The Effects of Capsaicin on Microbes Jacob Warden Freshman Central Catholic High School

Capsaicin Capsaicin is a chemical irritant to all mammals that is found naturally in chili peppers. Capsaicin is a naturally occurring chemical found in chili peppers, especially the flesh around the seeds. Used as a medicine in diluted forms.

Chemistry of Capsaicin Molecular FormulaC18H27NO3

Escherichia coli Rod shaped bacteria found in the lower intestines of mammals Some strains beneficial while others are pathogenic Serves as a common prokaryotic cell model

Previous Studies Journal of Ethnopharmacology 1999: Antimicrobial properties of alkamides present in flavoring plants traditionally used in Mesoamerica: affinin and capsaicin.

To determine what effect capsaicin extract has on E. coli survivorship Purpose To determine what effect capsaicin extract has on E. coli survivorship

Hypotheses Null hypothesis: Capsaicin extract will not significantly affect the survivorship of E. coli. Alternative hypothesis: Capsaicin extract will significantly affect the survivorship of E. coli.

Materials LB agar plates Escherichia coli Capsaicin Extract (liquid form) Micro and Macro pipettes + Sterile tips Spreader bars LB agar plates (1% Tryptone, 0.5% Yeast extract, 1% NaCl) Escherichia coli DH5-alpha (E.Coli) (Obtained from Doonan Lab, CMU) Bunsen Burner Sidearm Flask Vortex Incubator Gloves/goggles Klett Spectrophotometer SDF (Sterile Dilution Fluid)(100mM KH2PO, 100mM K2HPO4, 10mM MgSO4, 1m NaCl) Sterile Test Tubes Ethanol

Procedure Escherichia coli was grown overnight in sterile LB media. A sample of the overnight culture was added to fresh media in a sterile sidearm flask. The culture was incubated at 37°C until a density of 50 Klett spectrophotometer units was reached. (Cell density approximately 10^8 cells/mL). The culture was diluted in a sterile dilution fluid to a concentration of approximately 10^5 cells/mL. The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 mL.

Procedure continued 6. 0.1 mL. of cell culture was then added to the test tubes, yielding a final volume of 10 mL. 7. The solution was mixed by vortexing. 8. After vortexing to evenly suspend cells, 0.1 mL aliquots were removed from the tubes and spread on LB agar plates. 9. The plates were incubated at 37°C for 24 hours. 10. The resulting colonies were counted. Each colony is assumed to have risen from one cell.

Table of Concentrations Column1 Tube 1 Tube 2 Tube 3 Tube 4 Tube5 Capsaicin Variable X 0ml 0.001ml 0.01ml 0.1ml 1ml Sterile Fluid 9.9ml 9.899ml 9.89ml 9.8ml 8.9ml Microbe Final Volume 10ml Final Concentration X 0% 0.01% 0.10% 1% 10%

The Effects of Capsaicin on Microbes P-value: 0.000107

T-Critical = 3.90 (significant) Dunnett’s Test T-Critical = 3.90 (significant) Alpha = .05 Concentration of Capsaicin Extract T-Value Significant? 0.01%  1.91 No 0.1%  0.58 1%  2.36  No 10% 4.73   Yes

Conclusion The null was rejected for the 0%, 0.01%, 0.1%, and 1% concentrations. The null failed to be rejected for the 10% concentration. The results and the statistical analyses indicate that 10% capsaicin extract did adversely affect E. coli survivorship.

Limitations The spread plating was not perfectly synchronized One exposure time Only one type of bacteria was tested Limited number of concentrations tested

Extensions Different types of bacteria would be tested More concentrations of the capsaicin extract would be tested Synergistic effects would be tested Multiple exposure times would be used

References CSID:1265957, http://www.chemspider.com/Chemical- Structure.1265957.html (accessed 20:45, Jan 27, 2016) COPY "Capsaicin." - Wikipedia, the Free Encyclopedia. N.p., n.d. Web. 22 Jan. 2016. "Escherichia Coli." - Wikipedia, the Free Encyclopedia. N.p., n.d. Web. 22 Jan. 2016. "Antimicrobial Properties of Alkamides Present in Flavoring Plants Traditionally Used in Mesoamerica: Affinin and Capsaicin." Antimicrobial Properties of Alkamides Present in Flavoring Plants Traditionally Used in Mesoamerica: Affinin and Capsaicin. N.p., n.d. Web. 27 Jan. 2016.

Data Replicants #1 #2 #3 #4 Tube 1: 0% 248 300 244 212 Tube 2: 0.01% 352 392 180 408 Tube 3: 0.1% 166 280 174 284 Tube 4: 1% 172 112 132 Tube 5: 10% 17 65 28 81 Concentrations Averages 0% 251 0.01% 333 0.10% 226 1% 149.5 10% 47.75

ANOVA One-Way ANOVA SUMMARY Group Count Sum Average Variance C1 4 1004 251 1326.67 C2 1332 333 10958.7 C3 904 226 4194.67 C4 598 149.5 1091.67 C5 191 47.75 912.917 ANOVA Source SS df MS Ftest P-value F-Critical Between 186743 46685.8 12.6283 0.000107 3.05557 Within 55453.8 15 3696.92 Total 242197 19 Conclusions Ho: μ1=μ2=μ3=... Ha At least 2 μ's differ F-Test Alpha 0.05 P-Value Decision: Reject Ho