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The Comparison of Strawberry Extract on the growth of two Different
Gram-negative Bacteria. Formatting
Mohammad Dadkhah, Amin Najmabadi
Department of Biological Sciences, Saddleback College, Mission Viejo, California
Abstract
Strawberry is a rich source of bioactive compounds, such as phenolics and organic
acids, which have antimicrobial activities against human pathogens. Their antimicrobial
activity has gained importance as phenolic berry extracts inhibit the growth of selected
Gram-negative intestinal bacteria. Two types of Gram-negative bacteria were chosen for
this experiment. Escherichia coli are a common type of Gram-negative bacteria that can
get into food, such as beef and vegetables. Salmonella Typhimurium is a gram negative
bacterium that causes systemic infections and typhoid fever in humans. The aim of the
present study was to determine the effects of strawberry extract on selected pathogenic
bacteria such as E. coli and Salmonella Typhimurium to identify antibacterial activity. One
control group for E. coli and one for Salmonella Typhimurium and an experimental group
for each bacterium. 10ml of each bacteria were obtained from the colonies grown and
were placed on to the Petri dishes by a 0.25 lawn spread. Salmonella with DI water
0.30mm  0.13mm. E.coli with Strawberry had a zone of inhibition of 14.65mm 
0.39mm and with DI water 0.25mm  0.11mm. We suggest that there is no significant
difference when comparing Salmonella Strawberry with Salmonella DI water but there is
a significant difference when comparing E.coli Strawberry with E.coli DI water. There is
also a significant difference when comparing Salmonell strawberry with E.coli
Strawberry.
Introduction
Flavonoids are common substances in the daily diet. These polyphenolic
compounds are widely found in various types of edible plants, especially in vegetables,
fruits, tea and wine. Over 4000 different favonoids have been described and they are
categorized into several subgroups (Puupponen-Pimia et al. 2004). Flavanones are
typically present in citrus fruit, and favanols in green tea. Berries, which are traditionally
a part of the Finnish diet, are good sources of favonols while the predominating group of
favonoids, especially in red berries, is anthocyanins (Puupponen-Pimia et al. 2004).
Strawberry is a rich source of bioactive compounds, such as phenolics and organic acids,
which have antimicrobial activities against human pathogens.
Berry phenolics possess many interesting biological activities. Their antimicrobial
activity has gained importance as phenolic berry extracts inhibit the growth of selected
Gram-negative intestinal bacteria. Strawberries contain ellagitannins or Ellagic acid and
citric acid. Strawberry ellagitannins slow the growth of abnormal colon cells in humans
(Ulanowska et al2007). These ellagitannins protect human cells against cancer-causing
agents in tobacco smoke, food additives, and petroleum-based substances. Ellagitannins
act as scavengers to “bind” cancer-causing chemicals, making them inactive. The
ellagitannins inhibit the ability of other chemicals to cause mutations in bacteria.
Strawberry ellagitannins also protect DNA by blocking carcinogens from binding to the
DNA (Heinonen et al. 2000). Bacteria are a part of our everyday life. Most are harmless,
what do you mean harmless? a few are beneficial, how do we benefit? and some are
harmful pathogens. in what ways? Pathogenic bacteria or toxins produced by bacteria
often enter the human body via food or drink, causing symptoms or illness with several
mechanisms. E. coli is a common type of Gram-negative bacteria that can get into food,
such as beef and vegetables. E. coli is short for the medical term Escherichia coli. E. coli
normally lives inside your intestines, where it helps your body break down and digest the
food you eat. Unfortunately, certain types (called strains) of E. coli can get from the
intestines into the blood. This is a rare illness, but it can cause a very serious infection.
Salmonella Typhimurium is a gram negative bacterium that causes systemic infections
and typhoid fever in humans. This rod-shaped, flagellated organism’s sole reservoir is
humans. It has caused many deaths in developing countries where sanitation is poor and
is spread through contamination of water and undercooked food. Bacteria adhere to and
commonly penetrate through the epithelium of intestines. Most people recover from
salmonellosis spontaneously, but sometimes antibiotic treatments are needed. Studies on
flavonoids have attracted a lot of interest recently because they have a variety of
beneficial biological properties such as antioxidant and anti-cancer activities (Heinonen
et al. 2000; Daniyan et al. 2008; Ulanowska et al. 2007). The aim of the present study
was to determine the effects of strawberry extract on selected pathogenic Gram-negative
bacteria such as E. coli and Salmonella Typhimurium to identify antibacterial activity.
Such knowledge is important for the development of health-promoting functional foods
containing both probiotic bacteria and plant material, such as berries.
Methods
Forty of of? nutrient agar plates were made on November 4, 2009. Using a
mixture of 11.5g of nutrient agar powder with 500ml distilled water in two 2 Erlenmeyer
flasks. They were then placed in the autoclave for two hours at 120C. The solution was
poured into 40 Petri dishes and placed in the incubator at 37C for 24 hours. On
November 5th 2009 the plates were divided in to four groups of ten. One control group for
E. coli and one for Salmonella Typhimurium and an experimental group for each
bacterium. 10 ml of each bacteria were obtained from the colonies grown at Saddleback
College laboratory. The bacteria was placed on to the petri dishes by a 0.25 lawn spread.
Eighty 7mm chads were punched from 2cm Whatman filter papers and were placed in a
petri dish and autoclaved. These sterilized chads were dipped into DI water and placed on
the control groups and for the experimental groups they were dipped into strawberry
extract. Fresh strawberry extract was made by squeezing fresh strawberries in the juicer
in the student research laboratory. The 40 petri dishes were then stored in the incubator
at 37C for four days. On Monday November 9th 2009 the zones of inhibition were
measured in millimeters using a millimeter ruler. Microsoft Excel was used to calculate
the mean value for each group and an ANOVA test was preformed. The ANOVA was
verified using Bonferroni Correction Post Hoc test.
Results
16
14
12
10
8
6
4
2
0
Salmonella Strawberry
Salmonella DI water 1
E.coli Strawberry
E.coli DI water
Diffrent groups
Figure 1. Radius of zone of inhibition measured in Salmonella Typhimurium and
Escherichia coli with DI water vs. strawberry. Salmonella with strawberry had a mean
zone of inhibition of 0.45mm  0.16mm, Salmonella with DI water 0.30mm  0.13mm.
E.coli with Strawberry had a zone of inhibition of 14.65mm  0.39mm and with DI water
0.25mm  0.11mm. The result suggests that there is a significant difference when
comparing E.coli Strawberry with any of the other groups. (p=1.07 x 10-34, ANOVA).
Errors bars are  SEM.
A Post Hoc (Bonferroni Correction) analysis of the data suggests that there is no
significant difference when comparing Salmonella Strawberry with Salmonella DI water
but there is a significant difference when comparing E.coli Strawberry with E.coli DI
water. There is also a significant difference when comparing Salmonella strawberry with
E.coli Strawberry.
Discussion
The antibacterial effect of Strawberry was measured against two Gram-negative
bacteria, Salmonella Typhimurium and Escherichia coli. The results of our study show
that Strawberry has a different effect on different Gram-negative bacteria. Strawberry
extract inhibited the growth of E. coli but not Salmonella. Phenolic extracts of strawberry
disintegrated the outer membrane of examined E. coli because of phenolic compounds in
strawberry. Recent studies showed that phenolic compounds in strawberry was not
effective on Salmonella Typhimurium bacteria growth and most of the inhibition seemed
to originate from other compounds, such as organic acids which is not available in
strawberry extract (Puupponen-Pimia et al. 2004). Results showed that strawberry
extracts clearly showed that phenolic compounds, especially ellagitannins contain in
strawberry was responsible for the strong antibacterial effects against the E. coli bacteria.
Rephrase. There was no pH effects in the inhibition caused by the pure phenolic
fractions, because pH of the fractions was in neutral area. Main organic acids present in
most berries are benzoic acid, citric acid and malic acid (Ulanowska et al. 2007). In their
undissociated state (in pH below the pKa value of the acid) the acids may function as
permeabilizers of the Gram-negative bacteria outer membrane and may act as potentiator
of the effects of other antimicrobial substances. However Strawberry does not contain
benzoic and malic acids (Puupponen-Pimia et al. 2004). We would like to suggest that
the reason of growth of salmonella bacteria on experimental plates was because of
absence of these organic acids in the strawberries, which is supported by our preliminary
experiments. However this has to be further confirmed.
References
Katarzyna Ulanowska, Anna Majchrzyk1, Marta Moskot, Joanna Jakóbkiewicz-Banecka3
& Grzegorz W. 2007. Assessment of antibacterial effects of flavonoids by estimation
of generation times in liquid bacterial cultures. Department of Molecular Biology,
University of Gda´nsk. 2: 132—135
Keiko Iwashita, Masuko Kobori, Kohji Yamaki, and Tojiro Tsushida, 2000, Flavonoids
Inhibit Cell Growth and Induce Apoptosis in B16 Melanoma 4A5 Cells. Biosci.
Biotechnol. Biochem 64(9), 1813-1820
R. Puupponen-PimiaÈ, L. Nohynek, C. Meier, M. KaÈhkoÈnen, M. Heinonen,
A. Hopia and K.-M. Oksman-Caldentey. 2001. Antimicrobial properties of phenolic
compounds from berries. Journal of Applied Microbiology 90: 494-507
R. Puupponen-Pimiä, L. Nohynek, S. Hartmann-Schmidlin. M. Kähkönen, M. Heinonen,
K. Määttä-Riihinen and K.-M. Oksman-Caldentey, 2004. Berry phenolics selectively
inhibit the growth of intestinal pathogens. Journal of Applied Microbiology 98: 991-1000
S. Y. Daniyan and H. B. Muhammad, 2008, Evaluation of antimicrobial activities
and
phytochemical properties of extracts of Tamaridus indica against some
diseases causing bacteria. African Journal of Biotechnology 2451-2453
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s): Mohammad Dadkhah, Amin Najmabadi
Title: The Comparison of Strawberry Extract on the growth of two Different Gram-negative
Bacteria.
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.
The research that was done, was simple, they explained it very well and included many
details so I was able to follow along. Their goal was to see how much or how fast certain
bacteria would grow with strawberry extract present. They grew bacteria on agar, and
then placed a control which was just DI water and then the other was strawberry. The
strawberry contains ellagitannins which inhibits the growth of bacteria. Their graph was
clear, and they had many sources.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our
current state of knowledge.
I thought it was very good, I think they made sure they described everything in detail. I
kept looking for things they left out or things that were unclear and they did pretty well.
Grammar would be the only thing that might need some work. Other than that, I would
say good paper!
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical
errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the
manuscript.
This paper was a final version
(with a few corrections)