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CHAPTER ONE

1.0 INTRODUCTION
Yoghurt is a fermented dairy product obtained from
the lactic acid fermentation of milk. It is one of the most
popular fermented milk products in the world and produced
commercially at home. (Willey et al., 2008). In its
commercial production, non fat or low fat milk is
pasteurized cooled to 43c and are inoculated with known
cultures of microorganisms referred to as starter cultures.
The starter cultures may be a pure culture of a particular
species of Lactobacillus or a mixed culture of Streptococcus
thermophilus and Lactobacillus bulgaricus in a 1: 1 ratio.
The coccus which is the Streptococcus thermophilus grows
faster than the Rod which is the Lactobacillus bulgaricus
and is primarily responsible for acid production while the
rod adds favor and aroma. The growth of these
Microorganisms causes the transformation of milk's sugar,
lactose into lactic acid. This process gives yoghurt it's
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texture. The associative growth of the two organisms results
in acid production at a rate greater than that produced by
them individually.
Yoghurt is generally made from a standardized mixture
containing whole milk, partially defatted milk, condensed
skim milk cream and non fat dry milk. Alternatively milk
may be partly concentrated by removal of 15- 20% water in
a vacuum pan or by heating. While the microorganisms
fermenting milk confers on it certain health benefts
inadequate pasteurized milk may contain microorganism of
special importance to man. (Boor and Murphy, 2002). In
which its presence or absence in milk may refect success or
failure of good manufacturing practice (GMP) or cause
infection when consumed together with food. This is of
economic signifcance in Africa where the HIV/AIDS and
cancer scourge has left the public who consume milk
products immune suppressed and prone to bacterial and
fungi infection. (Boor,2001).
2
Health complications associated with consumption of
inadequately pasteurized milk products include serious
infections that are hard, to treat with antibiotics. This
becomes clinically signifcant if organisms isolated from an
assessed sample is resistant to conventional antibiotics.
Thus, can confer antibiotic resistance to the infected host
while providing no alternative drug (Gould, 1994). Heat
treated yoghurt do not contain lactic acid bacteria as these
are killed during post fermentation. Yoghurt manufacturing
companies mainly market "heat treat" yoghurt to prolong it's
shelf life (Hove et al, 1999). It is important however to
evaluate the microbial gravity of some milk products sold in
Nigeria. This project work aims at assessing the
antimicrobial susceptibility pattern of microorganisms
present in yoghurt sold in Enugu.
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CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 PRODUCT DESCRIPTION
Yoghurt is a smooth, fermented milk product that
evolved empirically some centuries ago through the growth
of thermophilic (heat loving). Lactic acid, bacteria,
Streptococcus thermophilus and Lactobacillus bulgaricus
which ferment the milks lactose to produce lactic acid. It
has a characteristic acidic taste possessing 0.95 -1.5% and
PH ranging from 3.7-4.2 with viable and abundant
fermenting microorganisms.
2.2 PRODUCTION OF YOGHURT
MATERIALS:
In yoghurt preparation the following essential
materials are use in processing the product: milk or
concentrated skimmed or partly concentrated skimmed milk
or milk product and the starter culture Lactobacillus
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bulgaricus and Streptococcus thermophilus. In. the absence
of pure culture one to two spoonful of commercially
purchased yoghurt can be used for the inoculation. Also,
there are optional ingredients like milk powder, skimmed
milk powder, favour, colours, sugar, wheat, edible casein,
preservatives, stabilizers (gelatin, locust bean gum, pectin,
starch) etc.
EQUIPMENTS:
They include refrigerator or cooler, boiler or heater,
thermometer.
PROCEDURES:
The milk to be fermented is frst heated to 70c for 15 -30
minutes the concentrate or skimmed milk powder is added
and the mixture is heated to about 80c with continuous
stirring for 5 minutes so as to kill the microorganisms which
are contaminants, lower the redox potential of the mixture
and produce factors and condition favourable to the
development of the bacteria to be inoculated. Also, the
5
added skimmed milk prior inoculation raises the nutritive
value of yoghurt and gives a product of better body and
consistency. The product is cooled to about 43c by dipping
in container containing cold water and inoculate with 3%
selected strains of actively growing microbial starter
(Lactobacillus bulgaricus and Streptococcus thermophilus) or
with 1.5% of each culture separately maintain the
temperature of 42 -44c for approximate 5 hours until
desired degree of acidity is achieved. Then cool rapidly to 8-
10c and refrigerate as you store till the next morning to
check for curd formation. The addition of - stabilizers,
favours, colour, sugar, fruit or honey etc may be added
before packaging if desired. The fnal product usually
contains some 10 cells per milliliter of each bacteria specie
while the' characteristic favour is due to the lactic acid and
trace amount of ethanol, dimethyl propanol, ethanoic acid
and other volatiles products by bacteria fermentation.
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2.3 VARIETIES IN YOGHURT PRESENTATION
Yoghurt has been described as a notoriously balanced
food, containing almost the nutrients present in milk but in
a more assimilable form. The can be produced from whole or
skimmed milk (Ojokoh, 2006). There are large ranges of
favours enhancer available commercially (Anthar, 1996)
that can be used in the production of yoghurt and yoghurt
is typically categorized as follows:
1. SET YOGHURT: This type of yoghurt is incubated and
cooled in the fnal package and is characterized by a
frm Jelly-like texture.
2. STIRRER YOGHURT: This type of yoghurt is
incubated in a tank and the fnal coagulum is "broken"
by stirring prior to cooling and packaging. The texture
of stirred yoghurt will be less frm than a yoghurt not
stirred which is some what like a very thick cream.
There is some slight reformation of the coagulum after
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the yoghurt has been packed, however this is slight
and can not be relied upon.
3. DRINKING YOGHURT: This type of yoghurt is very
similar to stirred yoghurt, having the coagulum
"broken" prior to cooling. In drinking yoghurt, the
agitation used to "break" the coagulum is severe. Little
care is applied if any reformation of the coagulum will
reoccur after packing.
4. FROZEN YOGHURT: This is inoculated and incubated
in the same manner as stirred yoghurt. However,
cooling is achieved by pumping through a
whipper/chiller/freezer in a fashion similar to the
cream. The texture of the fnished product is mainly
infuenced by the whipper/freezer and the size and
distribution of the ice crystals produced.
5. CONCENTRATED YOGHURT: This type is inoculated
and fermented in the same way as stirred yoghurt,
following the "breaking" of the coagulum. The yoghurt
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is concentrated by boiling of some of the water. This is
often done under vacuum to reduce the yoghurt often
lead to protein being totally denatured and producing
rough and gritty texture. This is called strained
yoghurt due to the fact that the liquid that is released
from the coagulum upon heating used to be "strained"
of in a manner similar to making of soft cheese.
6. FLAVOURED YOGHURT: Yoghurt with various
favours and aromas has become very popular. The
following are usually added at or just prior to flling
into pots. Common additions are fruits or berries,
usually as a pure or as whole fruit in syrup. These
additives often have" as much as 50% sugar in them.
However, with the trend towards healthy eating gained
momentum many manufacturers ofer a low sugar and
low fat version of their products. Low or no sugar
yoghurts are often sweetened with saccharin or more
commonly aspartame. The use of a "fruit sugar" in the
form of concentrated apple juice is sometimes found as
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a way of avoiding' "additional sugar" on the ingredients
declaration. This tends to be a market ploy and has no
real added benefts.
TABLE 1:
Typical Composition of Commercial Fruited Yoghurt.
S/N CONTENT PERCENTAGE COMPOSITION
(%)
1 Fat 0.1-3.5%
2 Lactose 3.4-5%
3 Milk solid non fat 11-18%
4 Stabilizer 0.2-0.4%
5 Fruit 10-20%
Source: NYA, (2000).
2.4 HEALTH BENEFITS OF YOGHURT
Dairy products such as yoghurt; contains probiotic
cultures e.g. Lactobacilli which are currently among the
10
best known examples of "functional food" (Oyeleke, 2009).
Their associated health claims include.
a.Yoghurt is easier to digest than milk and so many people
including children who can not tolerate milk, either
because of a protein allergy' or lactose intolerance can
enjoy yoghurt more digestible than milk. The live active
cultures create lactose, the enzymes lactose intolerant
people lack and another enzymes contained in some
yoghurts (beta-galactosidae) also improve lactose in
lactase defcient persons. Breaking down the milk sugar
lactose into glucose and galactose two sugars that are
easily absorbed by lactose intolerant persons. However,
bacterial enzymes created by the culturing process partly
digest the milk protein casein making it easier to absorb
and less allergenic (Witton, 2004).
b.Yoghurt contributes to colon health: There is a magical
truism that state "you are as healthy as your colon" when
we drink yoghurt we care for our colon in two ways:
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i) Yoghurt contains lactobacteria: Intestine friendly
bacteria culture that fosters a healthy colon and
lowers the risk of colon cancer by promoting the
growth of healthy bacteria and there by deactivate
harmful substances which can cause problem in the
colon.
ii) Yoghurt is rich in calcium which contributes colon
health and decreases the risk of colon cancer (Gray,
2007).
c.It improves the bioavailability of other nutrients culturing
of yoghurt increase the absorption of calcium, and
vitamins B, the presence of lactic acid in it aids the
digestion of le milk calcium, making it easier to absorb
Maltock, 2007).
d.Yoghurt can boosts immunity: The regular consumption
of live cultured yoghurt produces a higher level of
immunity boosting interferon as his bacteria cultures
stimulates infection fghting white cells in the blood
stream with anti tumor efects (Maltock, 2007).
12
e.Yoghurt aids healing: After intestinal, infection like
diarrhea which injures the lining of the intestines
especially the cells that produce' lactase which results to
temporal lactose mal- absorption problem, yoghurt
however because it contains less lactose and more lactase
is usually well tolerated by healing intestine and is a
popular healing food for diarrhea (Gray, 2007).
f.Protection against ulcers: Helicobacter pylori the
bacterium responsible for most ulcer, can be shut down
by yoghurt.
g.Yoghurt can decrease yeast infection eating eight ounces
of yoghurt that contains live and active cultures daily
reduces the amount of yeast colonies in the vagina and
decreases the incidence of vaginal yeast infection as show
from research (Gray, 2007).
h.Yoghurt is a rich source of calcium. An 8 ounce serving of
most yoghurts provides 450mg of calcium, one half of a
child's RDA and 30- 40% of the adult RDA for calcium.
Because the live active cultures in yoghurt increase the
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absorption of calcium, an 8 ounce serving of yoghurt gets
more into the body than the same volume of milk can.
i.It is an excellent source of protein. Plan yoghurt contains
around 10-14 grams of protein percent 8- ounces, which
amounts to twenty percent of the daily protein
requirement for most persons. i.e it contains more
percent than the same volume of milk also the culturing
of the milk protein during fermentation makes it easier to
digest.
j.Yoghurt can lower cholesterol -Daily consumption of
ounces (100g) of yoghurt signifcantly improved the
cholesterol while raising HDL (good) cholesterol which
may be because of the ability of the live culture in it to
assimilate cholesterol or because yoghurt binds the bile
acids which lower' cholesterol (Maltock, 2007).
k.Yoghurt help prevent and treat Arthritis; Lactobacillus a
probiotic (friendly) bacteria found in yoghurt ofers
"remarkable preventive and curative efects on arthritis.
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l.Improve fresh breath and a healthy mouth. Consuming
just 3.2 ounces of yoghurt twice a day not only lowers
levels of hydrogen sulfde and other volatile sulfde
compounds responsible for bad breath, but may also
eliminate tongue-coating bacteria and reduce dental
plaque formation, cavities and' risk for gingivitis,
Research backed by the international Association for
dental research shows that eating plain live -yoghurt for
six weeks can reduce levels of oral bacteria by up to 80%
so yoghurt is a traditional bad breath cure (Okpalugo et
al., 2008),
2.5 NUTRITIONAL PROFILE OF YOGHURT
Yoghurt as a dairy food can be consumed in form of
snack, thirst quenching beverages and as a desert, but it's
nutritional value cannot be over emphasized. It is a good
source of iodine, calcium, phosphorus, zinc, ribofavin,
vitamin B5 and vitamin B12. It is also nutritionally rich in
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protein, molybdenum and Pantothenic acid (Ensminger, et
al., 1986). The food rating system adopted as the
government standard for food labeling that are found in the
U.S food and drug administration allow yoghurt to be rated
as one of the world's healthiest food. Table II shows the
nutrient for which yoghurt is rated. The absence of a
particular nutrient does not necessarily mean that it is
absent, rather the nutrient is not provided in a sufcient
amount or concentration to meet the rating criteria.
TABLE 2:
Food rating for Yoghurt
S/N NUTRIENT
A
M
O
U
N
T

D
A
I
L
Y

V
A
L
U
E
(
D
V
)

(
%
)
N
U
T
R
I
E
N
T
D
E
N
S
I
T
Y

W
O
R
L
D

S
H
E
A
L
T
H
I
E
S
T
F
O
O
D

R
A
T
I
N
G

1 Iodine 87.22mcg 58.1 6.8 Very good
2 Calcium 447.37mg 44.7 5.2 Very good
3 Phosphorus 351.58mg 35.2 4.1 Very good
4 Vitamin B2 0.52mg 30.6 3.6 Very good
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[ribofavin]
5 Protein 12.86g 25.7 3.0 Good
6 Vitamin B12
[cobalamin]
1.38mcg 23.0 2.7 Good
7 Tryptophan 0.06g 18.8 2.2 Good
8 Potassium 572.81mg 16.4 1.9 Good
9 Molybdenum 11.27mg 15.0 1.7 Good
10 Zinc 2.18mg 14.5 1.7 Good
11 Vitamin B5
[pantothenic
acid]
1.45mg 14.5 1.7 Good
S/N
W
O
R
L
D

S
H
E
A
L
T
H
I
E
S
T
F
O
O
D

R
A
T
I
N
G

RULE
1 Excellent DV > = 75% Or Density > = 7.6 and DV > = 10%
2 Very good DV > = 50% Or Density > = 3.4 and DV > = 5%
3 Good DV > = 25% Or Density > = 1.5 and DV > = 2.5%
Source: USFDA, (2001)
2.6 FACTOR THAT ALTER THE QUALITY OF YOGHURT
1. MILK QUALITY: The milk used for yoghurt
manufacture should be of the highest bacterial quality
17
available. It should also have an absence of any material
that will impede or prevent the growth of the starter micro
organism, (anti-biotics, preservative, disinfectants,
bacteriophages).
2. BACTERIOPHAGES: Bacteriophages are a group of
virus that attacks the yoghurt starter organisms, a whole
range of defects can be attributed to the action of this
bacteriophage. Bacteriophage normally referred to just as
"phage" are the most likely cause of long or never-ending
incubations. Large manufacturers that have laboratory
facilities to check incoming milk will often eliminate the
possibilities of other starter inhibiting substances but
"Phages" are usually found in the drains and foor gullies of
a dairy producing any cultured product, poor hygiene and
lack of general house keeping increase the risk.
3. STARTER CULTURE: The starter culture is the term
generally applied to organisms used to ferment a cultured
product (Cheese, Yoghurt, Kefr). The micro organisms
selected for this purpose need to produce the desired efect
18
in the product. For normal commercial yoghurt the starter
must be capable of fermenting lactose and producing lactic
acid, little if any carbon dioxide is required and the favour
and aroma must be clean and fresh. Traditionally when a
suitable starter organism had been found on a large
quantity would be grown in a suitable nutrient medium and
small quantities would be used to inoculate each new batch
of yoghurt. This technique with a main batch of starter
culture is often referred to as using "bulk starter". The use
of a bulk starter is becoming increasingly uncommon
amongst commercial producers, mainly because of the risk
of "Phage" attack on the bulk starter, and the subsequent
lost time while a new batch of starter organisms are
prepared. A technique often referred to as Direct Vat
Inoculation (DVI) is becoming the industry norm. DVI
involves inoculating the yoghurt mix directly with a very
large number of freeze dried starter organisms. The
advantage of relative immunity to "Phage" attack for out
19
weigh the slightly longer incubation time required with this
technique.
4. FAT PERCENTAGE: The percentage of fat in the fnal
yoghurt has a signifcant efect on the "mouth feel", the
normal range of fat content is from 0.5% to about 3.5%,
however levels as low as 0% and as high as 10% are found
in some specialty products.
2.7 SOURCES OF MICROBIAL CONTAMINATION IN
YOGHURT:
Yoghurt as a fermented dairy product is produced from
milk which is a white female individual of the mammalian
and adopted for the nourishment of their young (Simpson
and Weiner, 1989).
The source of microbial contamination in yoghurt
could be traced to it's source, the "milk". Although yoghurt
may be fermented but some micro organisms that could
survive the fermentation and pasteurization process could
20
still strive and alter the quality of the product when this
happens, the food becomes unft for human consumption
(Jilon, 2001). The various sources of microbial
contaminants in yoghurt can be considered as follows:
1) Microbial contaminants from within the udder of
the cow:
Raw milk as it leaves the udder of healthy cows
normally contains very low number of micro organism and
generally will contain less than 1, 000 total bacteria per ml
(Kurweil, 1973). Milk contains relatively few bacteria when it
leaves the udder of a healthy cow and generally these
bacteria do not grow in milk under the usual conditions of
handling. However, Micrococci and Streptococci have been
recovered from aseptically drawn milk (Frazier and Westhof,
1995). In healthy cows, the teat cistern, teat cannal and the
teat apex may be colonized by a variety of micro organism
although microbial contamination from within the udder of
healthy animals is not considered to contribute signifcantly
21
to the total number of microorganism in the milk or to the
potential increase in bacterial number during refrigerated
storage natural fora of the cow have little infuence in the
standard plate counts while healthy udder should
contribute very little to the total bacteria count of bulk milk,
a cow with mastitis has the potential to shed large numbers
of micro organism into the milk supply and this might
signifcantly afect the products of the milk. Mastitis micro
organisms found to most often infuence the total bulk milk
count are Streptococcus spp. Most notably Streptococcus
agatatiae and Streptococcus aberis (Bramley and Mckinnon
1990; Bramley et al., 1984, Gonzalez et al., 1986, Jefrey
and Wilson, 1989).
Although other mastitis pathogens have the potential
to infuence the bulk tank count as well. Detection of
implied pathogens does not necessarily indicate that they
originated from cows with mastitis. Potential environmental
mastitis pathogens and similar organisms can occur in milk
as a result of other contributing factors such as dirty cows,
22
poor cleaning equipment of the milking machines and poor
cooling.
2) Microbial Contamination from Exterior of the
Udder.
In general, the direct infuence of natural inhabitants
as contaminants in the total bulk count of yoghurt is
considered to be small and most of the these micro
organisms do not grow competitively in milk, Teats and
udders of cow inevitably become soiled while they are lying
installs or when allowed to harbor large number of micro
organism. Total count often exceeds 10
8
to 10
10
per gram
(Bramley, 1982; Bramley and Mckinnon, 1990, Hogan et al.,
1989; Zehner et al., 1986). Organisms associated with
bedding materials that contaminate the surface of teats and
udders include Streptococci, Staphylococci, sporeformers,
coliforms and other gram negative bacteria both
thermoduric (bacteria that can survive pasteurization such
as Streptococci and Lactobacillus and psychotrophic (bacteria
23
that. grow under refrigeration) strains of bacteria are
commonly found on teat surfaces (Bramley and Mckinnon,
1990). Indicating that contamination from the exterior of
the udder can infuence Lab Pasteurization count (LPC) and
Preliminary Incubation Count of Milk. (Bramley and
Mckinnon, 1990, Pankey, 1989).
Generally through cleaning of the teat with sanitized
solution (Spray wet towel or dip) suggests that higher
coliform counts in bulk milk are more likely to occur due to
other factors such as utensils and milk contact surfaces are
inadequately cleaned, sanitized and dried. Bacteria may
develop on large numbers on the next milk to touch these
surfaces. Undesirable bacteria from these surfaces include
Streptococci, coliform bacteria, and psychotropic. Gram-ve
rods and thermodurics. Those which survive pasteurization
e.g Micrococci, Enterococci, Bacilli and Brevibacteria other
possible sources of contamination are the hands and arms
of the milker or dairy worker, the air of the barn or milk
priors and fies while other sources of contamination after
24
the milk leaves the farm includes the tanker truck, transfer
pipes, sampling and the equipment at the market milk plant
or other processing plant.
The defects that can occur in milk due to microbial
growth are of favours, lipolysis with development of
rancidity, gas product, fermentation to lactic acid with
souring, coagulation of milk protein, viscous or ropy texture
and discoloration.
2.8 INFLUENCE OF CLEANING EQUIPMENT AND
SANITIZATION
The degree of cleaning of the total bulk milk bacteria
count as much as, if not more than any other factor [Olsen
and Mocquat, 1980]. Milk residue left on equipment contact
surfaces support the growth of a variety of microorganisms.
Water used on the farm and during yoghurt production
might also be a source of microbial contamination,
25
especially psychrotrops that could seed soiled equipment
and milk [Bramley and Mckinnon 1990].
More resistant and thermoduric bacteria may endure
in low numbers on equipment surfaces and that are
considered to be efciently cleaned with hot water. Less
efcient cleaning; using lower temperatures and the
absence of sanitizers tend to select for the faster growing
less resistant organisms principally gram negative rods
coliforms and pseudomonads and Lactic streptococci.
Psychrotrophic bacteria tends to be present in higher count
in milk and often associated with occasional neglect or
proper cleaning in sanitizing procedures (Olson and
Mocquat, 1980) and poorly cleaned refrigerated yoghurt or
bulk tanks milk (Mackenzie, 1973).
Milk Storage Temperature
Under conditions of poor cooling with temperature
than 7.2
0
c (45
0
f), bacteria other than psychrotrophs are able
to grow rapidly and can become predominant in raw milk
streptococci have historically been associated with poor
26
cooling of milk- smears. These bacterial will increase the
acidity of milk, bringing about milk fermentation. Certain
strains are also responsible for a Malty defect that is easily
detected by its distinct odor. Storage temperature greater
than 15
0
c (60
0
f) tend to select for these types of bacteria
that grow and become signifcant in yoghurt or milk
products will depend on the initial fora of the milk (Bramley
and Mckinnon, 1990) and the degree of processing.
27
Figure 1:Microbial activities in raw milk at moderate
temperature.
Source: Cambell and Marshall, 1975.
Fig. 1. Shows the activity of micro organisms in milk at
moderate temperature. S. lactis and Lactobacilli grow and
multiply rapidly, ferment lactose and produce, lactic acid to
bring the reaction to PH 3.5 or Lower. Film yeast, molds, etc.
when introduced into milk as contaminant oxidize the lactic
acid and the PH rises permitting putrefying bacteria to grow,
thereby bringing about the spoilage of yoghurt. Mould and
yeasts are primary contaminants in yoghurt produced
commercially in Nigeria (Suriayarachichi and feet, 1981).
CHAPTER THREE
3.0 MATERIALS AND METHOD
3.1 MATERIALS
The media used in this work include Nutrient agar,
MacConkey agar, Peptone water, Petri dish, autoclave,
inoculating wire loop, forcep, Bunsen burner, Conical fask,
28
Antibiotic discs, Weighing balance, Test tube rack, plastic
pipette, wire loop, Microscope, Incubator, beakers, glass
slide, sterile cotton wool, test tube rack, universal container.
The composition of the media, their method of production is
presented in the appendix.
3.2 SAMPLE COLLECTION
Six diferent brands of bottle packaged yoghurt were
bought from hawkers and beverage stores in Enugu Urban.
Two samples of each brand were used and the brands were
designated A, B, C, D, E, F, giving a total of 12 yoghurt
samples. The samples were brought to the laboratory and
analyses within 6hours of collection.
3.3 ANALYSIS OF SAMPLE
Each sample was serially diluted using sterile distilled
water as diluents (Prescott et al., 2002). 9ml of distilled
29
water was measured out into test tubes, using separate
sterile pipettes, 1ml of yoghurt sample was measured out
into the frst test tube properly mixed. using a diferent
sterile pipette, 1ml from the frst test tube was pipette into
the second test tube already containing 9ml of distilled
water, this continued following the same procedure till the
last dilution (ie the last test tube).using the pour plate
method 1ml each of each sample unit from the test tubes
was pipetted into the sterile Petri dishes (using separate
sterile pipettes per sample) with their duplicates, then into
each Petri dish the prepared MacConkey agar was poured
aseptically and mixed by movement of the plate while fat on
the bench. This was also carried out on Nutrient agar media
was used. The plates were incubated at 37c for the 24hr.
After incubation the representative colonies on the plates
were subcultured on fresh nutrients agar to obtain pure
cultures of the isolates. The pure cultures were then
transferred into nutrient agar slants for biochemical
identifcation.
30
3.4 IDENTIFICATION OF ISOLATES
Identifcation of isolates were based on cultural,
morphological and biochemical characteristics following
standard methods.
Bacteria Identifcation
Gram Staining
The method used was that described by carpenter (1977)
and Thomas (1973). Smears of the isolates were prepared
and heat fxed on clean grease free slides. The smears were
stained for one minute with crystal violet. This was washed
out with a gentle running tap water. The slides were fooded
with dilute Gram's iodine solution. This was washed of with
water and the smears were decolorized with 95% alcohol till
the blue colour no more dripped out (about 30 seconds).The
smears were then counter stained with safranin solution
for about 10 seconds. Finally, the slides were washed with
31
tap water, air dried and observed under oil immersion
objectives.
Motility Test
This test was used to determine which of the isolates
were motile. Motility test is usually used to diferentiate
motile organisms from non-motile ones. For this test, the
hanging drop technique was employed and the technique
was carried out as described by Kirk et al., (1975). A little
Vaseline jelly was rubbed around the cavity of a hanging
drop slide. A drop of peptone water containing the pure
culture was placed on a cover slip. The hanging drops slide
was then placed over the drop of peptone water in such a
way that the center of the depression lies over the drop. The
slide was quickly inverted and viewed under the microscope,
using oil immersion objective.
3.4.1 BIOCHEMICAL TESTS
Urease Test
32
This test was used to demonstrate the ability of the
isolates to produce the enzyme urease which splits urea
forming ammonia. The test is usually used to diferentiate
organisms like proteus from other non urease positive
organisms, (Baker and Breach 1974). The method used was
that described by speck (1971). A loop full of the isolates
was used to inoculate a tube of urea-agar. The tubes were
incubated at 37
o
C. a change in colour from yellow to red
confrmed the presence of urease.
Catalase Test
This test was used to demonstrate which of the isolates
could produce the enzyme catalase that release oxygen from
hydrogen peroxide. This test is usually used as an aid to
diferentiate Staphylococci from Streptococci and to
diferentiate other catalase positive organism from catalase
negative (Barker 1976). The method employed here was that
described by Speck (1976).
33
A loopful of the pure colony was transferred into a
plane, clean glass slide. The sample was then mixed with a
drop of 3% v/v hydrogen peroxide. The reaction was
observed immediately Gas production indicated by the
production of gas bubbles confrmed the presence of
catalase.
Methyl Red Test
This test was used to detect which of the isolates could
produce and maintain sufciently a stable acid product
from glucose fermentation. The test is usually used as an
aid in the identifcation and diferentiation of the
Enterobacteriaceae (Baker 1976). This test was carried out
as described by Kirk et al (1975). Tubes of bufered glucose-
peptone broth were lightly inoculated with the isolates. The
tubes were incubated at 37C for not less than 48hours.
About 5 drops of the methyl red reagent was added into 5ml
of the culture. The production of a bright red colour
34
immediately on the addition of the reagent showed a positive
test, Methyl red test indicator consists of 0.lg methyl Red,
300ml of 95% ethyl alcohol.
Voges -Proskeur Test (V.P. test).
This test was used to detect which of the isolates were
able to produce a neutral end point acetyl methyl carbinol
(acetoin) from glucose fermentation or its reductive product
butylene glycerol. The test is usually used to diferentiate
between Gram negative organisms especially members of the
Enterobacteriaceae, (Baker, 1976). The test was carried out
as described by Kirk et; al (1975). Tubes of bufered glucose
peptone broths were lightly inoculated with a young culture
of the isolates. The tubes were incubated at 37C for not
less than 48 hours. Burrits reagent was used for the test.
0.6% v/v of solution A and 0.2ml of solution B were added
into 1ml of the culture in turns. The mixtures were shook
well after each addition.
35
Positive reaction was indicated by a pink colour that
appears immediately or within 5 minutes at the topmost
part of the tube.
Solution A contains 5g of naphlhol,
100ml of absolute ethyl alcohol,
Solution B contains 100ml of Distilled water,
40g of Potassium hydroxide
The alkali oxidized the acetyl methyl carbinol (acetone) to
diacetyl which gives the pink colour.
Indole Test
This test was used to determine which of the isolates
has the ability to split indole from tryptophan present in
bufered peptone water. -he test is usually used as an aid in
the diferentiation of Gram negative, Bacilli especially those
of the Enterobacteriaceae (Baker 1976). The test was carried
36
out as described by Kirk et al., (1975). Tubes of peptone
water were inoculated with young culture of the isolates.
The tubes were incubated at 37c for 48hrs. About 4 drops
of Kovac reagent were added into 1ml of each of the culture
tubes. Positive test was indicated by a red colour that
occurs immediately at upper part of the test tube.
Kovac's reagent consists of the following:
150ml of Amyl alcohol
10g of D-Dimethyl aminobenzaladhyde
50ml of concentrated hydrochloric acid.
Citrate Utilization Test
This test was used to identify which of the isolates can
utilize citrate as the sole source of carbon for metabolism.
The- test is usually used as an aid in the diferentiation of
organisms in the Enterobacteriaceae and most other genera.
(Baker1976). The medium used for this test was the Simon's
citrate agar. Slant tubes of Simon's citrate agar were
37
inoculated with young culture of the isolates. The
inoculation was done by stabbing the medium on the tubes
using sterile straight inoculating wire containing the
culture. The tubes were then incubated at 37C for about
24hours. Change in colour from green to blue after about
24hours of incubation indicated positive result.
Sugar Fermentation
Each of the isolates was tested for its ability to ferment
a given sugar with the production of acid and gas or acid
only. Since most bacteria especially Gram negative bacteria
utilize diferent sugars as source of carbon and energy with
the production of both acid and gas, or acid only the test is
used as an aid in their diferentiation. The growth medium
used was peptone water and the method used was that
described by Kirk et al., (1975). Peptone water was prepared
in a conical fask and the indicators bromocresol purple was
added. The mixture was dispensed into test tubes
containing Durhams tubes. The tubes with their content
38
were sterilized by autoclaving at 121c for 15 minutes. 1%
solution of the sugar was prepared and sterilized separately
at 115C for 10 minutes. This was then aseptically
dispensed in 5ml aliquot volume into the tubes containing
the peptone water and indicator. The tubes were inoculated
with young culture of the isolates and 1ncubated at 37C.
Acid and gas production or acid only were observed after
about 24 hours of incubation. Acid production was
indicated by the change of the medium from light green to
yellow colour, while gas production was indicated by the
presence of gas in the Durham's tubes. The control tubes
were not incubated.
Coagulase Test
Slide and tube method was used (carpenter 1977). In
slide test, a loop full of the isolate was mixed with human
plasma and allowed to stand for some minutes. Particles
indicating agglutination was used as indication of coagulase
reaction.
39
In tube method, plasma was added into a culture of
the isolate in peptone water in bijou bottles. The bottles
were incubated at 37C for 24hours. A
clumping/agglutination of the plasma were used to indicate
presence of coagulase.
Spore stain
The malachite green staining method was used. The
staining was carried out as described by Carpenter (1977).
Smears of the pure isolates were made on grease-free glass
slide and heat fxed. The slides were fooded with 5% v/v
malachite green solution. The slides were famed in such a
way that the stain steamed but did not boil. The slides were
then allowed to stand for 5min. The stain was then washed
out in running tap water. The smears' were counter' stained
with safranin for 30 seconds. This was stained with
safranin for were blotted, dried and examined under the oil
immersion objective. The spores stained green while
vegetative cells stained red.
40
3.5 ANTIBIOTIC SENSITIVITY TEST.
The Isolated organisms were tested against routinely
used commercially available antibiotics. The multi
susceptibility, disc for Gram positive and Gram negative
organisms were used. Each of the potentially pathogenic
organisms, isolated from the yoghurt was tested against the
antibiotics. A 24hrs old culture of each of the isolates was
heavily inoculated on a plate of nutrient agar. A strip of the
multi- susceptibility disc was then placed on the agar plate
with sterile forceps. The strips were made to be in good
contact with the medium. The plates were then incubated
for 24hrs at 37
o
c. Zone of inhibition around each strip of
paper discs containing the antibiotic was then measured
with a ruler and recorded.
41
CHAPTER FOUR
4.0 RESULT
The physicochemical properties of bacteria isolated
from the yoghurt samples are shown in Table 3. The
bacteria isolated include Klebsiella sp. Streptococcus sp,
Escherichia coli, Bacillus sp, Staphylococcus aureus,
Pseudomonas sp, Enterobacter sp and Proteus sp. The
antibiotic resistance patterns of the isolates are presented in
Table 5. The isolates showed varied antibiotic resistance
pattern. The isolates were resistant against many of the
antibiotics tested.
42
TABLE 3: THE IDENTITIES OF MICROORGANISMS ISOLATED FROM YOGHURT
SUGAR FERMENTATION
Probable
organisms
I
s
o
l
a
t
e

G
r
a
m
s

r
e
a
c
t
i
o
n

C
i
t
r
a
t
e

M
e
t
h
y
l

r
e
d

V
o
g
u
e
s

p
r
o
s
k
e
u
r

H
y
d
r
o
g
e
n

S
u
l
p
h
i
d
M
o
t
i
l
i
t
y

C
o
a
g
u
l
a
s
e

I
n
d
o
l
e

C
a
t
a
l
a
s
e

U
r
e
a
s
e

S
p
o
r
e

s
t
r
a
i
n

S
t
a
r
c
h

h
y
d
r
o
l
y
s
i
s
L
a
c
t
o
s
e

G
l
u
c
o
s
e

F
r
u
c
t
o
s
e

S
u
c
r
o
s
e

M
a
n
n
i
t
o
l

M
a
l
t
o
s
e

1 -RODS + - - - - - + - - - + AG AG AG AG AG
AG Klebsiella sp
2 +COCCI
in chain
- + - - - - - - - ND - - A A - -
- Streptococcus sp
3 -RODs - + - - - - + - - - + AG A AG AG AG
A Escherichia coli
4 +RODs + + - + + - - + + + - - A AG AG AG
AG Bacillus spp
5 +COCCI + - - + - + + + - - + A A A A AG
A Staphylococcus
aureus
6 -RODs - + + - + - - - - ND + A A A A A
A Pseudomonas sp
40
7 -ROD + + + + - - - + - ND + - A - - AG
AG Enterobacter sp
8 +RODS - - - - + - - - + - + AG AG AG AG AG
AG Lactobacillus
bulgaricus
9 -RODS - - + - + - - + + + + - AG - - -
- Proteus sp
Key: +=positive, -=negative, A=acid production, AG= acid and gas production, ND=not determined
41
TABLE 4: BACTERIA ISOLATED FROM DIFFERENT
YOGHURT BRAND
BACTERIA ISOLATED Yoghurt brands
A B C D E F
Klebsiella sp
+ - - + + -
Streptococcus sp
+ + + - + -
Escherichia coli
- + + - + -
Bacillus spp
+ - + - - +
Staphylococcus aureus
- + - + - -
Pseudomonas sp
+ + - - - -
Enterobacter sp
- + + + - -
Proteus sp
+ - - - + -
Key: + = Present
- = Negative
42
Table 5: Antibiotic resistance pattern of bacteria
isolated from yoghurt
BACTERIA
ISOLATED
INHIBITION ZONE (MM)
D CX NB GN E AC AX SXT CIP CD
Klebsiella sp 0 2 6 0 0 9 2 2 0 0
Streptococcus sp 0 3 7 0 2 3 0 0 0 0
Escherichia coli 0 4 10 0 0 6 0 3 2 0
Bacillus spp 0 2 0 0 0 6 0 0 0 0
Staphylococcus
aureus
0 4 6 0 0 6 0 0 2 0
Pseudomonas
sp
0 3 4 0 0 4 0 0 0 0
Enterobacter sp 0 4 8 4 0 6 2 4 0 0
Proteus sp 0 3 7 3 0 6 0 3 0 0
Key:D - Drovid NB - Norfoxacin
CX - Cephalexin AX - Amoxil
GN - Gentamycin SXT- Septrin
E - Erytromycin CIP - Ciprofoxacin
AC - Ampiclox CD - Clindamycin
43
CHAPTER FIVE
5.0 DISCUSSION/CONCLUSION
The result showed that many of the Yoghurt samples
were contaminated by microorganisms that have public
health implications. The isolates especially the potentially
pathogenic ones showed resistance against many of the
antibiotics tested (Table 5). Staphylococcus showed
resistance against Drovid, Amoxil, Septrin and Clindamycin
and were slightly sensitive to cephalexin, ciprofoxacin but
sensitive to Ampiclox and Norfoxcin.
Pseudomonas was also resistance to many of the
antibiotics tested. Some of the antibiotic patterns reported
in this work were also reported by Enabulele and Orikpete
(2009). Their isolates however showed higher resistance
pattern probably because they were isolated from clinical
patients. The sensitivity pattern shown by Escherichia coli in
this work is similar to that reported by Njoku (2009).
44
Conclusively, evaluated milk products clearly pose
some yet undefned risks. This is of clinical signifcance in
immuno suppressed people who may consume these
product6. These groups of people should be conscious when
consuming milk products as they may" ingest isolates
resistant to some broad spectrum antibiotics. This is
because the concentration of bacteria isolated from locally
produced yoghurt in the area sample difers from one local'
producer to the other, and lack of standardization makes it
hard to be sure of the quality of their products. The
relatively high level of resistance to antimicrobial agents
constitutes a major threat to public health as it may spread
bacterial resistance among the populace who come in
contact with such milk products. Since the analysis of the
work carried out assessed local producers in Enugu City, it
is important that the food monitoring bodies provides a
standard for the local producers in order to reduce the risk
that their products might pose to consumers
45
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rd
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53
APPENDIX 1
COMPOSITION AND PREPARATION OF MEDIA
Nutrient Agar (NA)
This medium was used for the enumeration of bacteria
cells and also to maintain pure cultures. Nutrient agar is a
general medium. It was therefore used here on the
assumption that as many organism as are on the samples
will grow.
Composition
The medium is composed of the following
'Lab -lemco powder 1g
Yeast Extract 200g
Peptone 50g
Sodium chloride 5.0g
Agar No.3 15g
pH. 7.4.
Preparation
54
The powered form was used and it was prepared as
directed by the manufacturer. Twenty three grams of the
powdered nutrient agar (Oxiod) was suspended in 1000ml of
freshly prepared distilled water and made to dissolve by
heating. This was autoclave at 121
o
C for 15 minutes. The
sterilized medium was allowed to cool down to about 45
o
C
and was then poured into sterile petri dishes in about 20ml
aliquots. The medium was allowed to solidify, on these
plates and were thereafter used.
2. MacConkey Agar (M C A)
This medium was used primarily to diferentiate
lactose fermenters from non lactose fermenters and also to
suppress the swarming activity of proteus and other
spreading organisms.
Composition
Peptone 20g
Lactose 10g
55
Bile salt's 5g
Neutral red 0.075g
Agar No.3 12g
Distilled water 1000ml
pH 7.6
Preparation:
The powdered form was used and it was prepared as
directed by the manufacturer. 52g of the powdered
MacConkey medium (Oxoid) was suspended in 1000ml of
freshly prepared distilled water and dissolved completely by
heating. The medium was then sterilized by autoclaving at
121
o
C for 15 minutes. The medium was allowed to cool to
about 45C before being poured into sterile petridishes in
20ml aliquote. The medium was allowed to solidify in these
plates and were thereafter used.
3. Peptone Water (PW)
56
This medium was used to enrich and develop the
inoculums that were used to inoculate the agar plates. It
was also used to maintain the culture for some biochemical
tests.
Composition
Peptone 10g
Sodium Chloride 5g
Distilled Water 1000ml.
pH 7.6
Preparation
The powdered medium was used and it was prepared as
directed by the manufacturer. Fifteen grams of the powdered
medium (Oxoid) was dissolved in 1000ml of distilled water.
The medium was sterilized by autoclaving at 121C for
15minutes.
4. Urea Agar(U.A)
This medium was primarily used for urease test.
57
Composition
Peptone 1g
Sodium Chloride 5g
Potassium dihydrogen- Sulphate K H
2
PO
4
2g
Glucose 5g
Agar powder 20g
Distilled water 1000ml.
Preparation
The powdered urea agar (oxoid) was used and was
prepared as directed by the manufacturer. Urea agar was
prepared by suspending 2.4g of the powdered medium in
9Sml of distilled was dissolved by boiling. The medium was
sterilized by autoclaving at 115C for 20 minutes. The
medium was cooled to about 50C and 5ml of 40% v/v
sterile urea solution was added. The medium was dispensed
into culture tubes in 15ml aliquote and were allowed to
solidify in slant positions. They were thereafter used.
58
5. Simon Citrate Agar (SCA)
This medium was used for the diferentiation of
Enterobacteriaceae. Based on the utilization of citrate as the
sole source of carbon.
Composition
Magnesium sulphate 0.2g
Sodium ammonium sulphate 0.8g
Ammonium dihydrogen sulphate 0.2g
Sodium citrate tribasic 2.9g
Sodium chloride 5.0g
Bromoethyl molblue 0.08g
Agar No.3 15g
Distilled water 1000ml.
pH 6.9
Preparation:
The medium was constituted as above and sterilized by
autoclaving at 121
o
C for 15 minutes. Two drops of
59
concentrated hydrochloric acid (HCL) were added to the
medium to adjust the pH of the medium to the accepted
level. About 15ml aliquote of the medium was dispensed
into culture tubes and the medium was allowed to solidify in
these tubes in slant positions. They were thereafter used.
60