sss

INSTITUTE OF BIOTECHNOLOGY
DEPARTMENT OF BIOTECHNOLOGY

SENIOR PROJECT PROPOSAL ON THE:

ASSESMENT OF MICROBIAL QUALITY OF SELECTED CANNED FOODS
SOLD IN GONDAR TOWN, NORTHWEST ETHIOPIA

Senior Project proposal submitted to Department of Biotechnology, Institute of Biotechnology,

University of Gondar in Partial Fulfillment of the Requirements for Bachelor of Science in
Biotechnology

By:
NAME ID. No.
1. Meles Sisay………………………………………………………..…02554/09

Adviser: Mr. Aragaw Zemene (MSc.)

Submitted to: institute of biotechnology department of
biotechnology

Submission Date: 2021

Gondar, Ethiopia

Table of Contents Page

Acknowledgements..................................................................................................................................... i
Abbreviations.............................................................................................................................................. ii
Abstract......................................................................................................................................................iii
1. Introduction.............................................................................................................................................1
1.2 Statement of the problem...................................................................................................................2
1.3 Significance of the study...................................................................................................................3
1.4 Scope of the study..............................................................................................................................3
2. Objective of the study.............................................................................................................................4
2.1 General objectives.............................................................................................................................4
2.2 Specific objectives.............................................................................................................................4
3. Literature review.....................................................................................................................................5
3.1 Concepts of spoilage canned foods....................................................................................................5
3.2 Sterilization and Pasteurization..........................................................................................................5
3.1.1 Sterilization:...............................................................................................................................5
3.1.2 Pasteurization:............................................................................................................................5
3.3 Advice given to consumers regarding canned foods..........................................................................6
3.4 Nutritional Aspects............................................................................................................................6
3.5 Bacteria in canned food.....................................................................................................................7
3.6 Safety of Canned Foods.....................................................................................................................7
4. Materials and Methods............................................................................................................................9
4.1 Study area..........................................................................................................................................9
4.2 Study design and period.....................................................................................................................9
4.3 Sample collection..............................................................................................................................9
4.4 Microbiological enumeration.............................................................................................................9
4.5 Further identification of isolates........................................................................................................9
4.6 Determination of Growth Potential of pathogens in canned foods...................................................10
4.7 Expected outcome............................................................................................................................11
7...............................................................................................................................................References
...............................................................................................................................................................14
 DECLARATION
We, declare that the work presented in this research, entitled ‘assessment of microbial quality of
selected canned foods sold in Gondar town ,north west Ethiopia canned foods collected from
around University of Gondar, collage and Arada was prerequisite for the award of
undergraduate Degree of Biotechnology, submitted to Institute of biotechnology at University of
Gondar. We also declared that, this research here by submitted to the University of Gondar
complies with the regulations of the University and meets the accepted standards withrespect to
our own original work, and all the sources that we have used accordingly accredited.

NAME ID. No.

s
1. Meles Sisay………………………………………………………..…02554/0

Signed by Examining Committee

Examiner: ___________________________________signature _________ Date ____________
Advisor: ______________________ssssss_____________ signature_________
Date_____________

Acknowledgements
Foremost thanks to gods and his mother. We would like to thanks our deepest gratitude goes to
our advisor Mr. Aragaw Zemene (MSc, Asst. Prof) who helped us starting from title selection
and preparation to the production of this senior project proposal and for his unreserved support
and continuous comments and suggestions, intellectual guidance, close supervision, and devotion
of his time in criticizing this paper. Thanks and appreciation for the laboratory assistants

and Mr. Bogale Damtew, Mr. Sintayehu Mamuyeand Mr. Tilahun Gezawfor
guiding usduring laboratory work.We have special thanks to Mr. Bogale Damtew
he has been extra helpful specially, at the time of media preparation

Our special thanks to Institute of Biotechnology, Department of Biotechnology for

the providing necessary materials, giving us Laboratory work place and chemicals
to do this research.

We would also like to thank collage of veterinary medics allowing to get sheep
blood supplement for preparation of blood agar media.

We would also like to express our pleasure to thank Mr. Addisu Nega (PhD Candidate) for
providing such an opportunity of practicing on how to develop our own proposal upon the
theoretical knowledge generously guided by him during the course work of scientific writing in
research methodology.

Last but not least, we are cordially thankful to our families and friends for their financial
contribution & motivational support without which we couldn’t be at this level of success today.
Abbreviations
ACF-……………………………………………………………………….. Acid Canned Foods

BPW………………………………………………………………………. Buffer Peptone Water

BPA………………………………………………………… Bisphenol Peptone Agar

EMB…………………………………………………………………. Eosin Methylene Blue

ETB-……………………………………………………………………….Ethiopian Birr

FAO……………………………………………………… Food and Agriculture Organization

ICMSF……………… International Commission on Microbiological Specifications for Foods

LACF……………………………………………………………… Low Acid Canned Foods

pH………………………………………………………………. Power of Hydrogen

PRAS……………………………………………………… Pre-Reduced Anaerobically Sterilized

KOH………………………………………………………….. Potassium Hydroxide

WHO…………………………………………………….. World Health Organization

USDA………………………………………….. United State Development for Agriculture

Abstract
The canned foods have an important role in the conunter side and urban of many developing
countries in meeting the food demands of the city dwellers. They are composite foods sold to
consumers for consumption which do not require significant further processing except re-heating
or achievement of baking process. However, these foods may contain likely potential hazard to
human health due to non observance with food safety regulations by food handlers.

This study is therefore aimed at evaluating the bacteriological quality and associated factors of
some selected ready-to-eat canned foods sold in Gondar town and its vicinity, North West
Ethiopia. An experimental based cross-sectional study will be used to conduct the study at
Molecular Biology laboratory of Institute of Biotechnology, University of Gondar from March to
June, 2021. A total of randomly selected 2 samples of canned foods containing 2 each of ,
vegetable, tomatoes and milk shall be collected from Gondar town super markets and shops
there by the microbial load, when stored at ambient and refrigerator temperatures, is to be
assessed. The collected samples shall be prepared according to ICMSF.

Then for microbial record thirty five grams of each sample will be aseptically put into sterile
flask contained 350 ml of sterile peptone water (0.1%) and thoroughly mixed by using sterile
blender for 2 minutes to provide a homogenate of 10 dilution. Then after, record will be
followed by isolation and identification of different anaerobic bacterial count which may include
Actinomyces, Bacteroides, Clostridium, Fusobacterium, and other gram positive coliforms under
examination. The data will be recorded and stored in Microsoft Excel 2010 spread sheet and
statistically analyzed using tables and graphs.

To the end, this study is expected to be used as an asset in providing scientific information and
future direction upon the use of canned foods so that consumers, stakeholders and others who
involve in the market chain shall be benefited as a whole. The study is expected to be finalized
within 5 months with the estimated cost of 4,000 ETB.

Keywords: Anaerobic; Bacteriological quality; Canned foods; Gondar town.

 INTRODUCTION

1.1Back ground
Canned foods are a significant component of the diet of most individuals in developed and
developing countries, donation food in a suitable form with year-round availability. The canning
process relies on heat treatment for the destruction of microorganisms and preservation of the
food, which is then generally considered to have an unclear microbiological shelf-life providing
that pack integrity is maintained. (Charbonneau, 2002).

Canned foods are sterilized before being sited on the grocery shelf but if the sterilization has
been unsuccessful, contamination or food spoilage may occur (Desrosier, 2004). Swollen cans
most times do occur and usually contain gas produced by members of the genus Clostridium.
(Glass, 2008).

acerbic spoilage without gas is called flat-sour spoilage (Mc Clell and Bessie, 1945). Lactobacilli
are responsible for acid spoilage where they break down the carbohydrates. The different kinds
of food which can be preserved by canning include fruits, vegetables, milk, ,other prepared
items. In the context of this write up, canning is the method of interest. During canning process,
air is driven from the container and a vacuum is formed as the jar cools and seals. (MacDonald
and Reitmeier, 2017).

According to the United States, Department of Agriculsture (USDA), the only two methods of
canning approved are water-bath canning and pressure canning. The idea of canning is to make
food available and edible long after the processing time. The heating process during canning
appears to make dietary fibre more soluble and therefore more readily fermented in the colon
into gases and physiologically active by-products. Canned tomatoes have higher available
lycopene content (Springer, 2002).

To improve food safety for those who consume canned foods, government have enacted laws
requiring alphanumeric codes being put on food cans during manufacture indicating information
relevant to health, such as the date of canning. “Open Dating” (use of a calendar as opposed to a
code) on a food product is a date stamped on a product package to help the store determine how
long to display the product for sale. It can also help the purchaser to know the time limit to buy
or use the product at its best quality. Consequently, foods that have nasty odor, flavor or
appearance should not be eaten. Apart from missing out on key nutrients, one is also prone to
food borne diseases like botulism caused by the bacteria Clostridium botulinum, Listeria
infection etc. (Dainty, 2004).

The occurrence of spoilage in canned foods is low, but when it occurs it must be investigated
properly. Swollen cans often indicate a spoiled product. During spoilage, canning may progress
from normal to flipper, to springer, to soft swell, to hard swell (Landry et al., 1988).

However, spoilage is not the only cause of abnormal cans. Overfilling, buckling, denting, or
closing while cool may also be responsible. Microbial spoilage and hydrogen, produced by the
interaction of acids in the food product with the metals of the can, are the principal causes of
swelling. High summer temperatures and high altitudes may also increase the degree of swelling.
Some microorganisms that grow in canned foods, however, do not produce gas and therefore
cause no abnormal appearance of the can; nevertheless, they cause spoilage of the product (Vosti
et al., 1961). This article will consider the changes that can occur during canning and their effect
on the quality of the product (Pither, 2003).

1.2 Statement of the problem

In Ethiopia, various canned foods have been reported to carry anaerobic bacteria, Staphylococcus
aureus, Salmonella and Shigella (FAO/WHO, 1997). A FAO/WHO joint Expert Committee on
Food Safety concluded as early as 1983 that illness due to contaminated food is perhaps the most
widespread health problem in the contemporary world (Kannan, 2002). Data published since
then by various countries confirm this statement and indicate that the problem has been on
increase since then (Mensah et al., 1999). Canned foods contribute significantly to the diet of
many people in the developing world (Mensah et al., 1999).

Although governments throughout the world are attempting to improve the safety of the food
supply, the occurrence of food-borne disease remains a significant health issue in both developed
and developing countries.
Food-borne diseases represent a major concern in developing countries and also Ethiopia.
Diarrheal disease represents the second leading cause of death in Ethiopia (Muleta, D. and M.
Ashenafi, 2001). Typhoid fever is responsible for 2.8% of deaths in children aged 5–9 years,
8.9% in the 10–14 age group, 3.2% in the 15–19 age groups and 1.5% in the 20–49 age groups
(Moyo et al., 2007). In addition, in December 2016, Gondar University hospital students have
endured a cholera epidemic and since then the disease became endemic in the town. Studying the
microbial quality and safety of ready-to-eat canned foods has paramount importance to
understand the public health importance of these foods. Food safety is more importantly a public 2
health issue as it plays a significant role in health development.

In the past few years, canned foods are flourishing in major towns of Ethiopia. There are many
people who get involved in the preparation and sale of canned foods. It becomes common
practice to observe them around school, bus stations and other places where several people
found. In Ethiopia, the majority categories of people are consuming canned foods; while some
are protected from using these foods fear of contamination. However, information on the
microbial load and safety of these foods and associated factors in Gondar town is still remaining
limited. Therefore, with respect to bridging the existing gap, this study shall attempt to assess the
microbial quality and associated factors of selected canned foods and their public health
implications in Gondar town, North West Ethiopia.

1.3 Significance of the study

Assessment of microbial quality of spoilage to canned foods has many importance.

The main significance of this study is to identify the spoiled canned foods from un spoiled ones
so that facilitate for consumers to get the better ones, identify the type of microbe that cause the
spoilage to permit the growth of that microbe by adjusting the environmental condition that is
not favorable to the microbes,

understand the mechanism of microbes that spoil the canned food so easily can control the
growth, produce canned foods that are free from harmful microbes and free from contamination
at all.
1.4 Scope of the study
The study will be conducted in Northwest Gondar, University of Gondar, Institute of
Biotechnology in Atse Tewodros campus. The scope particularly targeted to study on the
assessment of microbial quality of some selected spoilage canned food commercialized in the
study area. That may cause several illnesses to human being especially live in Gondar town.

Geographical Benefit Methodogy

Based on aproches

2. Objective of the study

2.1 General objectives
3
 The general objective of this study is to assess the microbial quality of selected canned
sfood commercialized in Gondar town its vicinity, Northwest Ethiopia.

2.2 Specific objectives

 To isolate the potential microbes that cause canned foods to spoil in Gondar town
 To characterize the type of anaerobic microorganisms present in canned foods
 To assess the associated factors promoting the growth of microbes in canned foods
 To create awareness among the consumer about the microbial status of canned foods sold
in Gondar town and its surroundings.
3. Literature review
4

3.1 Concepts of spoilage canned foods

Canned foods may contain toxic bacteria While it's extremely rare that weren't processed
properly may contain hazardous bacteria known as Clostridium botulinum. Consuming
contaminated food can cause botulism, a serious illness that can lead to paralysis and death if left
untreated.

Most cases of botulism come from foods that have not been canned properly at home. Botulism
from commercially canned food is rare. It's important to never eat from cans that are stuffed
dented, cracked, or leaking. Canned foods that weren't processed properly may contain deadly
bacteria, but the risk of contamination is very. It can be a nutritious option when fresh foods
aren't available. They provide essential nutrients and are incredibly convenient. That said canned
foods are also a significant source of BPA, which may cause health problems. Canned foods can
be a part of a healthy diet, but it's important to read labels and choose accordingly.

3.2 Sterilization and Pasteurization

For canned foods, heat treatment generally falls into the following two categories:

3.1.1 Sterilization: Heat treatment of low acid foods (pH>4.6) that leads to commercial sterile
products. Heat processing is at high temperatures (±121°C), under pressure, with the objective of
killing all viable micro-organisms, including the spores of pathogenic bacteria (Abdul et al.,
1999).

3.1.2 Pasteurization: Heat treatment of acid foods (i.e. pH<4.6) that together with low pH, leads
to commercial sterility. The process temperatures are slightly lower than sterilization (usually 70
–100°C) and are designed to kill the vegetative forms of micro-organisms. These temperatures
have little to no effect on the spores of bacteria, which are kept in check by the acid environment
(USFDA, 2001).

5
3.3 Advice

given to consumers regarding canned foods

Once opened the food will have the same expected shelf life as any other cooked food and any
unused portions should be stored refrigerated in clean containers for three to four days maximum
(Charbonneau, 2002).

Don’t purchase, or consume, bulging, rusted, leaking, or deeply dented cans. Discard heavily
rusted cans. Cans that are heavily rusted can have tiny holes in them, allowing bacteria to enter.
If you open the cans and there is any rust inside, do not eat the food.

A sharp dent on either the ends or side seam can damage the seams and allow bacteria to enter
the can. Discard any can with a deep dent on the seams. If a can containing food has a small
dent, but is otherwise in good shape, the food should be safe to eat. Discard deeply dented cans.
A deep dent is one that you can lay your finger into. Deep dents often have sharp points.

Store canned foods and other shelf stable products in a cool, dry place: temperatures 15 – 25°C
are recommended. Never store them above or beside the stove, under the sink, in a damp garage
or basement, or any place exposed to high or low temperature extremes. Check your pantry or
storage cupboard every few weeks and use the canned foods that you have within the
recommended shelf-life. High temperatures (over 40°C) are harmful to canned foods. The risk of
spoilage increases as storage temperatures rise. Canned goods designed for use in tropical areas
are specially manufactured to take the high ambient temperature into account.

3.4 Nutritional Aspects

One of the incorrect, negative perceptions about canned food is that it is not as nutritious as other
types of food. The original canned foods were made for military rations and it revolutionized the
military at that time. At that time anything was better than nothing.

3.5 Bacteria in canned food

Canned foods are divided into two main categories: Low Acid (LACF) and Acid Canned foods
(ACF). The dividing line is pH 4.6, low acid foods having a pH greater than this, and acid food
having a pH lower than 4.6 (Landry et al., 1998).

6
The significance of this pH value is that Clostridium botulinum can grow at pH above 4.6. C.
botulinum is able to form very heat resistant spores and can grow in the anaerobic conditions in
canned food. Since C. botulinum also produces a lethal toxin, it is essential to destroy the
spores. So we heat the food in the cans to temperatures above boiling point (in fact we heat to
the equivalent of 121.1C for 2.52 minutes and this is called a 12D process). Properly processed
LACF is safe. When we look at acid foods, we find that C. botulinum is incapable of growth
under these acid conditions and so no toxin can be produced. Since we don't need to deliver a
12D process, we can heat for a shorter time or at lower temperature. This has the advantage that
the food is changed less and it costs less to deliver the process. Depending on the food and the
degree of acidity, we really need only destroy vegetative (i.e. non-sporulating) pathogens and
spoilage microorganisms. This will then destroy Listeria (Featherstone, 2015).

3.6 Safety of Canned Foods

Food begins to spoil soon after it is harvested or slaughtered. This spoilage is caused by
microorganisms or by internal c hemical changes which are caused by enzymes. Microorganisms
can be destroyed and enzymes can be deactivated by heat treatment and this is the reason why
thermal processing is the focal point in canning technology. Bacteria types vary and display
different preferences for temperature at which they will grow or die. Understanding bacteria
behavior is crucial for a better understanding of heating and cooling processes (Foster, 1997).

Food safety is nothing else but the control of bacteria. To control them effectively we have to
first learn how bacteria behave. Let's make something clear, it is impossible to eliminate bacteria
altogether. Life on the planet will come to a halt. They are everywhere; on the floor, on walls, in
the air, on our hands and all they need to grow is moisture, nutrients and warm temperature.
They all share one thing in common: they want to live and given the proper conditions they will
start multiplying. They don't grow bigger, they just divide and divide and divide until there is
nothing for them to eat, or until conditions become so unfavorable that they stop multiplying and
die (Foster, 1997).
4. Materials and Methods
4.1 Study area

the study was conducted in Gondar town, North West Ethiopia. The study included two areas in
Gondar town (College, and Arada ) where the number of canned food supermarkets and their 8
customers are high (Central Statistical Authority, 2007).

4.2 Study design and period

Experimental based cross-sectional study design shall be employed to undertake this senior
sproject work from March to June, 2021 in Gondar town, North West Ethiopia

4.3 Sample collection

tomato pasta and bobo milk were bouth

The test canned food samples was collected randomly from open shops in the open market sites
College and Arada of Gondar town, North West Ethiopia. A total of 2 samples of canned
foods comprising milk and tomato was collected from Gondar , northern west supermarkets
and shops. Date of production and expiration, country of production and ingredients of
each sample shall be in the meanwhile recorded.

The samples then shall be kept at ambient temperature until further analysis. Before opening,
cans will be visually examined for presence of double seam and side seam defects, flaws
and physical damage and pertinent observations will be recorded. The non-coded end of the
metal Can will also be cleaned with alcohol-socked towel. This shall be flamed using a
laboratory burner (Robert et al., 1996).

Then the contents was s mixed by shaking cans prior to opening. A sterilized opening device
shall going to be used to cut the desired size entry hole. Under aseptic conditions, 35 g of
samples were removed from the centre and placed into a sterile stomacher bag, 350 ml of
sterile bacteriological peptone water (BPW) shall be added to it and homogenized for 2 minutes
using a stomacher (Model 400, Seward). The homogenized samples were serially diluted.
4.4 EQUIPMENTS AND CHEMICALs

in order to conduct the research the equipments and chemicals

that had been used the following oven ,measuring balances,
flasks, shaker, measuring cylinder, auto clave ,test tube, laminar
air flow, incubator, Bunsen burner, micropipette,
beaker ,spatulas,petridish,spread plat ,anaerobic jar, test tube
rack,refrigerater plat counting agar, 70% alcohol macconskey
gar, nutrient agar, blood agar, disinter , disitelwater

4.4 Microbiological enumeration

Appropriate dilution (0.1 ml) of homogenized samples was surface plated in duplicates on pre-
dried surfaces of the following media for microbial enumeration:. anaerobically sterilized) media
using anaerobic jar that will be provided by University of Gondar specialized . Upon the use of
this jar, the plates will be incubated under anaerobic conditions for 1 to 2 days at 30-32°C.
After colony counting, 10 to 15 colonies will be randomly picked from countable plates 9

for further characterization.

4.5 Further identification of isolates

Colonies that will grow on the different agar media shall be streaked in Levine’s eosin
Methylene Blue (EMB) Agar and incubated at 37°C for 24 hours. The presence of small
nucleated colonies with greenish metallic sheen indicates Actinomycetes, while absence of the
polish with large nucleated colonies indicate Enterobacter sp. Growth on the Nutrient Agar was
streaked in Mannitol Salt Agar and incubated inverted at 37°C for 24 hours. The presence of
yellow pigment will indicate Staphylococcus auras. Also, the bacterial pure culture will be
streaked in Blood Agar; the presence of hemolytic properties indicates Streptococcus species
while growth with swarming characteristics suggest Proteus species.
s Biochemical test including gram reaction, citrate, urease, catalase, oxidase, Indole, coagulase,
motility, methyl red shall be carried out using the scheme of Cheesbrough and Benson. The
resultant microbial species from biochemical test in this study will be compared with those of
known taxa using scheme of Cheesbrough and Bergey’s Manual of Determinative Bacteriology
(Holt et al., 1994).

4.6 Determination of Growth Potential of pathogens in canned foods

This study will be conducted to asses the potential hazards related to possible kitchen
contaminations after cans are opened and left-overs are kept for later consumption. The growth
potential of Salmonella typhimurium and Shigella flexneri will be assessed in canned samples
taken from the study sites.

Two cans of each food item will be opened aseptically and separately innoculated with
overnight culture of the test strains to give an initial inoculum level of 102-103 cfu/gm. The
inoculated foods will be mixed thoroughly in their respective cans aseptically and incubated at
ambient (20-25oC) and refrigeration (4oC) temperature for 48 hours and 5 days, respectively. To
determine the initial inoculums level, freshly inoculated foods (10g each) will be homogenized
separately into 90 ml of BPW and 0.1 ml of appropriate dilutions will be spread plated on 10

McCaskey Agar. Portions of inoculated food items (10g each) will further be sampled
aseptically at six-hour intervals for 48 hours for ambient temperature and at one-day
intervals for five days for refrigeration temperature.

4.7 Expected outcome

It is believed that this study was giving relatively brief information on the microbial quality
status of canned foods of Gondar town, North West Ethiopia. This will in turn have great
positive affection for saving the lives of many consumers by inspiring researchers to explore
more about the area with due attention. Moreover, this study was used as a baseline to conduct
further research activity in particular and to revise policies related to public health care issue at
large. we were test with four media but we can’t get any micro organism

1. maccocky agar
2. plate counitg agar
3. nutrient agar
4.blood agar
 6.CONCLUSION
canned foods is available in every developing and developed country and every people eat it
but they do not have knowledge how much it is save for eating and what types of microorganism
found in it and which one is the most dougreases or the nature of bacteria is it aerobically or
anerobicically if it is anaerobic what kind is of disease found it but this research give answer
generally this two canned foods are fiery from anaerobic bacteria

13
 7.RECOMMENDATION
The outcome of this research project shows, there are many issues we recommend for
further study. Due to environmental factor, we didn’t optimize every parameter of
further identification of anaerobic microorganisms that found in canned foods that
cultured from canned foods mostly tomato and bobo milk needs precisely optimized
protocols considering the specific environmental factors. Hence,we would like to
recommend the following suggestions. canned foods like tomato and bobo milk should
be utilized for that are eaten in our country at any direction . There should be an
intervention mechanism on save our society from spoilages foods from improper
packaging of food during production and distributions. Further researches have to
be done on both save our society and give awareness.
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