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 µl 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. ControlPos. Control10% Var.*50% Var.* Amylase solution 0ml1ml Distilled water5ml4ml3.5ml1.5ml Variable0ml 0.5ml2.5ml Total5ml 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.)

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

ANOVA Statistical Analysis ANOVA Source of VariationSSdfMSFP-valueF crit Between Groups E Within Groups Total

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

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.. December 14, “Antacids”. eMedTV.com.. December 14, Bassiouny, M.A.; Yang, J. "Influence of drinking patterns of carbonated beverages on dental erosion”. General Dentistry, May-June, vol. 53, no. 3, “Comparing Means with a Control”. Davidmlane.com.. January 17, “Listerine”. Wikipedia.org.. December 14, “Uses of Iodine”. Pleasantridge.k12.ca.us.. December 14, Wang, Nam Sun. “Starch Hydrolysis by Amylase”.. December 20, 2008.