1. www.myoops.org/.../CourseHome/index.htm EXPERIMENT TO TEST THE ANTIMICROBIAL PROPERTIES OF SPICES ON THE GROWTH OF Saccharomyces cerevisiae

2. Team of Investigators Foram Dave Hamzeh Abuyounis Patricia Karedeis

3. Taxonomic classification Kingdom: Fungi Phylum: Ascomycota Class: Hemiascomycetes Order: Saccharomycetales Family: Saccharomycetaceae Genus: Saccharomyces Saccharomyces cerevisiae is a unicellular fungus that typically grows asexually by simple cell division or by pinching of small “bud cells.” Also known as the “Baker's” or “brewer’s” yeast since it has been used to produce alcoholic beverages and raise bread for thousands of years. S. cerevisiae is used extensively in batch anaerobic fermentations to convert sugars to ethanol. http://neofronteras.com/wpcontent/photos/saccharomyces_ cerevisiae.jpg

4. Determine whether or not the spices, Syzygium aromaticum (clove) and Capsicum annum (chili) powder, have antimicrobial inhibitory effects on cultures of Saccharomyces cervisiae. www.nutrasanus.com/clove-oil.html www.global-b2b-network.com/b2b/63/64/174/1197... Purpose

5. Cloves have been shown to exhibit strong inhibitory antimicrobial effects (Matan et al., 2006; Bennis et al., 2004; Snyder., 1997). This is due to the strong germicidal phenolic chemical, eugenol, found in cloves (Belitz and Grosch., 1999; Snyder, 1997). Cloves have approximately 16-18% essential oil content which contains the antimicrobial compound eugenol. Eugenol structural formula www.ChemIDplus.com Figure 1.

6. Capsicum annum or chili powder is also known to have antimicrobial properties. C. annum consists of the known inhibitory substance capsaicin (Cichewicz and Thorpe., 1996). http://moleculeland.wordpress.com Figure 2. Capsaicin Structural Formula

7. As stated previously, cloves possess strong inhibitory antimicrobial effects due to their content of the phenolic chemical, eugenol (Matan et al., 2006; Bennis et al., 2004; Snyder, 1997; Belitz and Grosch., 1999). Cloves consist of approximately 16-18% of essential oil content which contains eugenol. Here in, it is hypothesized that cloves will have a potent inhibitory effect on the growth of S. cerevisiae in tissue culture, while chili powder will have a weak effect. Hypothesis

8. Five sterile Petri dishes were utilized in this experiment: one containing nutrient agar and heavily embedded with cloves and inoculated with S. cerevisiae one containing nutrient agar and heavily embedded with chili powder and inoculated with S. cerevisiae one containing nutrient agar and inoculated with S. cerevisiae two possessing nutrient agar alone and used as controls for air and agar contamination. Following the preparation of the above experimental and control plates, all Petri dishes were incubated at 37° C for five days. Experimental plate : Experimental plate: Experimental plate: Control plate: Control plate: cloves/nutrient agar chili powder/nutrient agar nutrient agar alone air agar Figure 3. Experimental and control plates used for S.cerevisiae tissue culture experimentation.

9. The agar control consisted of an unopened nutrient agar plate to rule out contamination of the nutrient agar itself. It was incubated along side the experimental plates. The air control, consisted of a nutrient agar plate which was opened only during the inoculation phase of the experiment. Importantly, this plate was not inoculated, and like the agar control, it was incubated along side the experimental plates.

10. Figure 4. Lawn method : a) Streak micro-organism on nutrient agar in a back and forth fashion to cover the entire dish then, b) swab at right angles to the previous streak. ( Morgan and Carter, 2002) a)b)

11. All observations were made using a stereoscopic microscope. To find the percent of yeast colony growth on each inoculated experimental plate, the area of a Petri dish itself was calculated. The area of the dish was found in two ways and the mean was taken to eliminate any experimental error. Method # 1 : The approximate radius of the Petri dish was found and then used in the formula Area= π X r² A= 3.14 X 3.8 The area by this method was found to be 45.34 cm² Method # 2: The area of the Petri dish was found by placing a piece of transparent graph paper over a Petri dish and counting the total # of squares, each square equaling 1 cm². With 44 squares counted the area was determined as follows: 44 X 1 cm² = 44 cm²

12. To determine the area of the Petri dish covered by the yeast the following calculations were performed: 1) Total # of Petri dish squares – # of squares left uncovered after incubation = # of squares covered by yeast. 2) # of squares covered by yeast X 1 cm² = area of Petri dish covered by yeast.

13. After five days of incubation at 37 ° C, the clove embedded agar plate showed no S. cerevisiae growth. Figure 5.Photograph of the clove embedded agar plate.

14. After five days of incubation at 37 °C, chili powder embedded agar plate showed substantial amount of yeast growth. The calculation of the area covered by yeast colony is displayed below in Table 1. Total number of squares. (one cm X one cm) Total number of squares uncovered by yeast Total number of squares covered by yeast Area of Petri dish Uncovered Area Area of yeast colony 44271745 cm²61%39% Note: The bacteria found growing in the air control was eliminated from the above data. Figure 6.Photograph of chili powder embedded agar plate Table 1. Area of yeast colony calculation

15. After five days of incubation at 37 ° C, surprisingly the streaked experimental agar plate showed no S. cerevisiae growth. Figure 7.Photograph of the nutrient agar experimental agar plate

16. After five days of incubation at 37 ° C, the air control plate showed two species found of contaminating bacteria. Their colony characteristics are displayed below in Table 2. Species #ShapeMarginSurfaceColorsize AIrregularLobatesmoothPale yellow5mm B roundsmooth Off white3mm Species A Species B Figure 8. Photograph of air control agar plate. Table 2. Colony characteristics of species growing on air control plate

17. After five days of incubation at 37 ° C, the unopened agar control plate showed no microbial growth. Thus, agar contamination was ruled out. Figure 9. Photograph of agar control agar plate

18. Figure 10. The results are plotted on a 3-D graph. The area of the Petri dish covered by yeast was calculated and converted to a percentage and was plotted on the Y-axis (ordinate) and the environment tested was plotted on the X-axis (abscissa). This graph displays and compares the variation in area covered by the yeast colony in each environment.

19. 1) The data supports the hypothesis that cloves have an inhibitory effect on the growth of S. cerevisiae in tissue culture, while chili powder will has a weak effect. 2) The data suggests that cloves maybe a strong inhibitor of S. cerevisiae growth and thus its eugenol content acts as a antimicrobial agent. 3) The data suggests that chili powder is weak inhibitor of S. cerevisiae growth and thus its capsaicin content acts as a weak antimicrobial agent. 4) However, it is important to be noted, that since S. cerevsiae did not grow in nutrient agar alone (built in control), the suggestion has to be made that yeast do not generally grow well on nutrient agar itself. It is possible then that something in chili powder, like an added polysaccharide or amino acid, is acting as food for the yeast. 5) In reviewing all the data and the above interpretations, it is recommended that this experiment be repeated using a “malt” agar which after research we have learned is the optimal agar for S. cerevisiae growth in vitro.

20. 1) The fact that S. cerevisiae is a yeast that was grown on agar plates ideal for bacteria (nutrient agar), may have affected or altered the results. 2) The transparent graph method used to obtain the area of the Petri dish was not very accurate. 3) Future experimentation will include using malt agar instead of nutrient agar. Additionally, use of computer based software program to better quantitate area of circle will be employed. 4) Additionally, as an added component to our next investigation eugenol will be added directly to the malt agar to analyze its direct effect.

21. 1) Bennis, S., Chami, F., Chami, N., Bouchikhi., & Remmal., (2004). Surface alteration of Saccharomyces cerevisiae induced by thymol and eugenol. The society for applied microbiology, 38: 454-458. 2) Cichewicz, R. H., & Thorpe, P. A., (1996). The antimicrobial properties of Chile peppers (Capsicum species) and their uses in Mayan medicine. Journal of Ethnopharmacol. 52:61-70. 3) Cowan, M., (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews, p. 564-582, 12:546-582. 4) Dombek & Ingram., (1987). Applied Environ Microbial, 53(6):1286–1291. 5) Ghosh, R., Nadimity, N., Fitzpatrick, J.E., Alworth, W., Slaga, T., & Kumar, A., 2005. Eugenol causes Melanoma growth suppression through inhibition of E2F1 transcriptional activity. Journal of Biol. Chem., 280(7) :5812-5819. 6) Matan, N., Rimkeeree, A., Mawson, A., Choompreeda, P., & Haruthaithanasan (2006).Antimicrobial activity of cinnamon and clove oils under modified atmosphere conditions. International Journal of food microbiology, 107(2): 180-185. 7) Morgan, I. G, and Brown Carter, M.E., Investigating Biology. Benjamin/Cummings Publishing Co., Inc. 2002. 8) Snyder, P. (1997). Antimicrobial Effects of Spices and herbs. Retrieved October 17, 2007, from Hospitality Institute of Technology and Management, St.Paul, Minnesota Website: http://www.hi-tm.com/Documents/Spices.html. 9) Walsh, S.E., Maillard, J.Y., Russell A.D., Catrenich, C.E., Charbonneau, D.L., & Bartolo, R.G., (2003). Activity and mechanisms of action of selected biocidal agents on Gram- positive and –negative bacteria. Journal of applied microbiology, 94: 240-247. 10) Capsaicin structure. Retrieved October 22, 2007 from http://antoine.frostburg.edu/chem/senese/101/features/capsaicin.shtml 11) Eugenol structural formula. Retrieved October 19, 2007 from www. ChemIDplus. com