1. Endospores are formed as an extreme survival strategy employed by
gram positive bacteria, such as Bacilli. Due to their highly resistant
characteristics, they pose high concern in food and medical
industries. Promising results from preliminary studies have shown
that green tea polyphenols work in inhibiting the germination of
Bacillus cereus endospores. The first goal was to test what minimal
treatment time was necessary to result in a 95-100% rate of
inhibition. B. cereus endospores were first harvested over a ten-day
period, purified and boiled at 100°C for 20 minutes, and treated
with two types of lipophilic green tea polyphenols: LTP and EGCG-S at
1% and 5% concentrations for 5-, 10-, 15-, and 30-minutes. The
average inhibition rate for the 15-minute treatment time was 98.7%
for 1% LTP, 99.6% for 1% EGCG-S, 99.9% for 5% LTP and 100% for 5%
EGCG-S. These results suggest that LTP and EGCG-S play a significant
role in inhibiting endospore germination at a 5% concentration for a
15-minute minimal treatment time. The second goal of this
experiment was to test the effect of 5% EGCG-S in food and milk. B.
cereus endospores were placed under six different conditions at 1-
and 24-hour treatment times. Each sample was serially diluted,
plated onto standard method agar plates, and subsequently
incubated at 37°C for 24 hours. The colony forming unit for both
experiments was determined. The analysis of the inhibitory effects of
EGCG-S carries great importance due to the prevalence of B. cereus in
food and beverage spoilage.
`
Presenters: Chinweude Okani, *Bushra Ali, Brianne Cravello
Mentor : Dr. Lee H. Lee
Biology Department
Lipophilic Green Tea Polyphenol Can Inhibit Bacteria Growth and
Endospore Formation in Food and Milk
Camellia sinesis, commonly known as green tea, is richly made up of
Catechins, a class of polyphenols. Some of these green tea
polyphenols are EGCG and EGCG-Stearate. Various reports have
suggested that certain green tea polyphenols have anti-bacterial,
anti-viral, anti-diabetic, anti-carcinogenic, anti-inflammatory, and
antioxidant properties. In a preliminary study, the application of
crude and pure hydrophilic and lipophilic green tea polyphenols were
tested on inhibiting endospore germination on three different
Bacillus sp. at 1%, 5%, and 10% treatment concentrations for a 2-hour
treatment time. The results of that study show that all of the green
tea polyphenol treatments used are effective in controlling the
germination of B. cereus, B. megaterium, and B. subtilis endospores.
It has also showed that their inhibitory capabilities are just as strong
at a 5% concentration as compared to a 10% concentration. The next
step was to run a time-kill study to find the minimal treatment time
needed to result in a range of 95%-100% of inhibition. The study
demonstrated promising inhibitory effects of B. cereus endospore
germination by lipophilic green tea polyphenols best at a 5%
treatment concentration and at a 15-minute minimal treatment time.
With this information, we ran an experiment to determine the effects
of green tea polyphenols on the natural bacteria found in food. Our
overall goal is to apply these green tea polyphenols as a natural
preservative in food and beverages to prevent spoilage. In addition,
we aim to incorporate the tea polyphenols into disinfectant sprays
and wipes in order to prevent contamination of devices in the health
field.
.
Blanco, Anna Rita, Simona La terra Mule, Gioia Babini, Spiridione Garbisa, Vincenzo Enea, and Dario
Rusciano. “(-) Epigallocatechin-3-gallate Inhibits Gelatinase Activity of Some Bacterial Isolates from Ocular
Infection, and Limits Their Invasion through Gelatine.” Biochimica Et Biophysica Acta (BBA) – General
Subjects 1620.1-3 (2003):81. Print.
Caipo, M.L., S. Duffy, L. Zhao, and D.W. Schaffner. “Bacillus Megaterium Spore Germination Is Influenced by
Inoculum Size.” Journal of Applied Microbiology 92.5 (2002): 879-84. Print.
Shigemune, N., Nakayama, M., Tsugukuni, T., Hitomi, J., Yoshizawa, C., Mekada, Y., Kurahachi, M., &
Miyamoto, T. (2012). The mechanisms and effect of epigallocatechin gallate (egcg) on the germination and
proliferation of bacterial spores. Food Control, 27(2), 269-274. Print.
Wuytack, E. Y., J. Soons, F. Poschet, and C. W. Michiels. “Comparative Study of Pressure and Nutrient-
Induced Germination of Bacillus Subtilis Spores.” Applied and Environmental Microbiology 66.1 (2000): 257-
61. Print.
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Sponsored by SHIP
INTRODUCTION
REFERENCES
CONCLUSION / FUTURE STUDY
EXPERIMENTAL DESIGN
STAINS & PHOTOGRAPHS
ABSTRACT
Patent EGCG-Stearate
Scanning Electron Microscopy
Bacillus cereus endospores
treated with 5% EGCG-S for
15 minutes.
Bacillus cereus
endospores untreated
for 15 minutes.
Lettuce Dilution
Dilution Protocol:
Transfer 10 g of lettuce into a sterile blender
with 90 ml DI water. Blend for 5 min. to obtain
1:10 dilution. Plate 0.1 ml of 1:10 solution
onto SMA plate to obtain 1:100 dilution.
Transfer 1 ml of 1:10 solution to 99 ml DI
water, and plate 1 ml of solution to obtain
1:1000 dilution. Transfer 0.1 ml of 1:1000
solution onto a SMA plate to obtain a 1:10000
dilution.
Ethanol-tea
solution (20%
tea concentrated
solution)
0.03 grams
of EGCG-S
Mix in 150
uL of EtOH
Lettuce-water
(LW) solution
10 grams of
lettuce
Blend with
90 mL of DI
water
150 uL Lettuce-water
solution
Add 50uL EtOH
Treat for 1 hr. room
temperature
Create 1:1, 1:10, 1:100,
and 1:1000 dilutions
150 uL Lettuce-water
solution
Add 50uL EtOH-tea
solution
Treat for 1 hr. room
temperature
Create 1:1, 1:10, 1:100,
and 1:1000 dilutions
1:1,0001:1 1:10 1:100
10 uL 10 uL 10 uL
UNTREATED TREATED
100 uL of solution 
(1:1 tube has 100 uL
solution, and 1:10,
1:100, and 1:1000 tubes
contain 90 uL water)
Plate 100 uL of 1:100 dilutions
of the treated conditions and
1:1000 dilutions of the
untreated
conditions on SMA plates.
Incubate at 37°C for 24
hours.
Repeat for chick
peas, and pasta
salad
RESULTS
LETTUCE LETTUCE +
TEA
STOCK 10-1 392 36
TREATED
10-2
-- 2
UNTREATE
D 10-3
1 --
LETTUCE CHICK
PEAS
PASTA
SALAD
AVG. CFU AVG. #
bac/mL
1:10 TNC TNC TNC -- --
1:100 TNC 188 TNC 188 1.8x105
1:1000 190 90 TNC 140 1.5x105
1:10000 16 11 562 196.3 1.963x107
1 mL
0.1 mL
1 mL
0.1 mL
1
mL
1:10,000
1:1,000
1:100
1:10
 This study has demonstrated promising inhibitory effects of natural
bacteria found in lettuce by lipophilic green tea polyphenols at a 5%
treatment concentration.
 Lettuce untreated with the EtOH-tea solution resulted in an average
growth of 100%. Lettuce treated with the EtOH-tea solution resulted in
an average growth of 9%. The study yielded an average inhibition of
91%
 Future studies include repeating this experiment with the use of
hydrophilic green tea polyphenols, repeating this experiment to treat
spore-forming bacteria (e.g. B. cereus), testing the inhibitory effects in
milk, and testing each treatment to see if changes in food such as; pH,
color, and flavor have any significant effects in EGCG-S inhibitory
properties.
1:10,000
1:1,000
1:100
1:10
Food Dilution
Lettuce EGCG-S Treatment
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
UNTREATED TREATED
%ofGrowth
% of Growth: Untreated vs. Treated