The Comparison of Strawberry Extract on the Growth of Two Different Gram-Negative Bacteria

The Comparison of Strawberry Extract on the growth of two Different Gram-negative Bacteria. Formatting

Mohammad Dadkhah, Amin Najmabadi

Department of Biology, Saddleback College, Mission Viejo, California

Abstract

Strawberry is a rich source of bioactive compounds, such as phenolics and organic acids, which have antimicrobial activities properties 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 and Salmonella Typhimurium. Escherichia coli are is a common type, of Gram-negative bacteria that can get into infect various food items, like 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 is [by using the term present study you are implying that it is current and therefore you would use present tense and not past tense to describe it] to determine the effects of strawberry extract on selected pathogenic bacteria such as E. coli and S. Typhimurium [you can shorten this one as well] to identify antibacterial activity. One control group for E. coli, and 1 one [use the same formatting for the entire paper so if you write a number out do not use the numerical symbol for it also, besides it is far more professional to write it out; also employ the use of commas and avoid using the word “and” so many times] for Salmonella Typhimurium and as well as an experimental group for each bacterium. 10ml of each bacteria were was obtained from the colonies grown and were was placed on to the Petri dishes by a 0.25 lawn spread. Salmonella with DI water 0.30mm ± 0.13mm [below you describe that there are four experimental setups however you neglect to describe the measurements for Salmonella Strawberry, although you refer to it below] . 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 Salmonella [remember to spell the subject of the study correctly and also to italicize it] 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 [make sure you spell things correctly] have been described and they are categorized into several subgroups (Puupponen-Pimia et al. 2004). Flavanones are typically present in citrus fruit, and favanols [spell correctly] in green tea. Berries, which are traditionally a part of the Finnish diet, are an excellent source good sources of favonols [spell correctly] while the predominating predominant [use this form instead] group of favonoids [spell correctly], 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. [verbatim from above, okay to use but change activities to properties]

Berry phenolics possess many interesting biological activities properties. Their antimicrobial activity has properties have 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, a few are beneficial, and some are harmful pathogens. 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 infect food, like 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 the 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 is 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 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 the plates were divided into 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 was obtained from the colonies grown at in the Saddleback College laboratory. The bacteria were 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 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 the Bonferroni Correction Post Hoc test.

Results

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.

[be sure to make the font size under the figure smaller than the rest of the text]

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 extract 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). Result shows that strawberry extracts clearly showed exhibited that phenolic compounds, especially ellagitannins contained in strawberry was responsible for the strong antibacterial effects against the E. coli bacteria. There was were no pH effects in the inhibition caused by the pure phenolic fractions, because pH of the fractions was determined to be in the neutral area range. The main organic acids present in most berries are include 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 dose does [incorrect word] not contains benzoic and malic acids (Puupponen-Pimia et al. 2004). We would like to suggest that the reason of for the growth of salmonella bacteria on experimental plates was because of due to the 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 and 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 experiment which was performed was of great educational value, and the results are quite interesting. The use of strawberry extract as a bacterial growth inhibitor was a unique choice. As well as all the background information as to why the researchers selected strawberries as their growth inhibitor as opposed to another type of berry. The execution of the experiment by using a control group with DI water was also a great idea and the way in which they described the use of this makes the reader understand its, importance. Overall I think the message of the paper is very interesting and informative and the authors really do convey interesting results.

General Comments

Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of knowledge.

The paper’s weaknesses included several spelling errors throughout such as in the first half of the introduction where “flavonoids” and all its derivatives were spelt incorrectly numerous times. I consider these issues to be quite serious especially at the level at which we are operating.

The title is also weak and I would suggest changing it to something such as “The Effect of Strawberry Extract on the Growth of Gram-Negative Bacteria” as this encompasses the research that was performed however it doesn’t limit the scope of the research either.

As far as the strengths go, the use of outside references was good, as well as the overall description of the experiment itself. In addition to this the background information utilized throughout the paper was also very good as it gives the reader a broader scope of the way in which this research is important.

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 This paper was a rough draft

I thought the organization of the paper was very good and I thought it flowed very well. Also the message that the authors were trying to convey seems quite clear. All errors have been highlighted in red so the authors may correct them. The majority of the errors were spelling errors with a few errors in regards to tense as well as some grammatical errors.

Also the authors would be wise to take a look through the past Saddleback College Journal to familiarize themselves with the format of their names, as well as the address of the institution and how it should be presented within the paper.