1. Consumable Liquid Effects on Amylase Activity By: Pat Ebbert Pittsburgh Central Catholic PJAS 2009

2. 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 Introduction

3. 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

4. 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

5. What affects Amylase Activity? Is amylase activity affected by the presence of consumable liquids in the oral cavity?

6. Variable #1: Diet Pepsi Diet Pepsi contains no sugar. Ingredients: Carbonated water, aspartame, phosphoric acid, potassium benzoate, caffeine, citric acid

7. Variable #2: Mylanta Antacid Mylanta Antacid- used to neutralize stomach acid that leads to upset stomach and nausea. Ingredients: Aluminum hydroxide, magnesium hydroxide, simethicone.

8. 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

9. Purpose To determine the effect of common consumable liquids on salivary amylase activity.

10. 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. Hypothesis

11. 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

12. 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 Important Property of Iodine

13. 1. Sterile starch agar plates were generated, employing an autoclave to prepare and sterilize. 2. Eight different test tube solutions were created to be used in experiment (explained on following slide). 3. 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). 4. 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). 5. After one hour, iodine solution was sprayed onto each plate and zone of starch-breakdown was measured in millimeters. Procedure

14. Neg. ControlPos. Control10% Var.*50% Var.* Amylase solution 0ml1ml Distilled water5ml4ml3.5ml1.5ml Variable0ml 0.5ml2.5ml Total5ml 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) Procedure (cont.)

15. P-value received from single-factor Anova= 2.76 x10 -29 P-values for all variables less than.01 found using Dunnett’s Test

16. ANOVA Statistical Analysis ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups432.2857672.0476292.25612.76E-292.246408 Within Groups49.2630.780952 Total481.485769

17. Dunnett’s Test Results Variable compared to Positive Control (14.6mm avg.) t-valueInterpretation Diet Pepsi 10% (11.9mm avg.) 6.83Significant Variance Diet Pepsi 50% (8.8mm avg.) 14.67Significant Variance Mylanta 10% (10.5mm avg.) 10.37Significant Variance Mylanta 50% (6.5mm avg.) 20.49Significant Variance Listerine 10% (10.4mm avg.) 10.63Significant Variance Listerine 50% (7.9mm avg.) 16.95Significant Variance α =.01, t-crit = 4.08

18. 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.

19. Limitations and Extensions Limitations 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. Extensions The amylase could have 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.

20. References “Alpha-Amylase”. Wikipedia.org.. December 14, 2008. “Antacids”. eMedTV.com.. 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.. January 17, 2009. “Listerine”. Wikipedia.org.. December 14, 2008. “Uses of Iodine”. Pleasantridge.k12.ca.us.. December 14, 2008. Wang, Nam Sun. “Starch Hydrolysis by Amylase”.. December 20, 2008.