Transcript Christian Wawrzonek metabolic enhancer effects on yeast PJAS

The Metabolic Enhancer Effect on
Microbes
By: Christian Wawrzonek
Metabolic Enhancers
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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.
Glutamine Claims
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Decreases muscle deterioration during a
workout.
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Used as a fuel for cells.
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May increase protein metabolism.
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Can increase hormone production.
Glutamine
 One
of the 11 nonessential amino acids.
The body produces glutamine from glutamic acid and
ammonia.
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Found
in many protein rich food sources.
Glutamine Uses
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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.
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).
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Saccharomyces cerevisiae as a Model
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The most investigated cell in the world.
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Used in microbiology due to its ease of
manipulation and rapid growth.
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Eukaryotic, shares similar biochemistry,
cell cycle, and genetics with more
advanced organisms, including humans.
Purpose
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Do Metabolic Enhancers really work?
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To determine if the metabolic enhancer LGlutamine 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.
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Materials
Experiment 1
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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% [ ])
Experiment 2
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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)
Procedure #1
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6.
7.
Saccharomyces cerevisiae was grown overnight in sterile
YEPD media.
A sample of the overnight culture was added to fresh
media in a sterile sidearm flask.
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.
Glutamine powder was dissolved in sterile water at a
concentration of (4%).
The glutamine solution, yeast stock, YEPD media, and
sterile water were added to sterile side arm flasks as
follows:
The flasks were swirled to evenly dissolve glutamine
solution.
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
Glutamine
Solution
100µl
10µl
0µl
Sterile Water
4mL
4.9mL
5mL
YEPD Media
4.9mL
4.9mL
4.9mL
Total
10mL
10mL
10mL
Concentration
of 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
350
0%
0.10%
1%
Klett Units
300
250
200
150
100
50
0
0
30
60
90
120
165
210
Time (Minutes)
240
285 1200
Glutamine Effects on Yeast Respiration
140
P>.05
Sig.
CO2 Ev(mLs)
120
P>.05
Sig.
P>.05
Sig.
100
80
60
40
20
0
0%
0.01%
0.10%
[Glutamine]
0.50%
ANOVAs
Test
F value
Respiration 14.9587
Experiment
F critical
P value
Interp.
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
Dunnett's Tests#1
Time
Variable
Compared
α = 0.05
t critical = 3.22
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#2
α = 0.05
t critical = 3.73
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
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.
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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.
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Limitations and Extensions
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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
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http://www.online-medicaldictionary.org/L+Glutamine.asp?q=L+Glutamine
http://mental-health.emedtv.com/glutamine/doesglutamine-work.html
http://www.vitamins-supplements.org/aminoacids/glutamine.php
http://en.wikipedia.org/wiki/Glutamine
http://genome.wellcome.ac.uk/doc_wtd020808.html
http://biochemie.web.med.unimuenchen.de/Yeast_Biol/03%20Yeast%20Metabolism.pdf
http://www.bodybuilding.com/store/glutpep.html
http://mentalhealth.emedtv.com/glutamine/glutamine.html