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Phoenix Wilson
Junior Seminar
15 April 2016
The Effects of Disease on the Probiotic Properties of Dairy Bacteria
Introduction
The human microbiome consists of all of the microorganisms that live in relation to the
human body (Guinane and Cotter, 2013). Recently, there has been extensive research proving the
correlation between the microbiome and overall human health. The microbiota that constitute the
microbiome are necessary to maintain homeostasis, affecting metabolic activity, immune
response, and nutrition (Dethelefsen et al., 2008). Studies have shown the existence of beneficial
commensal microbiota that possess characteristics that provide health benefits to the host.
Bacteria are the most common microbial phylum present, with about 100 trillion cells existing on
and inside the human body (Guinane and Cotter, 2013).
In the history of medicine, bacterial pathogens were often treated with antibiotics, used
either to kill pathogenic bacteria or to disrupt the proliferation of the infection. While
exterminating pathogenic bacteria, antibiotics can also harm healthy bacteria and disrupt natural
systemic interactions (Dethelefsen et al., 2008). One major effect of antibiotic administration is
dysbiosis, which can lead to disease. Dysbiosis describes an imbalance in microbes, usually upon
the introduction of harmful pathogens, which can decrease the amount of necessary healthy
bacteria in a system. When there are not enough healthy bacteria to maintain homeostasis, the
body cannot function properly. Recent investigations have considered alternatives to antibiotics,
and probiotics may pave a new way for disease treatment.

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The term probiotic is applied to bacteria that have a positive effect on the body of the
host (Jungersen et al., 2014). In addition to serving as medication, probiotics are often introduced
in foods to aid digestion and some specific bacterial strains have been found to improve human
health. Probiotics are prevalent in the genera Lactobacillus and Bifidobacterium, which are lactic
acid bacteria, and live cultures of these genera can be found especially in dairy products
(Haghshenas et al., 2015). Lactic acid bacteria are considered to have profound health benefits,
such as cancer prevention, activation of immune response, and disease prevention (Amarii et al.,
2014).
In the proposed research, I will study live cultures of the most prevalent bacterial genera
that are advertised to be in Yoplait yogurt, the most popular yogurt brand in the United States.
This experiment is an attempt to determine their probiotic properties, which could potentially
indicate the effectiveness of each strain in different environmental conditions.
Proposal
I am proposing to examine how environmental changes caused by disease can potentially
affect the probiotic properties of Lactobacillus bulgaricus, Streptococcus thermophiles, and
Lactobacillus acidophilus against pathogenic bacteria. Each probiotic strain will be plated by
itself, and each strain will have a control plate that has not been interceded. I will be determining
the effectiveness of the three bacterial strains based on acid and bile resistance and nitric oxide
production (Haghshenas et al., 2015). For the acid and bile resistance, there is a test that can be
done by inoculating each bacteria strain in three MRS broths: one with a pH of 2 due to
hydrochloric acid, one with medium containing 0.2% of bile salts, and a control MRS broth. The
optical absorption of the acid and bile salt broths will be compared to that of the control broth.
The purpose of determining acid and bile resistance is to test the ability of the bacteria to resist

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environmental changes. Diseases that affect the gut environment can often lead to changes in
acidity levels and an increase in bile production (Amraii et al., 2014). Testing bacterial resistance
to different levels of acid and an increase in bile salts can demonstrate if the bacteria are actually
able to function in a diseased system. The results could potentially provide insight on the ability
of the bacteria to combat dysbiosis by resistance to condition changes, such as increasing acidity
or basicity in the environment.
The production of nitric oxide in bacteria is utilized for killing pathogens because they
are excreted from macrophages such as white blood cells as a form of immune response (Khani
et al., 2012). NO can be considered to be a natural antibiotic response from bacteria with cytoxtic
and cytostatic properties (Khani et al., 2012). Nitric oxide production can be measured using
ELISA plates and Griess reagent to compare the absorbance of the bacteria at 450 nm (Khani et
al., 2012). Each bacteria strain will also have three plates each for the acid and bile resistance
tests, along with one for the nitric oxide production test.
Statement of Relevance
I chose to undergo this experiment in order to increase the understanding of the effect of
disease on commensal bacteria. The bacteria of focus are from genera that are commonly found
in food items such as yogurt, which can evidence the profound effect that diet has on the human
system, especially immune response.
The properties that represent characteristics of probiotics were selected because they are
necessary for disease prevention. Acid and bile resistance are a requisite for bacteria that live in
the gastrointestinal tract, so that they can withstand changes to the environment, such as an
increase in acidity and bile production (Amraii et al., 2014). Nitric oxide is a chemical that can
be produced by bacteria that is able to eliminate pathogens and infected cells (Khani et al.,

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2012). Characteristics such as these are able to hinder the proliferation of pathogens and rid the
body of infection.
The larger effect of this project would be providing insight to the question, are these
probiotics effective against diseases? Hopefully the results of the experiment would be able to
show that the bacteria are shown to express the probiotic characteristics. The implications of this
research have the ability to show the role that probiotics have in disease progression. Since the
bacterial strains are present in yogurt, there can be connections drawn between the probiotic
effects of food items with live cultures and human health. If proving that the bacteria can express
disease-hindering properties, not only will the research demonstrate how probiotic bacteria can
prevent the pathogenesis, and prevention of diseases. Then, conclusions could be made relating
to the actual effect of taking in concentrated amounts of these bacteria when treating disease.
Specific Aims
There should be three stages to my research will be infection, property determination, and
statistical analysis. For the infection part of the experiment, the lactic acid bacteria (LAB), the
will be grown in separate MRS agar plates. Then once the LAB reach the end of their log phases,
or the end of exponential growth, then the property determination tests can be performed. This
whole process itself will take about a week.
The determination of the probiotic properties could occur about two days after the the
bacteria has grown. The purpose of the assays and determination tests are necessary in order to
determine certain probiotic traits that the yogurt bacteria may potentially possess. From their
results, I can then draw conclusions about the results, to see if they could have an effect on a
diseased system. Each of the acid and bile resistance and nitric oxide production tests can be
undergone in a matter of hours, using just absorbance spectroscopy and ELISA, so the results

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could be gathered in about a week each. From these results, I can compare the effectiveness of
the probiotic properties in the bacteria between the control and the experimental plates to
understand if the different pH values affected probiotic expression in each strain of bacteria.
Then I could compare the amount of NO production between each bacteria strain and its control.
So in total, the determination of properties aims could possibly take up a month alone, in order to
catalogue the probiotic properties of the yogurt bacteria, which will be the bulk of the
experiment. From the property determination of the experiment, conclusions of how the
commensal bacteria protect the gut environment from the pathogenic cells can be drawn. Then,
the amount of probiotic properties that the bacteria express compared between the strains to see
which bacteria are able to survive and also function in unfavorable conditions. If time permits,
then another level I would like to add to the experiment would be investigating the conditions of
temperature and pH by which each strain could produce the most nitric oxide, which would be a
way to relate the two properties further.
The last aim of the experiment will be the statistical analysis. I will apply one-way
analysis of variance (ANOVA) test to test the relationships of each strain population in the three
different acid and bile conditions. A Turkeys test could be utilized after to determine how
significant the observed differences are. The information garnered from these tests will
essentially compile my results so that I may be able to draw conclusions. These processes will
take about two weeks. The statistical data can be determined to see if the three environmental
conditions have a significant effect on the bacterial growth, in comparison to the uninhibited
bacteria strains in the control plate.

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Expertise/Institutional Support
Due to the nature of my experiment, the applications of cell biology and microbiology
would be necessary. The person that has the most experience in these fields is Dr. Barbara Merk,
who I hope will be my research advisor.
Budget
I will need:
MRS (de Man, Rogosa and Sharpe) agar plates and broth
Lactobacillus bulgaricus, Streptococcus thermophiles, and Lactobacillus acidophilus

strains
Three ELISA kits
Hydrochloric acid
Griess reagent
Absorbance spectrophotometer
pH monitor
PBS

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