A will used as solvents of the processes .The

A
Research proposal submitted to the Department of Pure and Applied Sciences in
partial fulfilment of the requirement for the degree of Industrial Microbiology
and Biotechnology of the Technical University of Mombasa.

 

 

 

 

NOVEMBER,
2017

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DECLARATION

This
Research Proposal is my original work and has not been submitted for Degree in
any other University.

 

 

Signature………………………………………………
Date………………………………………

 

ANTHONY MUSA

BTMB/023J/2014

 

SUPERVISOR

This
Research Proposal has been submitted for Examination purpose with my approval
as the university supervisor.

Signature………………………………………………………
Date………………………………

 

MR. NZARO MAKENZI

Department of Pure and Applied
Sciences.

 

ACKNOLEDGMENT

I would wish to thank the Almighty God for enabling me
to do this project proposal. I would also like to appreciate the technical
university of Mombasa especially my supervisor Mr makenzi and other lecturers
for equipping me with adequate knowledge and skills in this field.

Lastly I thank my family and friends they have been a
great motivationa and a stepping shoulder in difficult times.

 

Table of contents

Table of Contents

DECLARATION.. ii

ACKNOLEDGMENT. iii

Table of contents. iv

ABSTRACT. v

CHAPTER ONE.. 1

1.1BACKGROUND INFORMATION.. 1

1.2 STATEMENT OF THE PROBLEMS. 2

1.3 JUSTIFICATION.. 2

1.4 OBJECTIVES. 3

1.4.1 Main objectives. 3

1.4.2 Specific objectives. 3

1.5 HYPOTHESIS. 3

1.5.1 Alternative hypothesis. 3

1.6 RESEARCH QUESTIONS. 3

1.7 SCOPE OF THE STUDY.. 3

CHAPTER 2. 4

2.0 LITERATURE REVIEW… 4

2.1 ONION AND GINGER.. 4

2.2 BACTERIA.. 4

2.2.1 Gram negative. 4

2.2.2 Gram positive. 5

2.3 MECHANISM OF ANTIBIOTIC RESISTANCE.. 6

2.4 ACQUISTION OF RESISTANCE.. 7

 

 

 

ABSTRACT

Antimicrobial
potentials of Allium cepa and Zingiber
officinale revealed that the extracts from fresh samples show different ranges
of antimicrobial activities. Onion and ginger are widely used food spices for
both culinary and medicinal purpose .The study will aim at determining
antimicrobial activities of both onion and ginger in both individual extracts
and blend form. A blend of phytochemical extracts from onion and ginger will be
evaluated against gram positive and gram negative bacteria for antimicrobial
activity .The study will use two gram negative bacteria and two gram positive
bacteria such as; Escherichia coli, Salmonella typhi and Staphylococcus aureas ,Bacillus coagulans  respectively . Water and methanol will used as
solvents of the processes .The zone of inhibitions will tested and the diameter
of the zones measured and compared.

 

CHAPTER ONE

1.1BACKGROUND INFORMATION

The
efficacy of traditional medicinal herbs is evident from their continued use by
a significant portion of the world population. Use of traditional medicine and
medicinal plants in most developing countries for maintenance of good health
has been widely observed. Effort has been devoted over years to the search for
new antimicrobial materials from natural sources for food preservation. Natural
derived compounds and products may have application in controlling bacteria in
food. Increasing reliance on the use of medicinal plants in industrialized
societies has been traced to the extraction and development of several drugs
and chemotherapeutics from this plants which are used for herbal remedies.
Onion and ginger were consumed throughout in Europe in the Middle Ages and were
later thought to guard against evil spirit and plagues due to their strong
odor. Folk healers traditionally used for food and medicinal applications
having anti-inflammatory antioxidant effects in both in vitro and in animal
models.

Food
borne diseases is gradually becoming a serious risk to public health with a
number of cases increasing yearly as people become infected by either eating
food with microbes. Proper control of microbes and effective sanitation will
help to reduce the rate at which people become sick by eating food .spices in
general show both antimicrobial and anti-oxidants characteristics, inhibiting
the growth of bacteria and other pathogenic microbes. Scientists in the past
have conducted numerous experiments testing foodborne pathogens with
phytochemicals extracted from spices. The antimicrobial properties of active
compounds extracted from spices and other plants Have Shown to affect the
behavior of pathogenic bacteria and fungi in the agro food and medical sector.
This study investigates the antimicrobial activity of phytochemical extracted
from onion and ginger against E.coli ,S
typhi, B coagulans and S. aureus
using  a well diffused agar.

1.2 STATEMENT OF THE PROBLEMS

Medicinal
plants have been in use by both ancient and modern man of all cultures for
treating different ailments .A single plant processed in different formulations
can be used to cure a wide range of diseases and may show excellent results
against different pathogens .The historical role of medicinal herbs in the treatment
and prevention of diseases do not assume their safety of uncontrolled use by an
informed public. Several cases of diarrhea by students have been reported due
to consumption of food with pathogens at neighbor hotels within the school in
Tudor area. This food pathogens has led to food borne diseases which has led
students suffer from diarrhea stomach ache and other disease effect. Onion and
ginger  having some antimicrobial
activities which inhibit growth of some pathogenic microbes food in food, can
then be used as a source of inhibition to such pathogens such as S.aureus 
and E.coli thus reducing
the food borne diseases caused by this microbes and other gram positive and
gram negative bacteria. Onions and ginger been widely spread for sale within
the school area,example: Buxton and Kongowea at very low prices and been spices
mostly used in food and drinks consumed by students daily will be then be a
cheapest way of controlling the food borne diseases by raw consumption or added
as spices in more concentration to increase its bio-availability in the GI
tract.

1.3 JUSTIFICATION

Rampant
food contamination in many countries has been observed in research by WHO were
lots of people are ill due to consumption of contaminated food and other die of
food borne diseases. Food borne diseases impede socioeconomic development by
straining health care systems and interrupting national economies, trade and
tourism. Plant extracts of onion and ginger have been proved to have
antimicrobial activity and can inhibit bacteria by different mechanism since
they contain bioactive compounds, phenol molecule and inflammatory compounds
which lead to anti-inflammatory effects which can be used for both preservation
and medicinal purpose to inhibit the growth and replication of food pathogen
thus reducing the bacterial load and food borne diseases.

1.4 OBJECTIVES

1.4.1 Main objectives

To
investigate the antimicrobial activities of onion and ginger against gram
positive and gram negative bacteria.

1.4.2 Specific objectives

1.
To determine the rate of inhibition using different solvents example water and
ethanol

2.
To find out the antimicrobial activity in individual extract and in combined
against gram negative and positive bacteria.

3.
To determine the zone of inhibition diameter and compare.

1.5 HYPOTHESIS

1.5.1 Alternative hypothesis

1.
Gram negative are more resistant than gram positive against antibiotics because
it contains outer membrane with lipopolysaccharide and protein which makes the
membrane less permeable.

2.
The extracts have different rates of activity in different solvents.

1.6 RESEARCH QUESTIONS

1 .What are the methods
used in onion and ginger plant extracts?

2. Will the onion and
ginger extract show antimicrobial activity?

 

1.7 SCOPE OF THE STUDY

The
study will be focused on the daily used spices used within Mombasa region and
some of the food pathogens which cause food borne diseases in the region.

CHAPTER 2

2.0 LITERATURE
REVIEW

2.1 ONION AND
GINGER

Onion
has been revered throughout time not only for its culinary use but also for its
therapeutic properties. Consumption of onion is beneficial to human health as
it contains polyphenol molecules or phytonutrients which include; flavonoids,
tannins, as well as allicin, quercetin binds to bacteria DNA gyrase while
allicin inhibits certain thiol containing enzymes in the microorganisms by the
rapid reaction of thiosulfinates.

GINGER
has a long tradition of being very effective in alleviating symptoms of
gastro-intestinal distress. Ginger can also been used as herbal medicine since
it possesses numerous therapeutic properties similar to onion including
antioxidant effects, an ability to inhibit the formation of inflammatory
compounds and direct anti-inflammatory effects. The main antimicrobial agent is
gingerol a natural occurring phenol which disrupt the cell wall of bacteria
causing cytoplasmic leakage.

2.2 BACTERIA

2.2.1 Gram negative

They
give a negative results in gram stain. This type of bacteria retains the violet
stain after the decolourisation step. Alcohol is used to degrade the outer
membrane making the cell wall more porous. They have thin peptidoglycan
sandwiched in between inner cell membrane making them to take up the
counterstain and appear red or pink in a microscope.

2.2.1.1 Escherichia coli

E.coli
is a gram negative, straight rod bacteria of about 1.1-1.5µm by 2.0-6.0µm
occurring either singly or in pairs. They belong to class proteobacteria, the
order enterobacteriales, the family enterobacteriaceae and genus Escherichia .
They are motile by petrichous flagella and fulcatatively anaerobic having both
respiratory and a fermentative type of metabolism. Biochemically they are
oxidase negative and usually citrate negative. E.coli occurs in normal flora in the lower parts of the intestine,
optimal growth is about 37 degree Celsius but are known to grow at temperatures
of about 44.5 degree Celsius .E.coli
strains containing enterotoxins are known to cause diarrheal diseases due to
their invasion and colonization characteristics .They also cause urinary tract
infections and nosocomial infections including; septicemia and meningitis.

2.2.1.2
Salmonella typhi

Is
a subspecies of salmonella enterica, rod shaped, flagellated ,aerobic and a
gram negative bacteria which infect broad range of vertebrate hosts. They have
a number of serovars which are divided into two; one which cause usually cause
enteric infections and ones which usually cause invasive degradation of
anaerobic metabolic pathways. S typhi has
recently been an emerging disease in Africa. It’s associated with HIV, malaria
and malnutrition have contributed to spread of this disease and the need to use
expensive antimicrobial drugs in the poorest health services in the world.

2.2.2 Gram positive

It
gives positive results to gram stain by taking up the crystal violet stain then
appear purple in microscope. This is due to their thick peptidoglycan layer in
bacterial cell wall which retains the stain after wash.

2.2.2.1 Staphylococcus aureus

S.aureus
is a gram positive bacterium that is a member of firmicutes group .It also
belongs to class Cocci, order Bacillus, family Staphylococcaceae and genus
Staphylococcus. The cells are spherical about 0.5-1.5µm in diameter and in
clusters or pairs .The are non-motile non-sporing facultative anaerobic with
both respiratory and fermentative mechanisms. Colonies are usually white and
cream on general purpose agar such as TSA or yellow on differential media such
as MSA .Biochemically they are catalase positive and can grow in about 10%
sodium chloride with an optimum temperature of about 30-37 degrees Celsius
.They are mainly associated with the skin and mucus membrane of warm blooded
animal and serves as indicator of nasal contamination in foods. They are known
to be opportunistic pathogens in humans 
causing diarrhea by producing extracellular toxins .S. aureus is an important food borne pathogen  causing food poisoning which leads to
diarrhea.

 2.2.2.2 Bacillus coagulans

It
is gram positive lactic acid forming bacterial species. It mainly causes an
outbreak of coagulation in evaporated milk packed in a lower temperatures .It
comes from family of lactobacillae and bacillaceae .its catalase positive,
spore forming, motile and a falcutative anaerobe.

 

2.3 MECHANISM OF ANTIBIOTIC
RESISTANCE

Bacteria
can resist the effect of  antimicrobials
through a variety of mechanisms .some species of bacteria are innately
resistant to ? 1 class of antimicrobial agents.in some cases the resistance is
innate, but in many others it is acquired of greater concern are cases of
acquired resistance, where initially susceptible populations of bacteria become
resistant to an antimicrobial agent and proliferate and spread under selective
pressure of use of the urgent .several mechanisms of antimicrobial resistance
are readily spread through a variety  of
bacterial genera such as; drug inactivating enzyme were an organism acquires
gene encoding enzyme that destroys the antimicrobial agent  before being effective ,increased elimination
of drugs where through efflux pump detrimental compound are transported or
removed out of the system an alternation in the target molecule where minor
structural changes of the target molecule 
prevents the drug from binding thus not effective.

2.4 ACQUISTION OF RESISTANCE

Several
process in the body may lead to antimicrobial resistance through either
spontaneous mutation which alters new gene and acquisition of new gene which
may occur between different gene bacteria .spontaneous mutation may cause
resistance due to the following; altering the target protein by ant-microbial
agent by either modifying or eliminating the binding site ,production of
compounds that will inactivate the ant-microbial agent, down regulation or
altering an outer membrane protein channel that the drug requires for cell
entryexample; ompf  in E.coli and up
regulating pumps that expels the drug from 
the cell .Mechanisms of genetic exchange include conjugation ,
transduction and transformation. Each of these processes, transposons may
facilitate then transfer and in-cooperation of the acquired resistance gene in
to the host genome. Conjugation among gram positive bacteria is usually
initiated by production of sex pheromones by the mating pair which facilitate
the dumping of donor and recipient organisms, allowing the exchange of DNA.

During
transduction, resistance gene are transferred from one bacterium to another via
a bacteriophage. Finally in 
transformation bacteria acquire and incorporate DNA segments from other
bacteria that have released the DNA compliments in to environment after  cell lysis, can move resistance gene in to
susceptible strains mutation an selection, together with mechanism of genetic
exchange enable many bacteria species to adapt quickly to the introduction of
the antimicrobial agents in to the environment . Although single mutation in a
key bacteria slightly reduce its susceptibility of the host bacteria to that
antibacterial agent, it may just be enough to allow its initial survival until
it acquires additional mutation or additional genetic information resulting in
full-fledged resistance to the antibacterial agent.

 

CHAPTER
3

3.0
METHODOLOGY

3.1
STUDY AREA 

 

This
study will be carried in Mombasa county with a number of people population of
about 1.2  million ,thus the study will
be beneficial  to people living in
Mombasa county prevalence to onion and ginger antimicrobial activity
antimicrobial activity to some of the food borne pathogens gram positive and
gram negative bacteria

3.2 SAMPLING
METHOD    

The
onion and ginger bought from Kongowea market near the university will be used
to get the plants extracts then taken to universities lab for experimental
analysis.

3.3
SAMPLE SIZE

24 samples will be
obtained for the study.

3.4
SAMPLE PROCESSING

3.4.1
Sample collection

Onion
and ginger will be bought in Kongowea market by randomly collecting them from
different vendors .The samples from different vendors will be randomly picked
and grouped to one sample for ginger and onion ,then a random picked onion and
ginger will be mixed in one group that will form a blend .3 grouped samples
each of 1/4Kg will be used for sample exaction.

3.4.2  Sample preparation

The
process of extraction is by hydrolysis, where 5g of onion and or ginger will be
homogenized in different places using a pestle and a mortor.20ml of water will
be added to form a solution of each sample. The solution is then sterilized and
filtered to remove the residues. The solution of the extract will be evaporated
using a rotary evaporator to form a dry residue which will be measured for each
of the individual extract and the blend extract then stored for antimicrobial
analysis.

The
same procedure will be used for extraction of both onion and ginger using
methanol. The homogenate will be dissolved in 20ml of methanol. Thereafter the
extract will be sterilized filtered to remove residues.

3.5
BACTERIAL STRAINS AND CULTURING

Staphylococcus
aureus and Escherichia coli will be obtained from the microbiology laboratory
at the technical university of Mombasa. The bacterial strain will be then be
suspended in Triptych soy broth and incubated at 37 degree Celsius for
24h.Diffusion method will be used using Mueller Hinton agar. Kirby-Bauer disc
for Bacterial strain control will be used to show the susceptibility of the
onion, ginger and onion-ginger blend extracts and their effectiveness on the
test microorganisms.

3.6
ANTIMICROBIAL TESTING

Kirby-Bauer
disc diffusion method will be used for determination of antimicrobial
activities of onion and ginger extracts. Bacteria in a soft nutrient agar
cultured for 24h at 37 degrees will be spread on the agar plate medium of
thickness between 5ml with a sterile glass spreader and rotated several times
.The plate will be left for 30mins to ensure evenly distribution of the
inoculum. The extract will be dissolved in three different measures; 0.1g,
0.01g and 0.001g for each of the three extracts to get nine samples dissolved
in 5ml 0f water and other nine samples dissolved in 5ml of methanol thus having
eighteen total dissolved extract samples.

The
holes will be punched out of the agar by using a sterile cork borer of 6mm
diameter then will be filled with 100µl of the sterilized supernatants of each
extract. The plates are incubated until zones of inhibitions will be measured
to nearest millimeter using transparent ruler. The antibacterial activity of
the extracts will be then compared to the activity of the antibiotics on
Kirby-Bauer disc.

3.7
DATA ANALYSIS

The
results will be recorded in a data entry book. The susceptibility results of
the bacterial isolates will be classified into three; susceptible, intermediate
and resistant based on the standard interpretation table.

 

REFFERENCES

Murray PR, Rosenthal KS,
Pfaller MA 2009 Medical Microbiology 6th edition

Nester, Anderson,
Pearsall Microbiology, a Human perspective 4th edition

D Acharya, S.
Trakulsomboon, S.K Maldhup, and S. Korbsrisate, (2012). Antibiotic susceptibility
pattern and indicator of decreased ciprofloxacin susceptibility of salmonella
enterica serovar typhi from Dhulikel Hospital, Nepal, jpn J Infect Dis, 65)264
267.

Serthi S, Dutta A, Gupta
B, Gupta S (2013) Antimicrobial Activity of spices against isolated food borne
pathogens. International Journal of Pharmacy and Pharmaceutical Sciences
260-262.

Facaultaviely Anaerobic
Gram-Negative rods (2000) In: Holt J (Ed), Bergeys Manuel of determinative
Bacteriology (9th edn), Williams and Wilkins, Baltimore, USA,
pp.532.

Gram positive cocci
(2000) Bergeys Manuel of determinative Bacteriology (9th edn), In:
Holt (Ed), Williams and Wilkins, Baltimore, USA, pp.532.

Azu N ,Onyegha R (2006)
Antimicrobial properties of Extracts of Allium
cepa (onion) and Zingiber officinale
(ginger) on Escherichia coli, Salmonella typhi and Bacillus subitilis ,Internet scientific
publications 3(2).

British Society of
Antimicrobial Chemotherapy, Disc diffusion method for antimicrobial
susceptibility testing, BR Soc Antimicrob Chemother; 2:1-46, (2002).

 

3.9
BUDGET

ACTIVITY/ITEM

AMOUNT

Raw
materials

500

Project
writing and Binding

2500

Reagent
and media

6000

Transport

1000

Miscellaneous

2500

TOTAL

12500