Consumable Liquid Effects on Amylase Activity

By: Pat Ebbert

Pittsburgh Central Catholic

PJAS 2009

Amylase is an enzyme found in the body that breaks down starch into simple sugars.

Amylase is found in the saliva and in the pancreas of humans, breaking down the starch from the foods we consume into simpler sugars, which are later converted into glucose needed for energy.

Introduction

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 because it can act on any portion of the substrate making reactions occur much faster.

To the right is a diagram showing the structure of the Alpha-Amylase enzyme.

The green dot represents a chloride ion, the pale yellow a calcium ion. Both are used to help the enzyme bind to the substrate.

Structure of Alpha-Amylase

What affects Amylase?

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 is used to neutralize stomach acid that leads to upset stomach and nausea.

Ingredients: Aluminum hydroxide, magnesium hydroxide, simethicone.

Variable #3: Listerine

Listerine is made to kill 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.

Null: The presence of Mylanta, Diet Pepsi, and Listerine will each 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.

Hypothesis

2% stock solution of 1,4-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-silicate test tubes

50ml conical plastic tube

Squirt bottle Sharpie marker Drinking straws Diet Pepsi Mylanta antacid drink Listerine mouthwash

Materials

Iodine has the property of binding to 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.

If amylase has broken down surrounding starch into simpler sugars, this area’s diameter can be measured to give a relative value of how much starch has been broken down.

An iodine atom

Important Property of Iodine

1. Eight different test tube solutions were created to be used in experiment (explained on following slide).

2. 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).

3. 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).

4. After one hour, iodine solution was sprayed onto each plate and zone of starch-breakdown was measured in millimeters.

General Procedure

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

This table represents what was present in the eight test tubes used in the experiment. After the solutions were prepared, 50µl of each tube were transferred into ten different starch-agar plate wells.

*The variable columns were repeated three times (Mylanta, Diet Pepsi, Listerine)

General Procedure (cont.)

Consumable Liquid Effects on Amylase Activity

0

2

4

6

8

10

12

14

16

Pos. Con. Neg. Con. DP10 DP50 MY10 MY50 LIS10 LIS50

Consumable Liquids

Avg.

Zon

e of

Sta

rch

Dige

stio

n in

mm

P-value received from single-factor Anova= 2.76 x10-29

P-values for all variables less than .01 found using Dunnett’s Test

ANOVA Statistical Analysis

ANOVA

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

Between Groups 432.2857 6 72.04762 92.2561 2.76E-29 2.246408

Within Groups 49.2 63 0.780952

Total 481.4857 69        

Dunnett’s Test Results

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

α = .01, t-crit = 4.08

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

Sources of Error, Limitations

The amylase chemicals could have degraded over time, yielding less effective binding to the starch.

While using straws to create wells, extra salivary amylase may have entered the plates, increasing starch degradation.

Measurements taken with the naked eye could be slightly off.

Extensions To increase accuracy, the

amylase could have been given more time to react with the starch-agar.

Also, more trials are always useful for more accurate results.

Precise measurements could have been used to ensure that the data was recorded correctly.

References “Alpha-Amylase”. Wikipedia.org. <http://en.wikipedia.org/wiki/Alpha-

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.