DEDICATION
This research is dedicated to
Almighty God
To my beloved parents
To dearest brothers and sisters
To all my friend
i
ACKNOWLEDGMENT
I express my heartfelt thanks to my supervisor Mrs. KARINE BERNARD who devoted a large
part of her time to the supervision of this work. Her guidance, comments, and advices led to the
successful completion of this dissertation.
I deeply express my gratitude to my beloved parents, my father Cajetan MUSEKURA, my
mother Laurence NARAME for their contribution to my education from birth to now. My
recognition also is extended to the family of Savio NDAYAMBAJE for their financial support
and encouragement.
I am grad to express my gratefulness to my colleagues and classmates for the assistance as well
as the friendly atmosphere we shared in the course of academic life. My special thank also go to
Deo MAHORO, UWIMANA J.M .V for his advices.
Finally, I would like to thank everybody who was important to successful realization of this
research as well as expressing my apology that I could not mention personally one by one.
ii
TABLE OF CONTENTS
Contents
DEDICATION ................................................................................................................................................... i
ACKNOWLEDGMENT ..................................................................................................................................... ii
TABLE OF CONTENTS.................................................................................................................................... iii
LIST OF ABBREVIATION ................................................................................................................................. v
ABSTRACT..................................................................................................................................................... vi
CHAPTER I: INTRODUCTION .......................................................................................................................... 1
Background of the study ........................................................................................................................... 1
1. Statement of the problem .................................................................................................................... 2
2. Objectives.............................................................................................................................................. 2
2.1. General objective ........................................................................................................................... 2
2.2. Specific objectives .......................................................................................................................... 2
3. Research questions ............................................................................................................................... 3
4. Hypothesis............................................................................................................................................. 3
CHAPTERII: LITTERATURE REVIEW ................................................................................................................ 4
II.1.Generalities on banana and traditional banana wine ........................................................................ 4
II.2.Classification of banana ...................................................................................................................... 4
II.3. Different types of banana cultivated in Rwanda ............................................................................... 5
II.3.1. Cooking bananas ......................................................................................................................... 5
II.3.2. Dessert bananas and plantains ................................................................................................... 5
II.3.3. Brewing bananas ......................................................................................................................... 5
II.4. Role of banana in Rwanda ................................................................................................................. 6
II.5 Preparation of traditional banana wine or processing ....................................................................... 7
II.5.1. Ripening or maturation of banana .............................................................................................. 7
II.5.2. Juice extraction ........................................................................................................................... 7
II.5.3. Fermentation of banana wine .................................................................................................... 8
II.6. Different types of traditional banana wines ...................................................................................... 8
II.7. Challenges to traditional banana wine production ......................................................................... 8
II.8. FERMENTATION ................................................................................................................................. 9
II.8.1. Definition .................................................................................................................................... 9
iii
II.8.2 Types of fermentations ................................................................................................................ 9
II.8. 3 factors influencing alcoholic fermentation ............................................................................... 10
II.8.4. Microorganisms using in fermentation..................................................................................... 12
II.8.6. Microorganisms that may affect traditional banana wine and producers ............................... 13
CHAPTER III: METHODOLOGY ..................................................................................................................... 15
III.1. Sample collection ............................................................................................................................ 15
III.2. Materials sterilization ..................................................................................................................... 15
III.3. Culture medium .............................................................................................................................. 15
III.3.1. Different culture medium used in experiment ........................................................................ 15
III.4. Serial dilution .................................................................................................................................. 16
III.5. Inoculation ...................................................................................................................................... 17
III.6. Incubation ................................................................................................................................... 17
III.7. Description of colonies ................................................................................................................... 17
III.7.1. Description ............................................................................................................................... 17
III.8. Microscopic observations ............................................................................................................... 18
III.9. Physicochemical properties ............................................................................................................ 18
III.9.1. Alcoholic degree....................................................................................................................... 18
III.9.2. pH ............................................................................................................................................. 19
CHAPTER IV: RESULTS AND DISCUSSION .................................................................................................... 20
IV.1. RESULTS .......................................................................................................................................... 20
IV.2. Discussion on microbial analysis..................................................................................................... 28
CHAPTER V: CONCLUSION AND RECOMANDATION ................................................................................... 31
V.1. CONCLUSION ................................................................................................................................... 31
V.2. RECOMMENDATIONS ...................................................................................................................... 31
REFFERENCES .............................................................................................................................................. 32
APPENDIX .................................................................................................................................................... 34
iv
LIST OF ABBREVIATION
EMB: Eosin Methylene Blue
FAO: Food and Agriculture Organization
K.I.E: Kigali Institute of Education
MICR: Microorganisms
MINAGRI: Ministry of Agriculture
M.T: Million tons
N.B: Nutrient Broth
RBS: Rwanda Bureau of Standard
SAB: Sabouraud Dextrose Agar
TBW: Traditional Banana Wine
v
ABSTRACT
The traditional banana wines manufactured in rural areas are of poor hygienic quality and the
mode of production is not standardized in the country. Therefore, this research entitled
microbiological analysis of traditional banana wine prepared by local population with feet and
hands in Musanze and Ngoma district has the objective of analyzing and compare the microbial
content of traditional banana wine manufactured in those areas. To achieve our objectives, 3
different samples from Ngoma sectors and 3 from Musanze sectors have been collected and
analyzed using Nutrient broth, Sabouraud and Eosin methylene blue agar media. The results
have shown that traditional banana wine made from Musanze and Ngoma contains many
microbes from different species among which possibly E coli, Streptococcus, Staphylococcus,
Lactobacillus, Enterobacter, Colostridium and Bacillus have been identified. The results showed
that the traditional banana wine lack hygienic condition which was worse in traditional banana
wine prepared by feet in Ngoma. The traditional banana wine made in Ngoma contained more
fecal coliforms 330/ml, total germs 302.3x107/ml, yeasts and molds 784x106/ml compared to
traditional banana wine made from Musanze containing 50/ml fecal coliforms 325/ml yeasts and
molds and 523x106/ml total germs. The total coliforms were the only category of
microorganisms to be more numerous in musanze traditional banana wine 117x104/ml compared
to 140x103/ml of Ngoma. Also, the smelling, color, pH and alcoholic degree were different
between traditional banana wine made from Ngoma and Musanze, the one of Musanze had high
pH and alcoholic degree compared to traditional banana wine made from Ngoma. Those made
from Ngoma had a shorter shelf life and bad smelling. Both traditional banana wines did not
respect the norms fixed by RBS, so according to that reason the producers of traditional banana
wine must improve the hygienic conditions and respect the norms of RBS.
vi
CHAPTER I: INTRODUCTION
Background of the study
In Rwanda, as elsewhere in the world there is production of various alcoholic beverages prepared
by local population which can be authorized or not by Rwanda bureau of standards. The
extraction of that juice from banana and fermentation to produce banana wine using traditional
methods is an important post-harvest activity in the family banana farming in eastern Africa. The
production and sale traditional banana wine has greater importance to local population because it
generates incomes to the family household [A2]. The most popular drinks are banana wine and
liquors. These drinks are different from each other by the raw material used in their production
and the method of manufacture [A16].
Traditional banana wine is a beverage obtained by fermenting banana juice using or not sorghum
flour as yeast source. These beverages produced are not controlled and hygiene might not be
sufficient depending on mode of production. That can change the properties of banana wine
because of presence of unwanted microorganisms. Those microorganisms appear using
unsterilized material, improper water and producers lacking hygiene in the production of that
traditional banana wine.
Hygiene and sanitation play a role for the producers as it can affect the flavor and the banana
wine shelf life which might result in fluctuation of income but also the consumers of banana
wine can become a target for infection diseases transmission. In Rwanda, banana wine is made in
different ways varying from one region to another. This leads to production of different kinds of
wine in terms of quality.
1
1. Statement of the problem
In Rwanda, banana wines produced by the local population in east and north are different. The
climate in these zones is not the same; the east being the hottest in the country and the north, the
coolest and the manufacturing process is different. Indeed, in eastern provinces, like Ngoma, the
local people use their feet during banana wine production while the ones in the north like
Musanze use their hands. It is known that the climate can have a great influence on food
preservation, as hotter climates food deteriorates in short time [B3]. Also knowing that microbial
flora of hands and feet is different, [B2,5] when no pasteurization process is done the microbial
flora of the people producing traditional banana wine can have an influence on the quality and
security of consumers health.
2. Objectives
2.1. General objective
To compare the microbiological quality of traditional banana wine made in cool and hot
conditions, using hands and feet
2.2. Specific objectives
 To compare the microbial content (total coliforms, fecal coliforms, total germs, yeasts
and molds) of those two different traditional banana wines made from Ngoma and
Musanze
 To view if the influence of climate must change the microbiological quality of different
traditional banana wine
 To characterize the influence of microorganisms in traditional banana wine prepared
using hands and feet
 To compare physicochemical properties (pH and alcoholic degree) of traditional banana
wines prepared using hands and feet
 To compare shelf life of traditional banana wines prepared using hands and feet.
2
3. Research questions
 In those different traditional banana wines made from east and north, which one has
greater microbiological quality of standard?
 What is the best method of production according to taste and shelf life of traditional
banana wine using hands or feet?
 Why in the north producers use the hands which differ from the east where they use the
feet to produce the traditional banana wine?
4. Hypothesis
 The microbiological quality of traditional banana wine from east and north will not be the
same because of different variables like climate which can cause the one of Ngoma to
have greater amount of microorganisms compared to the one of Musanze.
 The number and variety of the microorganisms in these two beverages must not be the
same because of different hygienic conditions and method of production.
 The pathogens which resist to high alcoholic degree and low pH can be present in
traditional banana wine made from east and north such as Staphylococcus
 The shelf life and taste of traditional banana wine made from Ngoma is shorter compared
to Musanze traditional banana wine.
3
CHAPTERII: LITTERATURE REVIEW
II.1.Generalities on banana and traditional banana wine
Banana is the common name for herbaceous plants of the genus Musa and for the fruit they
produce. It is one of the oldest cultivated plants. They are native to tropical south and Southeast
Asia, and are likely to have been first domesticated in Papua New Guinea. Today, they are
cultivated throughout the tropics and a few subtropical countries. They are grown in at least 107
countries, primarily for fruit, and to lesser extent to make fiber, banana wine and ornamental. Its
fruits, rich in starch, grow in clusters hanging from the top of the plant. They come in variety of
sizes and colors when ripe, including yellow, purple and red. All most all modern edible
parthenocarpic bananas come from two wild species Musa acuminata and Musa balbisiana
[B10].
In Rwanda, banana beverages are still among the fruit beverages processed by using traditional
methods such as the use of feet and spear grass to extract juice because banana juice and wine
remained on a homemade basis [A2]. However, some research groups such as (Byarugaba et al,
2008) are driving a research to come up with modern technologies to more effectively process
bananas and to make acceptable banana juice and wine which have the standard conditions [A2].
Banana is one of the major commodities in agricultural sub-sector in Rwanda used as cash and
food crop. The consumption of bananas in Rwanda is one of the highest in the Great Lakes
region. According to ISAR, 2007 banana is the top crop. Exclusively small-scale farmers grow it
and it occupies 35% of the country cultivated land. The perennial nature of banana crop is
important for food security and income generation in the peasant farming communities [A11].
II.2.Classification of banana
Banana is classified in the Kingdom of Plantae, Branch of Angiosperms, Class of
Monocotyledonous Order of Zingiberales, Family of Musaceae, Genus of Musa, Species of
M.acuminata [A2].
4
II.3. Different types of banana cultivated in Rwanda
II.3.1. Cooking bananas
According to the ministry of agriculture 2002 cooking banana has been experiencing market
growth in recent year’s especially in urban areas and occupies 30% of all bananas production in
Rwanda. This growth which was expected to continue increasing total market demand is
estimated at about 70000 tons per year. It reached its goal, as it occupied 35% of cooking
bananas in 2007 [B10]. The rural areas traded volume was estimated at 40000Million Tons (MT)
per year having increased from about 17000 Million Tones. However, Rwanda is still a major
importer of cooking bananas.
II.3.2. Dessert bananas and plantains
Production of dessert banana is small accounting for about 10% of the entire banana production.
However, this market is growing especially in urban areas and also it is of a much high value
than that of cooking bananas. Dessert banana mainly consist of apple banana (kamaramasenge)
and there is importation of both apple and Gros Michel. Apple bananas are mainly imported from
Uganda but also they are used for brewing industry while Gros Michel is imported from
Democratic Republic of Congo.Table1 shows 4 species of dessert bananas which are found in
Rwanda [B10].
II.3.3. Brewing bananas
Brewing bananas are the one used to brew banana wine and dominate 60% of country banana
production. As it is shown in table1, there are 6 species of brewing banana found in Rwanda
[A4].
5
Table 1: bananas cultivated in Rwanda
Dessert bananas
Cooking bananas
Brewing bananas
Kamaramasenge
Mbwaziruma
Intuntu
Gros Michel
Inganju
Intokatoki
Poyo
Ingumba
Igihuna
Ikinyangurube
Injongo
Indaya
Bakurura
Ingoromoka
Barabeshya
Kayinja
Incakara
Injagi
Inzirabahima
Ingenge
Intariho
II.4. Role of banana in Rwanda
Banana has greater importance to the society. It combats the hunger and leaves Rwandese
satisfied and feeling full. Banana is used for brewing the banana wine which is useful in the
ceremonies like weddings and baptism. Banana has an economic role and it participates in
poverty reduction.
Banana has a medical role, as it has the natural minerals, like potassium that plays a very
important role in keeping blood pressure at healthy level [B13].
6
II.5 Preparation of traditional banana wine or processing
II.5.1. Ripening or maturation of banana
To prepare banana wine, the chosen bananas are the ones which had been harvested when ripe, if
the ones taken are green. Various methods are used for ripening bananas, either by burial in pits
in the banana grove or by heating over the kitchen fire. Ripening of banana is induced initially by
continuous smoking over for three days. Fruit are covered with banana leaves to avoid
desiccation during smoking. This allow the ripening process underground where smoking raises
the temperature, and the banana fruits are covered with banana leaves and soil. Ripening of
banana continues on the third day onwards. And then soil is removed smoke is stopped but
leaves on the bananas remain and ripening continue. Bananas, viewed on the sixth day of
processing, are a yellow color. On the seventh day, the extraction of juice and its fermentation
into traditional banana wine starts.
II.5.2. Juice extraction
To obtain bananas juices, bananas are transferred to a large wooden mortar called (umuvure)
outdoors which is filled with a mixture of peeled, ripened banana. Juice is extracted by trampling
the banana and spear-grass mixture. Ripened, peeled bananas are pressed underfoot or worked by
hand in large wooden mortars to extract juice. Selected grasses are used to assist juice extraction.
The preferred grasses may differ from one region to another. Ishinge or fresh banana leaves are
most commonly used in juice extraction in those two different areas.
Banana juice is subsequently cleared by pouring through a filter built in small tree which tie
together, newly felled mature banana pseudo stems. Water is poured over the banana-pulp/grass
residue and the liquid obtained added to the pure juice.
7
II.5.3. Fermentation of banana wine
Roasted red sorghum, which is coarsely crushed and not ground, is added to the filtered juice.
Fermentation of the juice/sorghum mixture occurs in the wooden mortar, which is covered with
banana leaves during fermentation. After three days of fermentation, traditional banana wine is
filtered using funnel and poured into storage common in Rwanda such us plastic jerry-cans and it
is now drinkable. Maturation of banana, juice extraction and fermentation required 9-10 days
[A2].
II.6. Different types of traditional banana wines
In Rwanda there are 3 types of traditional banana wines which are:
Inkangaza: its banana wine that results from juice added with honey which is fermented
Butunda or indakumirwa: it’s a banana wine which is rich in alcohol and nutrient,
obtained from not diluted juice fermentation
Urwagwa: this wine is poor in nutrient and has a poor alcoholic degree obtained from
fermentation of banana diluted juice [A12].
II.7. Challenges to traditional banana wine production
There are many challenges encountered by the local people in producing traditional banana wine
such as soliciting packaging, packaging materials (crates and bottles).And in maintaining the
standard quality. All processors need to adhere to hygiene standards. However, most of locally
products have no quality control mechanism. This is a setback in marketing of products out of
the production areas [A10].
8
II.8. FERMENTATION
II.8.1. Definition
Fermentation is the metabolic process in which carbohydrates and related compounds are
partially oxidized with the release of energy in the absence of any external electron acceptors. It
can be described as a biochemical change, which is brought about by the anaerobic or partially
anaerobic oxidation of carbohydrates by either micro-organisms or their enzymes [A6].
II.8.2 Types of fermentations
II.8.2.1. Alcoholic fermentation
Alcoholic fermentation is the biological process in which sugars such as glucose, fructose and
sucrose are converted into cellular energy and produce ethanol and carbon dioxide as metabolic
waste products. This process is passed in absence of oxygen and performed by yeast such as
Saccharomyces cerevisiae that converts carbohydrates to carbon dioxide and alcohols. To the
point view of yeast, CO2 and alcohol are wastes and yeast continues to grow and metabolize the
sugar solution until the concentration of alcohol will became toxic.When it reaches a
concentration between 14-16% thereby killing the yeast cells. This is the reason why the
percentage of alcohol in wine and beer can only be approximately 16%. In order to produce
beverages with high concentration of alcohol the fermented product must be distilled [B8].
Yeast
C6H12O6
Sugar
2C2H5OH + 2CO2 + Energy (which is stored in ATP)
====>
Alcohol
+ Carbon dioxide gas + Energy
II.8.2.2. Lactic fermentation
Lactic fermentation is a biological process by which sugars such as glucose, fructose, and
sucrose, are converted into cellular energy and the metabolic by product lactate, by intervention
of bacteria such as Lactobacillus and Streptococcus and Bacillus, algae such as (Chlorella),
some water molds, protozoa, and animal cells, such as skeletal muscle cells, in the absence of
oxygen. The process of lactic acid fermentation using glucose is summarized below. In
homolactic fermentation, one molecule of glucose is converted to two molecules of lactic acid:
9
C6H12O6 → 2 CH3CHOHCOOH
In heterolactic fermentation, the reaction proceeds as follows, with one molecule of glucose
converted to one molecule of lactic acid, one molecule of ethanol, and one molecule of carbon
dioxide [B11]:C6H12O6 → CH3CHOHCOOH + C2H5OH + CO2
II.8.2.3 Malolactic fermentation
Malolactic fermentation takes place by intervention of lactic acid bacteria, such as Oenococcus
oeni, Lactobacillus and Pediococcus, which consume malic acid to liberate energy. This can
occur naturally. However, in commercial winemaking, malolactic conversion typically is
initiated by an inoculation of desirable bacteria. This prevents undesirable bacterial strains from
producing off-flavors bacteria. This reaction liberates oxygen [B1].
COOH-CHOH-CH2-COOH → COOHCHOH-CH 3+ CO2
II.8.2.4 Mixed acid fermentation
Mixed acid fermentation is a type of formic acid fermentation which resulted in the excretion of
ethanol and complex mixture of acids particularly acetic, formic, lactic and succinic acid. The
formic acid is degraded to hydrogen (H2) and carbon dioxide (CO2). Microorganisms involved in
this reaction are enteric bacteria belonging to Enterobacteriacae such as Escherichia,
Salmonella, and Proteus [A6].
II.8.2.5. Other fermentations
There are other fermentations such as methanol fermentation and acetone fermentation. The
various fermentation processes are all anaerobic. The CO2 produced by the process of
fermentation makes these beverages effervescent [A9].
II.8. 3 factors influencing alcoholic fermentation
II.8.3.1. Biological factors
Banana juice is submitted to different fermentations through the action of microorganisms. Some
of them are already present in fruits. The yeasts are used for alcoholic fermentation and bacteria
for malolactic fermentation, then those microorganisms use certain elements contained in juice in
order to live and grow transforming them into other elements [A5].
10
II.8.3.2. Temperature
Temperature affects fermentation, especially affecting yeast growth. The temperature range in
which fermentation is favored has been given in different ranges by different researchers. The
optimum fermentation temperature by most wine yeast is between 22oC and 27oC. For Marigalit
(1997) there is minimum fermentation temperature range where yeast does not work, and advises
are to not exceed 330C because fermentation may stop. These temperature ranges seem to not
apply to all fermentation depending on different wine style and conditions. The optimum
temperature for yeast growth has been reported to be close to 300C (Reed and Nagodawithana,
1993). According to vines.org the fermentation of banana wine depends on initial temperature of
the juice or sugar concentration [A9, 13].
II.8.3.3. Acidity and pH range
Acidity and pH must be maintained at different level depending on the wine wanted by producer.
Yeast’s cytoplasm is normally maintained at a pH between 5.5 and 5.75. Yeast prefers to live in
a mildly acid environment and yeast growth produces a range of organic acids which lowers the
pH of its environment. Acidification plays a role in limiting the growth of undesirable spoilage
microorganisms [A1].
II.8.3.4 .Carbon dioxide
According to Amerine, (1998) the greater quantity of CO2 during fermentation can stop yeast
growth. The carbon dioxide effect on yeast growth does not prevent alcoholic fermentation but
when CO2 become more sufficient, it cause stop of fermentation. The inhibiting effect of CO2 is
used to control fermentation rates.
II.8.3.5. Minerals
The normal process of fermentation requires minerals such us potassium, magnesium, zinc,
cobalt, iodine iron, zinc, calcium and anions of phosphorus, for growth of yeast require copper,
iron, magnesium, phosphorus and sulfur. Adequate amount of that minerals are supplied by
grape or banana apart from phosphate [A1].
11
II.8.4. Microorganisms using in fermentation
II.8.4.1. Different yeasts and molds found in traditional alcoholic beverages
II.8.4.1.1.Yeasts
Yeast is unicellular fungus that has a single nucleus and reproduces either asexually by budding
and transverse division or sexually through spore formation. Yeasts are dispersed naturally on
the plant leaves, and flowers, soil and salt water and yeasts are also found on the skin surface and
in the intestinal tracks of warm-blooded animals where they may live symbiotically as parasites
[A6].
The most known is Saccharomyces cerevisiae which plays greater role in brewing banana wine,
Most beers are fermented with bottom yeasts, related to Saccharomyces pastorianus, which settle
at the bottom of the fermentation container. The beer flavor also is influenced by the production
of small amounts of glycerol and acetic acid. Bottom yeasts produce beer with a pH of 4.1 to 4.2
and requiring 7 to 12 days of fermentation [A8].
In spoilage, yeasts found often belonging to Candida valida which is the most important yeasts
growth of these organisms occurs on the surface of wine, where a thin of film is formed. The
organism attacks alcohol and other constituent from this layer and create an appearance that
sometimes referred to as wine flower. This type of spoilage is characterized by increased volatile
acidity and later it caused spoilage of odor and taste. There are some species of yeasts that can
cause infection such as candidiasis this infection is caused by Candida albicans [A.6].
II.8.4.1.2. Molds
Molds are fungi that grow in the form of multicellular filaments called hyphae. Some species of
molds were isolated in sorghum beer made in Rwanda called Aspergillus Niger, Fusarium sp.
and Aspergillus sp [A7]
12
II.8.5. Source of pathogenic microorganisms in wine
II.8.5.1. Personal hygiene or handle
In preparation of traditional banana wine the hands, feet and clothing may be the source of
pathogenic organisms, because hands and feet of traditional wine producers are rarely washed
before squeezing and filtration which can lead to contamination of traditional banana wine with
many microorganisms like Staphylococcus.
II.8.5.2. Material used in fabrication
In traditional banana wine making the materials are not well washed and sometimes they use the
river water also the materials used are not sterilized also the container in which the traditional
banana wine is stored, transported, processed or merchandised.
II.8.5.3. The environment
Many microorganisms which are pathogenic which may affect the traditional banana wine it may
found in room preparation. The most types of wines poisoning bacteria may be spread by cross
contamination in the rooms by pests. The most are toilets and unwashed materials near the
rooms’ preparation.
II.8.6. Microorganisms that may affect traditional banana wine and producers
II.8.6.1.Total coliforms
Total coliforms are group of microorganisms which meet the different criteria like gram
negative, nonspore forming, ferment the lactose with gas and acid production in 24 to 48 hrs at
37oC. Coliform bacteria live in soil and water. Those organisms may be the sources of those
appear in water, especially in surface of water. Those coliforms can changes or modify the
quality of traditional banana wine because they produce a lot of gas. According to Lyamugabe et
al total coliforms found in sorghum beer is (32.30x106/ml) [B6, A7].
II.8.6.2. Fecal coliforms
Fecal coliforms are bacteria found in feces of human or animal. These organisms are a subset of
total coliform group. In addition to the criteria defining the coliforms, they are thermotolerent
coliforms are able to grow and ferment lactose with the production of acid and gas at 44oC. The
presence of those microorganisms in water or other beverages like banana wine has been shown
to have a general excellent in correlation with fecal contamination although some members of
13
this group (e.g Klebsiella) can be isolated from environmental samples where no fecal pollution
can be found [A14]. Among them, some are also pathogenic infectious agent. The most known
are Escherichia coli, Shigella, Salmonella.
II.8.6.2.1. E coli
It is one of the most prolific microorganisms in human intestinal tracks. It is so common and so
easily cultivated. E coli produce toxin that can cause gastrointestinal disturbances, collectively
termed E.coli gastroenteritis. E.coli has been found in traditional sorghum beer made in southern
Rwanda with account of 21.90x106/ml [A7].
II.8.6.2.2. Shigella
The causative agent of human shigellosis, Shigella causes disease in primates, but not in other
mammals. It is only naturally found in humans and apes. During infection, it typically
causes dysentery. Family of bacteria that can cause sudden and severe diarrhea (gastroenteritis)
in human shigellosis, the illness caused by the ingestion of Shigella bacteria, is also known as
bacillary dysentery. Ingestion of fewer than 100 Shigellabacteria can cause food poisoning
[A14].
II.8.6.2.3.Salmonella
Facultative anaerobic that can cause disease such as Salmonella typhi, Salmonella typhimurium
and Salmonella enteritidis are transmitted to human via contaminated food, especially poultry
and dairy product water borne infectious is less frequent. Cause salmonellosis or salmonella
gastroenteritis characterized by fever, nausea, abdominal pain, diarrhea.
II.8.6.3. Staphylococcus aureus
Are bacteria that are mostly cause of infection to the skin of human body also and other
organisms that infection caused by invasion or toxin production from the minor skin results to
diseases such as pneumonia and meningitides. It has been found in traditional sorghum beer with
account of Staphylococcus aureus with account of 16.02x103/ml [A7].
14
CHAPTER III: METHODOLOGY
III.1. Sample collection
Three samples of traditional banana wine were collected in 3 different families from north
Musanze District, in different sectors such as (Rwaza, Muko, Muhoza) and 3 samples of
traditional banana wines were collected Ngoma District to be analyzed in laboratory of K.I.E for
total coliforms, fecal coliform, total germs, yeast and molds, pH and alcoholic degree. Those
samples were putted in separate clean bottles, brought to laboratory and kept in refrigerator less
than 24 hours before their analysis. For the microbial content analysis, the 3 samples collected
from Musanze were pulled together in order to form one mix of samples, and the 3 samples
collected from Ngoma were also pulled together in order to form one mix of samples.
III.2. Materials sterilization
All materials used for microbial contents analysis were washed with water, dried and sterilized in
autoclave at 1210C for 15 minutes apart for plastic Petri dishes and pipettes which were sterilized
using ethanol 70%. All techniques were done aseptically around the Bunsen burner. All of
working tables were cleaned and disinfected with ethanol 70% so as to prevent cross
contamination that could occur during operations.
III.3. Culture medium
A culture medium is solid preparations that have been used to grow and store microorganisms in
order to be used in identifying and isolating bacteria and fungi strains.
III.3.1. Different culture medium used in experiment
Eosin methylene blue agar (EMB) was used for isolation and differentiation of total and fecal
coliforms.
Nutrient Broth (NB) was used for isolation and differentiation of total germs.
Sabouraud dextrose agar (SAB) was used for isolation and differentiation of fungi, yeast and
lactic acid bacteria.
15
III.3.1.1.Culture medium preparation
III.3.1.1.1. NB preparation
3.9 grams was weight using the electronic balance and was added with 3grs of agar and
suspended in 300ml of distilled water, heated until completely dissolved and autoclaved at 1210C
for 15 minutes and poured carefully into Petri dishes.
III.3.1.1.2. SAB preparation
19.5 grams were weight using electronic balance of the medium and suspended in 300ml of
distilled water, heated until completely dissolved and autoclaved at 1210C for 15 minutes, and
poured into Petri dishes.
III.3.1.1.3. EMB preparation
7.2 grams were weight using electronic balance of the medium and suspended in 200ml of
distilled water, heated until completely dissolved and autoclaved at 1210C for 15minutes and
poured into Petri dishes.
III.4. Serial dilution
Dilutions were very important in order to get a number of colonies that could be countable.
Seven test tubes were filled with 9ml of sterilized traditional banana wine. By using a
micropipette, 1ml of the original sample was taken and put in 9ml (dilution10-1). After mixing,
1ml was taken from that tube and then put in the next tube (dilution10-2). The same process was
repeated up to the tube number seven.
Plate1 Serial dilution of traditional banana wine in K.I.E labolatory
16
III.5. Inoculation
The inoculation has been done by putting 100µl of the diluted suspension samples into Petri
dishes by using micropipette. Then the samples were spreaded on the agar media by using a
spreader.
III.6. Incubation
The Petri dishes were putted into incubator for 24 hours at 370C and 440C to enable total
coliforms and fecal coliforms to grow on EMB. For Petri dishes of NB and SAB, they were
putted for 72 hours at 28-300C to enable yeasts and fungi to grow.
III.7. Description of colonies
III.7.1. Description
In order to differentiate the microorganisms, the description of colonies is needed. The colonies
are described using different characteristics which are:
Size: diameter in millimeter
Form: Punctiform, circular, filamentous, irregular, rhizoid
Elevation: flat, raised convex, pulvinate, umbonate, umbilicate
Margin: entire, undulate, lobate
Color: white, yellow, black, buff, orange, pink
Density: opaque, translucent, transparent
Consistency: viscid, membranous, brittle, butyrins.
17
III.8. Microscopic observations
A small portion was taken from each type of colony and was putted on slide containing a drop of
methylene blue and well mixed. The slide was covered by cover slip and microorganisms were
observed at magnification of x1000. A drop of immersion oil was added on the cover slip to
clarify the image. Characteristics taken into consideration in microscopic observation for bacteria
were: - the form (cocci, rods, and spirillum), arrangement (cluster, pairs, tetrade, and chain), size
(big, small), mobility, presence of spores (terminal, subterminal, central).
For mold the characteristics taken into consideration were: the presence of septa or not in hyphae
and the mode of asexual reproduction (fission, arthrospores, chlamydospores, sporangiospores,
conidiospores or blastospores).
For yeasts, the characteristics taken into consideration were: shape (spherical, oval, cylindrical),
size (big or small)
III.9. Physicochemical parameters
III.9.1. Alcoholic degree
The vinometer used to measure the alcoholic degree of the banana wine samples during six days,
was hold vertically with the funnel upwards. Each sample of traditional banana wine was poured
one by one into funnel without air bubble allowed in capillary traditional banana wine column. In
looking that the vinometer end of capillary the vinometer was turn upside –down. Then
percentage was read directly from the graduation where the upper side of traditional banana wine
stays.
Plate 2 Alcoholic degree measurement done in K.I.E laboratory
18
III.9.2. pH
The pH was measured during 6 days using pH meter. The electrodes were washed using distilled
water and were putted into each sample of traditional banana wines. Then, the pH was readable
on pH scale.
Plate3 pH measurement done in K.I.E laboratory
19
CHAPTER IV: RESULTS AND DISCUSSION
IV.1. RESULTS
Descriptive and qualitative analysis of microorganisms grown in SAB, NB, EMB
Table 2: Descriptive and qualitative analysis of total coliforms grown on EMB at 370C
Types of
colonies
Colonies description
Microscopic
observation
Average of
microorgan
isms/ml
Musanze/ml
1
Circular,
umbonate,
entire,
purple,
glistening, opaque
Paired rods, short and
large,
non
motile
Probably Enterobacter
3x104
2x104
2
Circular,
pulvinate,
entire, green metallic,
dull, opaque
Single rods, some are
long other are short and
thin,
non
motile
Probably E coli
7.5x104
15x104
3
Spindle, convex, entire,
black in the center and
rose at the side, dull,
opaque
Single rods, short and
thin, motile. Probably
Klebsiella
0.5x105
1x105
_
4
Circular, convex, entire,
black,
glistening,
opaque
Paired rods, short and
thin , non motile
Probably Enterobacter
4.5x105
9x105
_
5
Circular,
umbonate,
entire,
purple,
glistening, , opaque
paired, Rods, short and
thin,
non
motile
Probably Enterobacter
0.5x105
_
1x105
Circular,
pulvinate,
entire, pink, glistening,
opaque
Circular,
pulvinate,
2
entire,
pink,
dull,
opaque
Circular,
pulvinate,
3
entire,
rose,
dull,
opaque
Number of microorganisms/ml
4x104
_
6.55x107
1.17x106
Yeast, oval, small
3x106
6x106
Yeast, oval, big
7.253x107
136x106
906.104
Yeast, spherical, big
53x106
106x106
_
1.28.53x108
2.48x108
9.06x106
Number of microorganisms/ml
1
Ngoma/ml
20
1.4x107
_
1
2
3
4
5
Total coliforms
1
2
Number of microorganisms per ml
Number of microorganisms per ml
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
3
yeasts
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
Total coliforms
Musanze tbw
Musanze tbw
Ngoma tbw
Figure1 Varieties and number of total coliforms
Yeasts
Ngoma tbw
Figure2Total coliforms in tbw Musanze vs Ngoma
On the above table 2 and figures 1 and 2, the total coliforms are respectively not the same in
traditional banana wine made in Musanze and Ngoma, Musanze has higher variety and number
of microorganisms. Among them, Enterobacter, E. coli and Klebsiella sp have been identified.
Total coliforms number was 1.17x106/ml compared to Ngoma which has total coliforms
number 0.14x106/ml. In the traditional banana wine brewed in both district, there are other
species probably called Candida sp, which were also higher in Musanze 2.48x108 /ml compared
to Ngoma banana wine which is 9.06x106/ml.
21
Table 3: Descriptive and qualitative analysis of fecal coliforms grown on EMB at 440C
Types of
colonies
Colonies description
Microscopic observation
1
Circular, umbilicate,
entire, purple, dull,
opaque
Irregular umbilicate,
lobate,
purple,
glistening, opaque
Irregular, umbonate,
undulate,
red,
glistening, opaque
Circular, flat, lobate,
red, dull, opaque
Circular, entire, dark in
center,
greenish
metallic, opaque
Paired rods, short, no
motile
Probably
Klebsiella
Single rods, short chain,
motile and thin Probably
Klebsiella
Single rods, short and
thin, motile
2
3
4
5
Single rods, chain, non
motile
Rods, motile probably E
coli
Circular,
pulvinate,
entire,
pink,
dull,
opaque
Yeast, oval
10
20
_
70
_
140
90
_
180
5
_
10
30
Ngoma/ml
_
50
7
330
_
70
1000
Number of microorganisms per
ml
1000
Number of microorganisms per
ml
Musanze/ml
190
Number of microorganisms/ml
1
Average
of
microorganisms
/ml
15
100
10
1
1
2
3
4
Fecal coliforms
Musanze tbw
5
1
Yeasts
Ngoma tbw
100
10
1
Fecal coliforms
Musanze tbw
Figure3 Varieties and number of fecal coliforms
Yeasts
Ngoma tbw
Figure4 Fecal coliforms in tbw Musanze vs Ngoma
On the above table 3, figures 3 and 4, the fecal coliforms are totally different in traditional
banana wine brewed in Ngoma and Musanze and Ngoma has higher amounts of microorganisms
compared to Musanze with 330 fecal coliforms/ml and 50 fecal coliforms/ml respectively.
22
Among them Klebsiella and E coli have probably been identified. Again, in banana wine made in
Musanze there is other specie which is probably Candida sp with70 yeasts/ml.
Table 4: Descriptive and qualitative analysis total Germs grown on NB medium
Types
of
colonies
1
Colonies description
Microscopic observation
Circular,
pulvinate,
entire, yellow, dull,
opaque
Cocci, pair, motile and
non
motile
Probably
Streptococcus
2
Circular, convex, entire,
yellow,
glistening,
opaque
Irregular, flat, lobate,
white, dull, opaque
3
4
5
6
7
8
9
10
11
12
Irregular, raised, erose,
buff, dull, opaque
Circular, convex, entire,
buff, glistening, opaque
Circular, convex, entire,
white,
glistening,
transparent
Circular,
pulvinate,
entire,
orange,
glistening, opaque
Circular,
pulvinate,
entire,
yellow,
glistening, opaque
Irregular,
umbonate,
lobate, white, dull,
opaque
Circular,
pulvinate,
entire,
yellow,
glistening, opaque
Irregular, flat, undulate,
white, dull, opaque
Circular,
pulvinate,
entire, white, dull,
opaque
13
Filamentous,
raised,
filamentous,
green,
dull, opaque
14
Filamentous,
raised,
filamentous,
white,
dull, opaque
Number of microorganisms/ml
Average
of
microorganis
ms/ml
109x106
Musanze/ml
Ngoma/ml
80x106
138x106
Cocci, clusters, tetrads
probably Staphylococcus
24.5x106
47x106
2x106
Single rods, motile with
spores
out
probably
Clostridium or Bacillus
Paired rods, short with
subterminal spores
Single rods, short with
terminal spores Probably
Bacillus
Single cocci, small non
motile,
15x106
3x106
27x106
11.5x107
23x107
_
7x107
14x107
_
135x104
_
270x104
Paired cocci, non motile
Probably Streptococcus
2x104
_
4x104
Cocci cluster, small non
motile
Probably
Staphylococcus
Paired rods, short and large
non motile
4.5x104
_
9x104
2x104
_
4x104
Cocci, cluster, non motile
probably Staphylococcus
Aureus
Single rods, short, non
motile
Probably
Lactobacillus
Yeast, spherical, Probably
Saccharomyces
120x107
Molds, hypha with septate,
clamydospores
Mold, hypha with septate,
conidiospores
Probably
Aspergullus
23
240x107
_
22.5x107
_
45x107
1.5x104
_
3x104
11.5x106
2x107
3x106
1.505x106
3.106
1x104
1.8x106
5.2x108
3.02x109
Number of microrganisms per ml
Number of microorganisms per ml
1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Total germs
Musanze tbw
1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
Total germs
Musanze tbw
Ngoma tbw
Ngoma tbw
Figure5 Varieties and number of total germs
Figure6 Total germs in tbw Musanze vs Ngoma
On the above table 4 and figures 5 and 6, the number of microorganisms is higher compared to other
medium especially in banana wine made in Ngoma which has 3.02x109total germs/ml and
Musanze has 5.2x108total germs/ml. Among the 14 different species identified, 5 belong to both
traditional banana wines. Lactobacillus, Staphyloccoccus, Colosridium, Bacillus, Streptococcus have
probably been identified.
24
Table 5: Descriptive and qualitative analysis total Yeasts and molds grown on SAB medium
Types of
colonies
Colonies description
Microscopic observation
1
Circular,
pulvinate,
entire, white, dull,
opaque
Filamentous,
pulvinate, filamentous,
white, dull, opaque
Yeasts, spherical, big
Probably Saccharomyces
2
3
Filamentous,
pulvinate, filamentous,
green, dull, opaque
Filamentous,
pulvinate, filamentous,
white, dull, opaque
Filamentous, convex,
filamentous, black in
the
center,
dull,
opaque
Circular,
pulvinate,
entire,
red,
dull,
opaque
Circular,
convex,
entire,
buff,
dull,
opaque
4
5
6
7
average
of
microorganism/
ml
366.5x106
Musanze/m
l
Ngoma/ml
280x106
453x106
Molds, septated hyphae,
chlamydospores
and
mixed with macroconidia
Probably Aspergillus
Mold
with
hypha
septated, macro conidia
1.5x106
2x106
1x106
1.5x106
3x106
_
Molds, septated hyphae,
long and thin, blastopores
10x106
20x106
_
Molds,
hypha
septate,
chlamydospores
with
long,
10x106
20x106
_
Probably
cylindrical, small
yeast,
1.5x107
_
3x107
The smallest are yeast,
oval, small
15x107
_
30x107
5.5x108
3.2x108
7.8x108
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
1.00E+09
1
2
3
4
5
6
Number of microorganisms per ml
Number of microorganisms per ml
Number of microorganisms/ml
7
Yeasts and Molds
Musanze tbw
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
Yeasts and molds
Musanze tbw
Ngoma tbw
Figure7 Number of varieties of yeasts and molds
Ngoma tbw
Figure8 Yeasts and Molds in tbw Musanze vs Ngoma
25
On the above table 5, figures 7 and 8, the number of microorganisms is higher in traditional
banana wine brewed in Ngoma 784x106yeast and molds/ml, while number of Musanze
traditional banana wine is higher, 325x106yeast and molds/ml. 2 types were found to belong to
both banana wines. Sacchoromyces and Aspergillus have probably been identified.
Table 6 pH and alcoholic degree from traditional banana wine of Musanze and Ngoma
samples
Day1for pH
Day 1 for Day3 for Day3 for Day6
alcoholic
pH
alcoholic
degree
Musanze
for Day6
pH
alcoholic
degree
9.03
4.68
degree
Average
4.82
color
Grey
Grey
Grey
smelling
Odor tolerable
Odor tolerable
Oder non tolerable

Release
10.5
4.70

9.1
_
of gases
Ngoma
Average
4.48
7.63
color
Clear kaki
smelling
Odor tolerable
4.1
8.3
7.66
Kaki
Bad smelling

Release
4.23
kaki
Bad smelling
_
_
of gases
- no gas
6
12
5
10
4
8
3
Musanze
tbw
2
Ngoma
tbw
1
Alcohol
pH
 presence of gas,
6
Musanze
tbw
4
Ngoma tbw
2
0
0
1
3
6
1
Days
Figure9 pH in tbw made from Musanze vs Ngoma
3
6
Days
Figure10 Alcohol in tbw made from Musanze vs Ngoma
26
for
On the above table 6 the average pH of traditional banana wine brewed in Musanze at day1
(4.84) is higher compared to pH of Ngoma (4.48) and alcoholic degree in Musanze traditional
banana wine (9.03) is higher than alcoholic degree of Ngoma (7.63). From day1 to day 3, the pH
in both traditional banana wine decrease to increase again at day 6. From day 1 to day 3 the
alcohol in both traditional banana wines increase then at day 6 decrease. Also the gas in Musanze
traditional banana wine was tolerable compared to Ngoma which was bad smelling.
Number of microorganisms between traditional
banana wine made from Musanze and Ngoma
1.00E+10
1.00E+08
1.00E+06
1.00E+04
Musanze tbw
1.00E+02
Ngoma tbw
1.00E+00
Total
coliforms
fecal
coliforms
Total germs Yeasts and
Molds
Figure11 Average of microorganisms in traditional banana wines made from Musanze vs Ngoma
27
IV.2. Discussion on microbial analysis
Both traditional banana wines made from Musanze and Ngoma contain varieties of
microorganisms such as total coliforms, fecal coliforms among which we have identified
probably Klebsiella, Escherichia coli and Enterobacter; Yeasts and Molds among them we have
identified probably Saccharomyces and Candida sp and total germs among which probably
Lactobacillus, Staphyloccoccus, Colosridium, Bacillus and Streptococcus have been identified.
These varieties of microorganisms found are similar with the ones identified in traditional
sorghum beer as it has been investigated by Lyumugabe et al. [A7].
The results presented in table 2 to 6 and figure 9 showed that in the traditional banana wine made
from Ngoma, the number and variety of microorganisms is higher compared to traditional
banana wine made from Musanze for all the microorganisms category found except for the total
coliforms. The cause of that high number and variety of microorganisms can be that the people
use their feet which are not well washed and river’s water or lake water, while in Musanze tap
water is preferably used.
As they lack the sense of hygiene that can have lead to high
contamination of traditional banana wine made from Ngoma.
Among the total germs, potential pathogens, general indicator of potential contamination have
been identified. Among them Staphylococcus, Streptococcus, Clostridium and Bacillus have
been identified in both banana wines but the number and variety was higher in the one from
Ngoma. Staphylococcus and Streptococcus are frequent commensals on nose, throat and on the
skin of healthy of people that may lead to the contamination of food stuff or drink in producing
enterotoxin which can poison the food. Clostridium and Bacillus are sporulating bacteria which
are found mostly in the soil, Clostridium being also found in the intestinal tract of humans [A8].
In food, they have also been found to be causal agent of food intoxication. Their presence
indicates that the producers were probably carrier of Staphylococcus and Streptococcus and
would have introduced these microorganisms in the banana wine due to unhygienic practices.
Also, these findings reveal that during the production of banana wine, the producer’s feet and
hands would have been in contact with soil containing Clostridium and Bacillus. Also, when the
banana are put in the pit for their ripening, the soil which is add to cover the banana might have
been contaminated with the bacteria which would have subsequently been introduced in the
banana wine.
28
Also, Lactobacillus probably identified have probably contributed to decrease pH as they
perform homolactic, heterolactic and malolactic fermentation producing lactic acid as well as
Clostridium sp which can perform propionic fermentation producing acid butyric. In these
fermentations, as different other products are also released, such as acetone and butanol, these
might have contributed to the banana wine aroma and to spoilage of wines [A8, B1].
Among the yeasts identified, it is known that Saccharomyces is used for alcoholic fermentation
to produce alcohol and might have been among the ones which have increased the alcohol
content of our banana wines. However, Candida sp which have also been identified might have
caused the spoilage observed after day 3 and as some are known pathogens, they might expose the
consumers to candidiasis.
Also number of coliforms indicated serious lack of hygiene during the preparation of traditional
banana wine either using feet or hands and these results are higher to the ones found in
traditional sorghum beer [A7] but similar to result found by (Twagirimana, 2010).
Those
coliforms might come from the endogenous microflora found into raw material used such as
(banana, sorghum, and banana leaves), in the water, the soil for incubation and material used.
The fecal coliforms found show that the environment, the water used in preparation, or hands
and feet of people making banana wine were contaminated by feces. Also, they might have
contributed to degradation and bad smelling noticed in traditional banana wines as they might
have produced different acids such as formic, succinic, acetic and lactic responsible for odors
and taste [A6].
The samples of traditional banana wine present different colors from clear kaki to grey and
smelling were from tolerable to bad odor as the days after the banana wine production were
increasing. Also, the banana wine of Musanze produced a lot of gas during six days compared to
Ngoma. The pH and alcohol degree results in traditional banana wine made from Musanze were
also surprising. It was expected that in Ngoma, as the number of microorganisms is high due on
one hand to the hot climate, and on the other hand to the material used, that the alcohol content
would have been higher and the pH lower. However, in Musanze, during the banana wine
preparation, the quantity of sorghum flour add is higher than in Ngoma, adding more nutrients.
This might have allowed the growth of microorganisms, among, them the yeasts, for a longer
period of time producing more alcohol as seen 3 days after the collection. Also, we can presume
29
that the yeasts found in Musanze were maybe higher alcohol performers and the big number of
other microorganisms found in Ngoma might have decrease the pH, cause spoilage, with less
alcohol production. Also the traditional banana made in Rwanda has high pH and alcohol
compared to traditional banana wine made in Tanzania [A15].
All those banana wine have microbiological pathogens that can cause diseases and the
requirement for traditional banana wine have not been set by RBS, so the producers must know
the standards of traditional banana wine in order to avoid those diseases.
30
CHAPTER V: CONCLUSION AND RECOMANDATION
V.1. CONCLUSION
Microbial Pathogencity of traditional banana wine has greater effect on human consumption
resulted from lack of hygiene, skills and equipment.
The results we have seen show health risk associated to traditional banana wine due its indicator
was much below the limit set by RBS. The result showed that there are many sources of
contamination of traditional banana wine they include air, water, soil, humans.
According to all those traditional banana wines all processing in production are not controlled
that leads to the contamination of microorganisms and cause some infectious diseases.
The consumption of the traditional banana wine must predicted to the local producer to obey and
follow microbial tolerance limits defined by RBS related to banana wine, number of microbes
can be minimized through proper handling through all process
V.2. RECOMMENDATIONS
The recommendation that can be given to the producers, consumers and other people in common
of traditional banana wine for better improvement of mode production because it constitutes the
source of revenue and it is consumable.
 Improvement of hygiene in process of fermentation in order to avoid unwanted
microorganisms such as bacteria
 Methods of using feet in production of traditional banana wine must be well improved in
district of NGOMA in order to get the good quality of banana wine
 Quality control measures should be explained to all traditional producers
 There must use pasteurization in order to kill the unwanted microorganisms and sterilize
the materials their use Local producer should be aware of standards set by RBS
31
REFFERENCES
A.BOOKS
1. Amerine M.A., Kunkee R., Ough K.C.S., Singleton V.L. and Webb, A.D. (1980). The
technology of wine making. (4th Edition). A.V.I, Westport, Connecticut.pp.183-703
2. Byarugaba B., and George W. ( 2008), The effect of enzymatic processing on banana
juice and wine, Stellenbosch university
3. Davies G.E. (1993). Domestic banana-beer production in Mpigi district, Uganda.
Infomusa 2(1): 12-1.
4. Gaidashova S.V., Okech S.H.O., Gold C.S. and Nyagahungu I. (2005). Why beer
bananas? The case for Rwanda. Infomusa 14(1): 2-6.
5. Habarugira I. (1998). Contribution a l´etude microbiologique du vin de banane
traditionnel au Rwanda. Universite nationale du Rwanda, Butare.
6. Prescott L.M., Harley J.P. and Klein D.A. (2005) Microbiology, (6th edition), Mc Grawhill, New York.
7. Lyamugabe F., Kamaliza G., Bajyana E. and Thonart P.H. (2010) Microbiological and
physico-chemical characteristic of Rwandese traditional beer “Ikigage”, African journal
of biotechnology vol. (27):4241-4246
8. Micheal T.M., John M.M., Paul V.D. and David P.C. (2005). Brock Biology of
Microorganisms (12th Edition). Prentice Hall, San fransisco.
9. Margalit Y. (1997) .Concept in wine chemistry. The wine appreciation guild ltd
10. Mgenzi S.R.B., Mshaghuley I.M., Staver C., and Nkuba J.M., (2009) Mussa processing
businesses and their support environment in Tanzania: it’s role in poverty alleviation to
rural community.Tanzania
11. Mpyisi E., Nyarwaya J.B., and Shyiringiro E. (2000). Statistiques agricoles: production
agricole, élevage, superficies et utilisation des terres. Année agricole 2000. MINAGRIFSRP-USAID. 43pp
12. Nikuze E. (2010) Chemical and microbiological analysis of premices wine fabricated by
COOTTRACO,KIE
13. Reed G. and Nagodawithana T. (1993) Enzymes in food processing, Academic
processing, inc. California.
32
14. Ryan KJ, and Ray CG. (2004). Sherris Medical Microbiology (4th Edition). McGraw
Hill.
15. Shayo N.B., Kamala A., Gidamis A.B. and Nako S.A.M. (2000). Aspects of
manufacture, composition and safety of orubisi: a traditional alcoholic beverage in the
north-western region of Tanzania. International Journal of food science and nutrition.
519(5): 395-402,
16. Sathiamoorthy S, Narayanan CK, Ramajayam D (2002). Scope of processing and value
addition in banana: Development of banana/plantain based food processing industry in
Kerala, Tiruchi-based National research center (NRC). Shingiro J. (2004) a survey on
banana wine making in Rwanda.
B. ELECTRONIC REFERENCES
1. http/www.vicino.com/ask the wine maker archive aspx? id=0
2. http://wiki.answers.com/Q/What_are_common_types_of_bacteria_found_on_hands
3. http://www.britannica.com/EBchecked/topic/212684/food-preservation/50551/Lowtemperature-preservation
4. http://www.doh.wa.gov/ehp/dw/programs/coliform.htm
5. http://www.nrcresearchpress.com/doi/abs/10.1139/w05-130?journalCode=cjm
6. http://www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/lab5coliforms.html
7. www.biology.clc.uc.edu/courses/bio104/cellresp.html
8. www.botany.hawii.edu/faculty/wrong/bot135/lect14html
9. www.liveandfeel.com
10. www.minagri.gov.rw
11. www.tempeh.info/fermentation/lactic acid fermenation.php
12. www.yeastgenome.org
13. http://www.whfoods.com/genpage.php?tname=foodspice&dbid=7
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APPENDIX
The plates a) is Musanze, b) is Ngoma
a)
b)
Plate3 Total germs grown on NB Dilution10-5 banana wine a) made in Musanze and b) in Ngoma
a)
b)
Plate4 Total coliforms grown on EMB at 37oC dilution10-5 banana wine a) made in Musanze and b) in
Ngoma
34