cerevisiae from the control.

Alternative Hypothesis: Glutamine supplementation will yield a faster rate of population growth and cellular respiration of Saccharomyces cerevisiae.

Materials

Sterile Side Arm Flasks L-Glutamine Powder Saccharomyces cerevisiae

(Lab Strain) Sterile Micro Pipettes Sterile Macro pipettes Klett Spectrophotometer YEPD media (0.5% Yeast

extract, 2% peptone, 2% glucose)

Sterile Deionized water Glutamine Solution (4% [ ])

Rapid Rise Saccharomyces cerevisiae (Cooking Strain), Red Star brand

Micro Pipettes Macro Pipettes Balloons (23 cm Unique

Industries) 125 ml Erlenmeyer Flasks Deionized Water Sucrose (1% final [ ]) 100mL Graduated Cylinders Plastic Tub Plastic Film Wrap L-Glutamine Powder (GNC

Pro Performance)

Experiment 1 Experiment 2

Procedure #11. Saccharomyces cerevisiae was grown overnight in sterile

YEPD media.2. A sample of the overnight culture was added to fresh

media in a sterile sidearm flask.3. The culture was placed in an incubator (30°C) until a

density of 150 Klett spectrophotometer units was reached. This represents the stock solution of yeast.

4. Glutamine powder was dissolved in sterile water at a concentration of (4%).

5. The glutamine solution, yeast stock, YEPD media, and sterile water were added to sterile side arm flasks as follows:

6. The flasks were swirled to evenly dissolve glutamine solution.

7. Klett spectrophotometer readings were recorded at times: (0,30,60,90,120,165,210,240,285, and 1200 minutes)

Table 1 Flask A(x5) Flask B (x5) Flask C (x5)

Yeast 0.1mL 0.1mL 0.1mL

GlutamineSolution

100µl 10µl 0µl

Sterile Water 4mL 4.9mL 5mL

YEPD Media 4.9mL 4.9mL 4.9mL

Total 10mL 10mL 10mL

Concentrationof Glutamine

1% 0.1% 0%

Procedure #2(1) Glutamine powder was dissolved in sterile water at a

concentration of (4%).(2) Sugar, water, and glutamine solution were added

to 12 flasks in the ratios as follows:

(3) 7 grams of Red Star rapid rise Cooking Yeast was added to each flask and balloons were immediately affixed to each flask.

(4) The flasks were transferred to a warm (30 C) water bath.

(5) After 90 minutes of incubation, each balloon was removed from the flask (careful to prevent any leakage of gas; each balloon was pinched at the neck and twisted off).

Procedure#2 Continued(6) A plastic tub was filled with water.

(7)Each graduated cylinder was filled with water to the brim and sealed with plastic wrap.

(8)The cylinder was inverted and immersed into the water and the plastic wrap removed.

(9) The balloon was placed into the water with the mouth placed into the cylinder.

(10) The mouth was slowly released and the air was pumped into the graduated cylinder. The volume of gas was then recorded.

Table 2 Flask A (x3)

Flask B (x3)

Flask C (x3)

Flask D (x3)

Yeast 7 grams 7 grams 7 grams 7 grams

Sucrose(10%)

4mL 4mL 4mL 4mL

Distilled Water

31mL 35mL 35.9mL 36mL

Glutamine Solution (4%)

5mL 1mL .1mL 0mL

Total Volume

40mL 40mL 40mL 40mL

[Glutamine] 0.5% 0.1% .01% 0%

Glutamine Effect on Yeast Population

0

50

100

150

200

250

300

350

0 30 60 90 120 165 210 240 285 1200

Time (Minutes)

Kle

tt U

nits

0%0.10%

1%

Glutamine Effects on Yeast Respiration

0

20

40

60

80

100

120

140

0% 0.01% 0.10% 0.50%

[Glutamine]

CO

2 E

v(m

Ls)

Sig.Sig.

Sig.P>.05P>.05

P>.05

ANOVAsTest F value F critical P value Interp.

Respiration Experiment

14.9587 3.8625 0.000764 Significant

Growth Experiment

Growth at 120 Min.

19.227 4.256 0.000563 Significant

240 Min. 12.845 4.256 0.002307 Significant

285 Min. 4.515 4.256 0.043851 Significant

1200 Min. 0.858 4.256 0.455861 Insignificant

Time Variable Compared

t value Interpretation

120 Minutes 1% vs. c. 3.88 Significant

120 Minutes 0.1% vs. c. 6.1 Significant

240 Minutes 1% vs. c. 0.038 Insignificant

240 Minutes 0.1% vs. c. 4.51 Significant

285 Minutes 1% vs. c. 0.861 Insignificant

285 Minutes 0.1% vs. c. 2.12 Insignificant

Dunnett's Tests#1t critical = 3.22α = 0.05

Dunnett's Tests#2

Variable Compared

t value Interpretation

0% [stock] to 0.01% [stock]

4.41 Significant

0.1% vs C 5.08 Significant

0% [stock] to 1% [stock]

7 Significant

α = 0.05t critical = 3.73

Conclusions #1 Alternative Hypothesis Accepted:

Glutamine powder yielded a faster rate of population growth of Saccharomyces cerevisiae at 120,240, and 285 minutes.

Failed to reject the Null Hypothesis at the time of 1200 minutes.

Dunnett’s test was significant for both concentrations at 120 and 240 minutes.

Conclusions #2 Accepted Hypothesis: Glutamine

supplementation significantly increased cell respiration (as measured by CO2 evolution) at all concentrations (0.5%, 0.1%, 0.01%).

Null Hypothesis Rejected Dunnett’s test showed all concentrations

significant.

Limitations and Extensions Much larger sample sizes, Might require a team of

researchers. Slight variations in timing of readings for growth

experiment (Only one Klett Spec available) Possible slight variations in optical properties of

side arm flasks. Variation in the balloon elasticity (trap the gas in

more quantitative manner). Balloons placed awkwardly on flask limiting CO2

intake. Properly place balloons.

References http://www.online-medical-dictionary.org/L+Glutamine.asp?

q=L+Glutamine http://mental-health.emedtv.com/glutamine/does-

glutamine-work.html http://www.vitamins-supplements.org/amino-acids/

glutamine.php http://en.wikipedia.org/wiki/Glutamine http://genome.wellcome.ac.uk/doc_wtd020808.html http://biochemie.web.med.uni-muenchen.de/Yeast_Biol/

03%20Yeast%20Metabolism.pdf http://www.bodybuilding.com/store/glutpep.html http://mental-health.emedtv.com/glutamine/glutamine.html