Transcript Ebbert consumable effects on amylase

Consumable Liquid Effects
on Amylase Activity
By: Pat Ebbert
Pittsburgh Central Catholic
PJAS 2009
Introduction
 Amylase is an enzyme found in the body that
breaks down starch into simple sugars
 Found in the saliva and in the pancreas of
humans
 Breaks down starch into simpler sugars
Alpha-Amylase
 Found in human saliva
and pancreas.
 Breaks down long
strands of
carbohydrates into
simpler sugars such as
maltose or glucose.
 Considered superior to
β-Amylase – acts on
multiple substrate sites
Structure of Alpha-Amylase
 The green dot
represents a chloride
ion, the pale yellow a
calcium ion. Both are
used to help the
enzyme bind to the
substrate.
What affects Amylase Activity?
 Is amylase activity affected by the presence
of consumable liquids in the oral cavity?
Variable #1: Diet Pepsi
 Diet Pepsi contains no
sugar.
 Ingredients: Carbonated
water, aspartame,
phosphoric acid,
potassium benzoate,
caffeine, citric acid
Variable #2: Mylanta Antacid
 Mylanta Antacid- used
to neutralize stomach
acid that leads to upset
stomach and nausea.
 Ingredients: Aluminum
hydroxide, magnesium
hydroxide, simethicone.
Variable #3: Listerine
 Listerine - kills bacteria
in the oral cavity that
cause plaque buildup
and bad breath.
 Ingredients: water,
menthol, methyl
salicylate, eucalyptol,
thymol, alcohol, benzoic
acid, sorbitol, sodium
benzoate
Purpose
 To determine the effect of common
consumable liquids on salivary amylase
activity.
Hypothesis
 Null: The presence of Mylanta, Diet Pepsi, or
Listerine will have NO significant effect on the
activity of enzyme alpha-amylase.
 Alternative: The presence of Mylanta, Diet
Pepsi, and Listerine WILL each have a
significant effect on the activity of enzyme
alpha-amylase.
Materials
 2% stock solution of 1,4-
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α-D-glucan
glucanohydrolase
(alpha-amylase) in
distilled water
3% stock solution of
iodine in distilled water
5ml macro-pipette
50-200µl micro-pipette
Micro-pipette tips
16 starch-agar plates
 8 13x100mm boro
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silicate test tubes
50ml conical plastic
tube
Squirt bottle
Sharpie marker
Drinking straws
Diet Pepsi
Mylanta antacid drink
Listerine mouthwash
Important Property of Iodine
 Reacts with starch to create a
dark blue color.
 If starch has been broken down
into simpler sugars, the iodine
cannot bind and therefore
leaves an area of no color
visible.
 Zone of clarity – indirect
measure of amylase.
An iodine atom
Procedure
1.
2.
3.
4.
5.
Sterile starch agar plates were generated, employing an
autoclave to prepare and sterilize.
Eight different test tube solutions were created to be used in
experiment (explained on following slide).
Five wells in each of 16 starch agar plates were filled with
50µl of amylase for eight different data sets (10 trials of each
set, 80 total wells).
Two data sets were positive and negative control sets, three
were 10% variable solutions, and three were 50% variable
solutions (all of these came from the test tubes).
After one hour, iodine solution was sprayed onto each plate
and zone of starch-breakdown was measured in millimeters.
Procedure (cont.)
Neg. Control
Pos. Control
10% Var.*
50% Var.*
Amylase
solution
0ml
1ml
1ml
1ml
Distilled water
5ml
4ml
3.5ml
1.5ml
Variable
0ml
0ml
0.5ml
2.5ml
Total
5ml
5ml
5ml
5ml
After the solutions were prepared, 50µl of each tube were transferred
into ten different starch-agar plate wells.
*The variable columns were repeated 10 times (Mylanta, Diet Pepsi,
Listerine)
Consumable Liquid Effects on Amylase Activity
P-value received from single-factor Anova= 2.76 x10-29
16
Avg. Zone of Starch Digestion
in mm
14
P-values for all variables
less than .01 found using
Dunnett’s Test
12
10
8
6
4
2
0
Pos. Con.
Neg. Con.
DP10
DP50
MY10
Consumable Liquids
MY50
LIS10
LIS50
ANOVA Statistical Analysis
ANOVA
Source of Variation
Between Groups
Within Groups
Total
SS
df
MS
432.2857
6
72.04762
49.2
63
0.780952
481.4857
69
F
P-value
92.2561
2.76E-29
F crit
2.246408
Dunnett’s Test Results
α = .01, t-crit = 4.08
Variable compared to
Positive Control
(14.6mm avg.)
t-value
Interpretation
Diet Pepsi 10%
(11.9mm avg.)
6.83
Significant Variance
Diet Pepsi 50%
(8.8mm avg.)
14.67
Significant Variance
Mylanta 10%
(10.5mm avg.)
10.37
Significant Variance
Mylanta 50%
(6.5mm avg.)
20.49
Significant Variance
Listerine 10%
(10.4mm avg.)
10.63
Significant Variance
Listerine 50%
(7.9mm avg.)
16.95
Significant Variance
Results and Conclusions
 The null hypothesis that the presence of
Mylanta, Diet Pepsi, and Listerine will each
have NO significant effect on the amylase
activity was rejected (p-value < .05).
 The alternative hypothesis that the presence
of Mylanta, Diet Pepsi, and Listerine WILL
each have a significant effect on the amylase
activity was therefore accepted.
Limitations and Extensions
 Limitations
 The amylase could have
 Extensions
 The amylase could have
degraded over time, yielding
less effective binding to the
starch.
 Straws were used to create
wells, possibly creating
crevasses or other
imperfections.
 Measurements taken with
the naked eye could be
slightly off.
been given more time to
react with the starch-agar.
 More time periods could be
recorded, allowing for a
better picture of activity over
time.
 More trials.
 More precise measurement
tools for quantifying activity
zones.
References
 “Alpha-Amylase”. Wikipedia.org. <http://en.wikipedia.org/wiki/Alpha
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Amylase>. December 14, 2008.
“Antacids”. eMedTV.com.
<http://gerd.emedtv.com/antacids/antacids.html>. December 14, 2008.
Bassiouny, M.A.; Yang, J. "Influence of drinking patterns of carbonated
beverages on dental erosion”. General Dentistry, May-June, vol. 53, no.
3, 2005.
“Comparing Means with a Control”. Davidmlane.com.
<http://davidmlane.com/hyperstat/B112114.html>. January 17, 2009.
“Listerine”. Wikipedia.org. <http://en.wikipedia.org/wiki/Listerine>.
December 14, 2008.
“Uses of Iodine”. Pleasantridge.k12.ca.us.
<http://www.pleasantridge.k12.ca.us/magnolia/elements/iodine/iodine2.
html>. December 14, 2008.
Wang, Nam Sun. “Starch Hydrolysis by Amylase”.
<http://www.eng.umd.edu/~nsw/ench485/lab5.htm>. December 20,
2008.