EXPERIMENT%20TO%20TEST%20THE%20ANTIMICROBIAL%20PROPERTIES%20OF%20SPICES%20ON%20THE%20GROWTH%20OF%20Saccharomyces%20cerevisiae

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EXPERIMENT TO TEST THE ANTIMICROBIAL PROPERTIES
OF SPICES ON THE GROWTH OF Saccharomyces
cerevisiae
www.myoops.org/.../CourseHome/index.htm
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Team of Investigators
Foram Dave Hamzeh Abuyounis
Patricia Karedeis
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Background Information

• 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 brewers 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/saccharom
yces_cerevisiae.jpg
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Purpose

• 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...
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Background Research

• 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.
• 
  
  
• 
  
  
  
  
  
  
  

• www.ChemIDplus.com

Eugenol structural formula
Figure 1.
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• 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
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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.
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Methods

• 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.
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Controls

• 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.

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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)
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Methods

• 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 p 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

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Methods

• 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.
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• 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.
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Results

• 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.
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Results

• 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
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Results
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
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• 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
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• 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.

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CONCLUSION

• 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.
• The data suggests that cloves maybe a strong
  inhibitor of S. cerevisiae growth and thus its
  eugenol content acts as a antimicrobial agent.
• The data suggests that chili powder is weak
  inhibitor of S. cerevisiae growth and thus its
  capsaicin content acts as a weak antimicrobial
  agent.
• 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.
• 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.

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Further analysis

• 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.
• The transparent graph method used to obtain the
  area of the Petri dish was not very accurate.
• 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.
• Additionally, as an added component to our next
  investigation eugenol will be added directly to
  the malt agar to analyze its direct effect.

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REFERNCES

• 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.
• Cichewicz, R. H., Thorpe, P. A., (1996). The
  antimicrobial properties of Chile peppers
  (Capsicum species) and their uses in Mayan
  medicine. Journal of Ethnopharmacol. 5261-70.
• Cowan, M., (1999). Plant products as
  antimicrobial agents. Clinical Microbiology
  Reviews, p. 564-582, 12546-582.
• Dombek Ingram., (1987). Applied Environ
  Microbial, 53(6)12861291.
• 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.
• 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.
• Morgan, I. G, and Brown Carter, M.E.,
  Investigating Biology. Benjamin/Cummings
  Publishing Co., Inc. 2002.
• 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.
• Walsh , S.E., Maillard, J.Y., Russell A.D.,
  Catrenich, C.E., Charbonneau, D.L., Bartolo,
  R.G., (2003). Activity and mechanisms of action
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  microbiology, 94 240-247.