Transcript EXPERIMENT TO TEST THE ANTIMICROBIAL PROPERTIES OF …

EXPERIMENT TO TEST THE
ANTIMICROBIAL PROPERTIES OF
SPICES ON THE GROWTH OF
Saccharomyces cerevisiae
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Team of Investigators
Foram Dave
Hamzeh Abuyounis
Patricia Karedeis
Taxonomic classification
Kingdom: Fungi
Phylum: Ascomycota
Class: Hemiascomycetes
Order: Saccharomycetales
Family: Saccharomycetaceae
Genus: Saccharomyces
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cerevisiae.jpg
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.
Purpose
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Determine whether or not the spices, Syzygium aromaticum (clove) and Capsicum
annum (chili) powder, have antimicrobial inhibitory effects on cultures of
Saccharomyces cervisiae.
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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.
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Figure 1. Eugenol structural formula
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).
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Figure 2. Capsaicin Structural Formula
Hypothesis
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.
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:
cloves/nutrient agar
Experimental plate:
Experimental plate:
chili powder/nutrient agar nutrient agar alone
Control plate:
air
Figure 3. Experimental and control plates used for S.cerevisiae tissue culture experimentation.
Control plate:
agar
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.
a)
b)
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)
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
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.
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.
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.
Figure 6. Photograph of chili powder embedded agar plate
Table 1. Area of yeast colony calculation
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
44
27
17
45 cm²
61%
39%
Note: The bacteria found growing in the air control was eliminated from the above data.
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
Species B
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 A
Figure 8. Photograph of air control agar plate.
Table 2. Colony characteristics of species growing on air control plate
Species #
Shape
Margin
Surface
Color
size
A
Irregular
Lobate
smooth
Pale yellow
5mm
B
round
smooth
smooth
Off white
3mm
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
% of Yeast Coverage
45%
40%
35%
30%
25%
20%
Saccharomyces cerevisiae
15%
10%
5%
0%
clove
chili
powder
nutrient
agar
alone
air
control
agar
control
Environment Tested
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.
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.
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.
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