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University Of Khartoum Faculty of Veterinary Medicine
Department Of Preventive Medicine
And Veterinary Public Health
Microbial Load of Pasteurized Milk Produced In Khartoum
State
Thesis Submitted To the Graduate College,
University of Khartoum, As a Partial Fulfillment of
Requirement for the Degree of Master of Tropical Animal
Health.
By
Suaad Elamin Al Elmagid Sulyman
(B.S c 1991)
Faculty of Veterinary Medicine
University of Khartoum
Supervisor:
Dr: Tawfig El Tigani Mohamed
May 2007
ii
Dedication
To The Sole of my father
To my mother
To my husband, my daughters
and sons and all
Of my family
iii
ACKNOWLEDAGEMENTS
My deepest gratitude is to my supervisor Dr.Tawfig
ElTigani for his priceless guidance, help, patience and
encouragement.
I would like to acknowledge the staff of the
Preventive Medicine Lab. Faculty of Veterinary Medicine
University of Khartoum especially Hussein AbdelRahim.
My warmest gratitude for my family, friends for
strong support and for bearance.
iv
ABSTRACT
This study was to investigate the range of contamination of pasteurized
milk samples which were marketed in Khartoum State shops, and identification
of different types of bacteria present in collected milk from various factories.
Sixty packaged pasteurized milk samples were collected. The samples
were examined microscopically and cultured to determine if there were
bacteria, and types of bacteria involved in milk samples were determined.
Twenty one (21) 35% of milk samples were found to be positive for presence
of bacteria where as (39) sample were negative for bacteria.
The isolated bacteria from pasteurized milk were enterobacter 42.8%,
proteus mirbilis 9.5%, pseudomonas aroginosa 14.2%, bacillus creus 23.2%and
actinobacillus lignersii 9.5%.
The present study concluded that that pasteurized milk which was distributed
in Khartoum State was of low quality.
Hence it was recommended that quality assurance program should be
started to ensure that good quality milk and milk products are produced and
consumed in the country.
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ملخص الدارسة
الملوثه لأللبان المبسترة األنواعأجريت هذه الدراسة لمعرفة وجود البكتريا ومعرفة
.بأسواق والية الخرطوم
بقاالت بوالية الخرطوم وفحصت تر من ـتون عينه من اللبن المبسـ سجمعت
.لعيناتمن جملة تلك ا هبعد أن زرعت لمعرفة أنواع البكتريا الموجودة في مجهرياً
عينة 39بينما لم تنمو في ، وجد بها نمو بكتيري %35 احدي وعشرون عينة من
%65من اللبن المبستر
واع البكتريا ـومن أن .اـواع اللبن من البكتريـو بعض أنـخلد ـا وجـكم
.Enterobacter, Proteus, Pseudomonas bacillus, Actinobacillus .المعزولة
ة ـوق بواليـذي يسـودة اللبن المبستر المنتج والـدني جـت إلي جـخلصت النتائ
.الخرطوم
. ودة ومراقبة اللبن المنتج للمستهلكـذا توصي الدراسة بوضع برنامج لصحة وجـل
. لضمان سالمتها بداية اإلنتاج واالستهالكوتوضح
vi
LIST OF CONTINETS
Page
Dedication ------------------------------------------------------------------------------i
Acknowledgement---------------------------------------------------------------------ii
English in Abstract --------------------------------------------------------------------iii
Abstract Arabic ------------------------------------------------------------------------iv
List of Contents ------------------------------------------------------------------------v
List of Table----------------------------------------------------------------------------ix
List of Figures--------------------------------------------------------------------------x
Introduction ----------------------------------------------------------------------------1
CHAPTER ONE: LITERATURE REVIEW
1. 1. Historical background ----------------------------------------------------------3
1.2. Nutritional value of milk --------------------------------------------------------3
1.3. Milk Composition----------------------------------------------------------------4
1.4. Cows Milk-------------------------------------------------------------------------4
1.5. Source of bacteria in milk-------------------------------------------------------4
1.6. Source of contamination in milk -----------------------------------------------5
1.7. Major types of bacteria in milk-------------------------------------------------8
1.8. Pathogenic bacteria in milk -----------------------------------------------------8
1.9. Milk quality control evaluation ------------------------------------------------10
1.9.1 Microbial measurement--------------------------------------------------------10
1.9.1.1 Bacterial count ----------------------------------------------------------------10
1.9.1.1.1. Standard plate count ( s.p.c) ---------------------------------------------10
vii
1.9.1.1.2. Additional tests procedures ----------------------------------------------10
1.9.1.1.2.1 Preliminary incubation count (Pi) -------------------------------------11
1.9.1.1.2.2 Laboratory pasteurized count ( LPC) ---------------------------------11
1.9.1.1.2.3. Bacteria species evaluation --------------------------------------------11
1.10 Pasteurization --------------------------------------------------------------------12
1.10.1 Methods of Pasteurization---------------------------------------------------13
1.10.2 Objectives of Pasteurization -------------------------------------------------14
1.11 Pasteurized milk -----------------------------------------------------------------14
1.12. Problems encountered with pasteurization system-------------------------15
1.13 Factors affecting Pasteurized milk quality -----------------------------------16
1.14 Spoilage of pasteurized milk---------------------------------------------------19
1.15 Bacteria survive pasteurization ------------------------------------------------19
CHAPTER TWO: MATERIALS AND METHODS
2.1 Collections of samples -----------------------------------------------------------22
2.1.1 Source of samples --------------------------------------------------------------22
2.1.2 Sampling method ---------------------------------------------------------------22
2.1.3 Samples for bacteriological examination------------------------------------22
2.2 Sterialization-----------------------------------------------------------------------22
2.2.1 Sterialization of equipments---------------------------------------------------22
2.2.2. Sterilization of culture media and solutions--------------------------------23
2.3 Cultural methods ------------------------------------------------------------------23
2.3.1 Cultural techniques -------------------------------------------------------------23
2.3.2. Incubation of culture ----------------------------------------------------------23
viii
2.3.3 Examination of culture---------------------------------------------------------24
2.3.4 Purification of cultures---------------------------------------------------------24
2.4 Identification of Bacteria --------------------------------------------------------24
2.5 Species differentiation based on biochemical reactions
for isolated bacteria -------------------------------------------------------------25
2.5.1. Catalase test---------------------------------------------------------------------25
2.5.2 Oxidase test----------------------------------------------------------------------25
2.5.3. Sugar fermentation-------------------------------------------------------------25
2.5.4 Oxidation fermentation test ---------------------------------------------------26
2.5.5 Citrate utilization ---------------------------------------------------------------26
2.5.6 H Sproduction test --------------------------------------------------------------26
2.5.7 Motility test----------------------------------------------------------------------26
2.5.8. Urease test ----------------------------------------------------------------------27
2.5.9. Indole production --------------------------------------------------------------27
2.5.10 Methyal Red ( MR ) reduction test -----------------------------------------27
2.5.11 Voges – proskauer test -------------------------------------------------------27
2.6 Preparation and formulation of culture media --------------------------------28
2.6.1 Solid media: blood agar base--------------------------------------------------28
2.6.2 MacConkey agar----------------------------------------------------------------28
2.6.3 Nutrient broth -------------------------------------------------------------------29
2.6.4.Pepton water --------------------------------------------------------------------29
2.6.5.Tryptone water -----------------------------------------------------------------30
2.6.6.MR-VP-medium ----------------------------------------------------------------30
ix
2.7.Reagents----------------------------------------------------------------------------31
2.7.1hydrogen peroxide---------------------------------------------------------------31
2.7.2Kovac’s reagent------------------------------------------------------------------31
2.7.3 voges-proskaur (VP test) ------------------------------------------------------31
2.7.4 Lugol’s Iodine-------------------------------------------------------------------31
2.7.5 Nitrate test reagents ------------------------------------------------------------32
2.7.6 Oxidase reagents ----------------------------------------------------------------32
2.8 Staining technique ----------------------------------------------------------------32
CAHPTER THREE RESULTS
3.1 Bacteriological findings----------------------------------------------------------34
3.2 Species of bacteria isolated from packed pasteurized milk samples -------34
3.3 Species of Gram –negative bacteria isolated from
pasteurized milk samples -------------------------------------------------------34
3.4 Species of Gram–positive bacillus isolated from pasteurized
milk samples -------------------------------------------------------------------35
CHAPTER FOUR DISCUSSION ------------------------------------------------50
Conclusion and Recommendation --------------------------------------------------54
REFERENCES. ----------------------------------------------------------------------55
x
LIST OF TABLES
Table no Contents page
1 The Milk chain supply in Uganda 6-7
2 Survival of psychrophiles in pasteurized milk 21
3 Bacteriological screening of packed pasteurized milk samples 36
4 Bacteria isolated from packed pasteurized milk samples 38
5 The results of biochemical reaction for identification of gram – negative
bacteria at genus level
40
6 Identification of enterobacter from different pasteurized milk samples 41
7 Classification of pseudomonas species 41
8 Biochemical test for identification of entero –bacteria species 42
9 Identification of bacillus species in pasteurized milk samples 43
10 Illustrate no of isolate in different factory 44
xi
LIST OF FIGURE
Figure no content page
1 The bacteriological screening of packaged pasteurized milk samples 37
2 Show the number of isolated milk samples 39
3 Show distribution of positive isolation percentage from different factories 45
4 Show the comparative representation of isolate from different factories 46
5 Show the percentage of bacteria isolate from Taza factory samples 47
6 Show the percentage of bacteria isolate from Kenana factory samples 48
7 Show the percentage of bacteria isolate from Best factory samples 49
1
INTRODUCTION
The dairy industry is large and dynamic segment of the agricultural
economy of many nations. Consumption of dairy products continues to
increase thought out the world receipts from milk marketing also increase.
The wide spread of dairy products and well publicized recent
epidemics of animal diseases has increases consumer concern about the
quality of foods of animal origin (Ruegg, 2001).
Highly specialize and intensive operations also produce milk in the
tropics along with feed (Horst, 1996).
Milk is a magnificent medium for growth of microorganisms and
these for the risk for quick microbial deterioration of quality is present from
time of milking to the time of use in the milk plants (International Dairy
Federation IDF, 1994).
There are many steps of fluid milk processing, the important part of
this process is testing and regulating milk supply. Milk and dairy products
should be among most strictly regulated foods because they are very
perishable. Milk must be safe disease free because it acts as vehicle of
infection of many diseases especially for children. So pasteurization of milk
is of significant value.
The main factors affecting the keeping quality of pasteurized milk
are raw milk quality, severity of heat treatment, post-pasteurization
contamination and storage temperature (IDF, 1986).
2
The hygienic quality problems of milk may arise from diseased
animals. Giovannini (1998), reported that milk must therefore, be protected
from contamination and deteriorations from of the production, farms to the
table of the consumers.
Coincident to these trends globalization has influenced the definition
of high quality milk, and consumer expectations are increasing affecting
animal management practices. Milk secretion and milk quality is necessary
to meet evolving consumer expectations (Ruegg, 2001)
In Sudan the distributed milk is never found the real quality control
measures needed to be precious food, as dairy industry in Sudan is week
and at early stage of development.
However, new private factories started processing of fluid milk and
some dairy products. They were faced with so many problems of which
quality control measures constitute an important concern.
Objectives of this study:
1. To detect range of contamination of marketable pasteurized milk.
2. To carry out the isolation and identification of types of bacteria
present in the collected samples.
3. Evaluation of the effect of storage conditions (temperature) on
keeping quality of pasteurized milk.
3
CHAPTER ONE
LITERATURE REVIEW
1.1 Historical background
The fundamental investigation on sources of bacteria in milk was
reported in 1889 by Conn, followed by a report by Russel at Madison in
(1895).
Progress in use of laboratory methods for controlling the sanitary
quality of milk dates from publication in (1892) by Sed wick and
Bactchelder.
Then information on sources of contamination and growth of
bacteria in milk, organized in (1901) by Park (New York City). (American
Public Health Association, 1960). Some bacteria historically associated
with milk include tuberculosis, brucellosis, and typhoid, hence
pasteurization develop to kill these types f bacteria (Winstone, 2003).
1.2 Nutritional value of milk
The value of milk is very clear as usually it’s a main source of
complete protein, calcium, vitamin riboflavin, essential fatly acids and
energy for rabid development of child.
Milk is an excellent source of important minerals, and contains water, fat
solute vitamins and essential trace elements (Foley et al; 1974).
4
Milk is a natural food with clear nutritional value (Faye and
Loseaue, 2002).
1.3 Milk composition
The main milk composition vary considerably depending on
individual animal, its breed, stage of lactation, age, health, herd
management practices and environmental condition (O'connor, 1995).
The gross composition quality is water fat, protein and lactose (Harding,
1999).
1.4 Cow’s milk contains
Water 87.4%
Total solids 12.6%
Solid hot fat 8.9%
Fat 3.7%
Protein whey protein 0.6%
Casein 2.8%
Lactose 4.8%
Minerals 0.7%
(Chandan, 1997)
1.5 Sources of bacteria in milk
Microbial contamination occurs generally from three main sources.
The udder exterior of the udder and from the surface of Milk handling and
5
storage equipment (Bramley and Mckinon, 1990). Hygiene of cow,
environment production, cleaning and storage conditions all influence
microbial number in milk (Murphy and Boor, 2000). They added that
temperature and length of storage time are important that may allow
microbial contamination to reproduce.
Bramley and Michinon (1990) reported that organisms associated
with soil and bedding material include streptococci staphylococci spore
former, coli forms and other gram-negative bacteria. Also they claimed
some microbes associated with environmental mastitis.
Ineffective cleaning hot water temperature and absence of sanitizer
found to be selected for faster growing less heat resistant organisms mainly
gram-negative rods, coli form, pseudomonas and lactic streptococci
(Jackson and Cleg, 1965).
Cows infected with streptococcus aglacia typically shed huge
numbers into raw milk since the anterior udder is only source of this
bacteria (Winston, 2003).
1.6 Source of contamination in milk
There are models to assure product safety in U.S.A Faye and
Loiseau, (2002) mention the results of the qualitative studies conducted by
scientists, for supply chains at milk in Uganda, are shown below
in Table (1)
6
Table (1): The milk chain supply in Uganda
Steps Hazards Risk factors Farms Fecal contamination
Contamination by environmental multiplication of bacteria on milking material. Contamination by pathogen bacteria Contamination by chemical residue Lypolysis and raw milk turning rancid Proteolysis , jellification Of UHT milk decreasing of cheese yield, appearance of sour component
Inhibition of lactic fermentation. Problem for milk processing
Transmission by hands and animal during milking. Inefficient cleaning and Disinfecting of material and or poor drying Healthy carrier animals Non respect of waiting time for veterinary medicine treatment Frequent and brutal decanting Collect milk with mastitis. Collecting milk from animals treated with antibiotics
Transport Growing of microbial flora Contamination by material
Carrying time too long at high temperature Inefficient cleaning and , or bad drying
Collecting Centers
Cross contamination
Cleaning and bad quality control of milk marketing
7
Continue table (1)
Dairy Plants
Human contamination Contamination by environmental germs Development of Psychotropic flora Development of coliform flora Lypolysis Cross contamination Recontamination by environmental germs Persistence of microorganism
Hand contamination with milk at time of sampling. Use of contaminated water for cleaning Temperature of cooling not regulated and lengthy storage Absence of cooling Manual filling of the tank from the top Bad quality control of milk Poor hygiene at packaging, absence of thermal treatment Consumption of contaminated raw milk Poor quality, high temperature and too long reservation
8
1.7 Major types of bacteria in milk
Bacteria of importance in milk categorized into three groups by
Tanwani and Yadera (1983). Organisms excreted in milk include
streptococci staphylococci, Brucella, Mycobacterium, Salmonella Listeria,
Candida, Anthrax, and Bacillus, Coryne bacteria Cryptococcus, Coxiella
and Nocardia.
Organisms of outside origin Bacillus, Escherchia, lactobacillus,
clostridium streptococcus, salmonella, pseudomonas acetobacter and
alcaligens.
Organisms, which excrete toxins: staphylococcus, streptococcus
Escherchia, clostridium and bacillus.
1.8. Pathogenic bacteria in milk
Outbreaks of diseases associated with milk and dairy products are
reported recently less than 10 years (American Public Health Association
A.P.HA, 1960) in United States an out break was caused by salmonella spp.
in commercial milk supply Shigella sonei a milk born out break (A.P.H.A)
England specific types of Escherichia coli causing infant diarrhea (A.P.H.A
1960) the presence coxiella bruentii has also been demonstrated.
Brucella species also are known to be pathogenic for man.
Brucellosis is one of milk borne diseases in many parts of Sudan (Mustafa
9
and Idris, 1976). They added that staphylococcus poisoning out break trace
able consumption of raw or boiled milk products were not rare.
Lund et al., (2002). Reported that mycobacterium avium subspecies
tuberculosis contributes to crohn's disease in human .
Bacteria types associated with milk
Bacteria Effect on milk
Pseudomonas Spoilage
Enterbacteriace Pathogenic and spoilage
Staphylococcus aureus Pathogenic
S.agalaciae Pathogenic
S.thermophilus Acid production
L.lactis ho. Acid production
L.lactis diacelylactis Flavor production
L.cremoris Acid production
Leuconostoc lactis Acid production
Bacillus cereus Spoilage
L.lactis Acid production
L.bulgaricus Acid production
L.acidophilus
Propaini bacterium Acid production
myco bacterium Tuberculosis pathogenic
International livestock research institute, Ethiopia, 1995.
10
1.9 Milk quality control evaluations
1.9.1 Microbial measurement
Measurement of bacterial numbers in milk is of interest because of
their direct role in milk spoilage and because they are indicator of poor
hygienic production process or infective pasteurization (Harding, 1999).
1.9.1.1 Bacterial count
A significant milk quality concern is bacterial in milk, potentially
represents a public health concern in addition to milk quality concern,
(Winiston, 2003).
The following counts performed are
1.9.1.1.1 Standard Plate Count (SPC)
Bacteria numbers in milk are determined by testing samples that are
collected. Sampling is mandatory, test result; provide an estimate of the
total number of bacteria present in the sample and the number is expressed
as bacteria /ml. A typical number for example may be 9000/ml. This
number represents the number of bacteria that had entered the milk from all
possible sources.
1.9.1.1.2 Additional tests procedures
Two other routine bacteria tests are often used in addition to SPC in
an effort to determine it the source of bacteria in milk is infected cows, dirty
cows or dirty systems.
11
1. 9.1.1.2.1 Preliminary incubation count (PI)
The preliminary incubation PI count attempts to determine the
presence of bacteria that tend to grow in cold condition such bacteria
originate from sources outside the udder.
PI count generally higher than when significantly higher 3-4 times it
suggests soil related bacteria failure to cool milk adequately and quickly
provide favorable conditions for bacteria to grow like pseudomonas.
It may be possible for some streptococcus non agalciae to elevate PI
when shed from infected quarters.
But typically elevated PI related to external sources. PI count
should be 20,000 per ml less.
1.9.1.1.2..2 Laboratory pasteurized count (LPC)
The LPC test determine the presence of bacteria that can survive
exposure to temperature of 145Fº for 30 minutes these temperatures kill all
typical mastitis causing bacteria, but certain environmental species may
survive grow and produce damaging enzymes.
LPC count should be less than 100/ml it elevated look at locations in
the system that failed to clean adequately.
1.9.1.1.2..3 Bacteria species evaluation
An additional help milk quality evaluation involves bacteria species
this evaluation determines the predominant bacteria species.
12
a) Streptococcus, aglacia: infected cows shed huge numbers into
raw milk since the interior of udder is the only source. This
bacteria is found in any quality.
b) Environmental streps: (Strep. Non-aglaciae species) when
different counts indicate high numbers of streptococcus, non, ag-
species it may represents several different issues. Cows with
bacteria or these bacteria thrive in the environmental of cow. A
target or goal for strep-non-ag species in bluk milk should be less
than 750/ml elevated counts require a look for infected cow or
environment. Inadequate cooling also is a factor. Milk held around
temperature 45 ºF for couple of hours allows these bacteria to grow
rabidly.
c) Cloiforms: coliforms are another group of bacteria that show up
in milkcoliform include group of genera like
Escherchia,Citrobacter ,Enterobacter and Klebsila (IDF, 1994).
The coliform count in raw milk should be less than 100/ml, and the
count typically is much less when things done properly coliforms count
100/ml indicates dirty cows being milked.
1.10 Pasteurization
The treatment of food beverage with mild heat, irradiation or
chemical agents to improve keeping quality or in active diseases causing
micro-organisms, when originally louis Pasteur observed spoilage of wine
13
and beer could be prevented by heating them to 122-140ºF for a few
minutes. Pasteurization as defined by IDFC (1994) is the heat treatment
process applied to a product to a void public heat hazard arising from
pathogenic micro-organism associated with milk.
1.10.1 Methods of pasteurization
There are two widely used methods of pasteurization according to
FAO (1984):
High temperature short time, (HTST) and ultra high temperature
short time, HTST: holding milk at temperature 72ºC (161ºF) for at least 15
seconds Ultra high temperature (UHV): involves holding milk at a
temperature 138ºC (280ºF) for a few seconds.
Pasteurization methods are usually standardized and controlled by
National Food Safety agencies in the United States, and Food Standard
Agency in United Kingdom.
HTST process must be designed so that the milk is heated evenly.
This milk has shelf life of two to three weeks.
UHT pasteurization is combined with sterile handling and container
technology. It can then store un refrigerated for long period of time.
Batch pasteurization involve heating large batcher of milk to lower
temperature 68ºC 155ºF other techniques called.
Higher heated short time: (HHST)
These lies between HTST and VTH
14
1.10.2 Objective of pasteurization
Pasteurization is of commercial value in reducing the total number of
bacteria and increasing the keeping quality of milk, it should not however
be considered a substitute for effort to produce high quality milk
(Henderson, 1971).
International Dairy Federation IDF applied it to milk with objective
of minimizing possible health hazard organizing from pathogenic micro-
organisms associated with milk.
Zall (1990) reported that the original objective of pasteurization to
prevent milk acting as carrier of human pathogen.
1.11 Pasteurized milk
Pasteurization as to applied fluid milk consists of heating milk to a
temperature sufficiently high and for a time sufficiently long to kill most of
the bacteria, the temperature based on the destruction of pathogenic bacteria
(Enright et al., 1957).
Pasteurization combined with refrigeration of milk is most
commonly used. Pasteurization does not kill spore but eliminates
pathogenic bacteria has been historically associated with milk
Pasteurization does not prevent spoilage, but milk can be essentially
sterilized by ultra high temperature UHT, in which milk heated to higher
temperature than normally used around 142ºC-150ºC (Sawaisgood, 1985)
15
for 3-6 second. This milk can be stored for months at room temperature
without spoilage.
Gallardo et al., (1998) tested 10 samples of pasteurized UHT treated
milk and sterilized milk. All conformed to the standard for mesophilic
aerobes. Enterobacteriaelae, Coliform, Escherchia coli, Salmonella,
Shigella Staphylococcus aerus, however, proteus spp. were detected in one
sample.
(Nadir et al., 1997) analyzed 140 pasteurized milk samples for
mesophilic bacteria and total coliform counts. He found 28 samples
(20.0%) were out side the legal standard, 19 samples 13.1% contaminated
by fecal coliforms (Guzmacher et al., 1999) reported that gram negative
psychotropic microorganism are destroyed by pasteurization and their
presence is due to post pasteurization contamination. A total of fifty
pasteurized milk samples were collected from two factories in Khartoum by
Nasshwa (2002). She found that an average of Bacillus species from all
samples and that was the largest isolated percentage (41.7%).
Baderia (2006) examined thirty packaged pasteurized milk samples
from Khartoum State shops, of which 26 samples (86.7%) of milk were
found to be positive for the percentage of bacteria.
1.12. Problems encountered with pasteurization system
(Sandra et al., 2003). Tightly controlled laboratory studies may have
demon started the efficiency of pasteurization to destroy various pathogens,
16
however some problems that could interfere with pasteurizer performance
may include: (Sandra et al., 2003)
I. Start with poor quality milk with a high degree of bacterial
contamination
II. Milk not heated to the correct target temperature HT ST 161 ºF , due
to no enough hot water available or in adequate plate cooler
function, pasteurizer mal function or not calibrated properly.,
cleaning failure build up of fat, protein or in organic films wile enter
here with heat transfer.
III. Milk is not maintained at target temperature for long enough
duration due to HTST, milk not circulated for full 15 second.
Operator error.
IV. Curding of milk it fermented (acid PH). Chill raw milk
V. Post pasteurization contamination of milk.
1.13. Factors affecting pasteurized milk quality (IDF, 1986)
1. Hygienic quality of raw milk
Processing and storage conditions, high temperature short time
thermal pasteurization is able to extend shelf life of milk around three
weeks, depending on initial microbial quality of raw milk (Richert et al.,
1992). Harding (1999) mentioned that when milk is stored at ambient
temperature and collected in cans, mesophilic bacteria which produce lactic
acid tend to predominate. Zall (1990) reported that. psychotrophs in raw
17
milk, pre-processing could be critical factor in keeps quality of pasteurized
milk.
2. Heat treatment and processing
Dumalisile et al., (2005) reported that different pasteurization
methods such as low temperature long time (LTLT). High temperature short
time (HTST) and post pasteurization play an important role in the survival,
distraction of different bacteria, in inoculated in UHT and were reported ,
survive after heating up to 30 minutes. Milk processed at 76ºC had the
lowest bacterial growth rate. Processing of milk with pulsed electric field
immediately after (HTST) pasteurization extends shelf life more than two
weeks (Simon and Hansen, 2001).
3. Packing
Mesophilic and psychotrophic bacterial count of milk were recorded
for milk samples in all packing materials for a given sampling during 17
day storage period (Zygoura et al., 2004). Flavor deterioration of packed
pasteurized milk were faster in standard milk, than packed barrier and foil
boards (Simon and Hanson 2001). Gram negative psychotropic bacteria had
grown to high level in various numbers for consumer packages after 7-11
days of incubation.(Eneroth et al., 2002)..
18
4. Cleaning and sanitation of plant and equipments
It is essential that contaminations from equipments used between the
cow milking and refrigerated storage unit be kept to a minimum level
(Harding, 1990).
Refrigerated storage unit be kept to a minimum level (Harding,
1990). Murphy and Boor (2000) reported that milk residue or equipment
support growth of micro-organisms. They also stated that cleaning and
sanitizing leave residual soil on equipments could increase number and
types of microbics which can growth on milk contact surfaces. Found that
pasteurizer machine could be a source of contamination when in adequately
cleaned or maintained. Also filling machine was a significant source of
contamination. However, proper cleaning followed by sanitizing with
chlorine increase milk shelf life.
5. Types of micro-organisms
There are many organisms mainly bacteria which have access to
milk which are classified to three temperature ranges according to their
optimum growth rate requirement psychophile optimum growth rate t
temperature (0-15ºC). mesophile need (20-40ºC) and a high temperature
(45-55ºC) is for the thermopiles. (Harding, 1999).
19
6. Storage conditions
Pasteurized milk storage affects bacterial growth rate, also storage
period, batches and location played significant roles in the bacterial growth
rate (Elmagli, 2004).
1.14. Spoilage of pasteurized milk
Pasteurized milk spoilage is either due to post pasteurization
contaminaton or growth of organisms that have survived heat treatment,
where as flora at spoilage is dominated by gram-negative rods.
Post pasteurization contamination is indicated with the presence of
gram positive organisms suggest that spore forming bacteria have survived
heat treatment (Banks and Dalgleish, 1990).
The most common spoilage micro-organisms are bacteria of gram-
negative rods.
Pseudomonas sp, coliforms, and gram+ve bacillus clostridium spp
and streptococcus spp (IDF, 1994).
1.15. Bacteria survive pasteurization
Two types of bacteria resist pasteurization at 145ºF which are
thermoduric and thermophilic thermodrric are spore former or non spore
former which resist temperature (Henderson, 1971).
Thermophilic bacteria can cause bacteriological problem in milk
which under goes pasteurization at 145ºF, in addition to spore forming
bacilli lactobacillus thermophilus also may in countered. Psychrophilic
20
bacteria which grow at low temperature most encountered genera are
pseudomonas. Achromobacter, flavi bacterium, and alcaligenes. Coliform
bacteria, can grow bellow 50º Ftheir presence is due to post pasteurization
problems psycophilic casue rancid flavor and odors.
In milk (American Public Health Assoiation, 1960). On going
researches lead to discovery of pathogens able to survive pasteurization, in
particular mycobacterium avium sub spp. paratuberculosis (MAP) which
cause John's disease in cattle of cause Crohn’s disease in human , found in
USA and UK MAP in USA also found due to post pasteurization
contamination.(Lund et al., 2002)
In an experiment done for behavior of listeria monocytogenes in
pasteurized milk at different storage temperatures, it was found that at
survival of L.monocytogenes at different in storage temperature.
pasteurized milk . Annamalai et al., 2002).
The effect of pasteurization on survival in milk of four strains of
psychrophilic pseudomonas and one strain of alcaligenes was determined
by Macau Lay et al., (1963). They reported that the survival of
psychrophilic bacteria in pasteurized milk depends on the initial number of
organisms and partly on length of time of storage at 3-5ºC as shown in the
Table (2) bellow.
21
Table (2): Survival of psychrophiles in pasteurized milk:
Initial no. cells per ml Culture
106 105 104 103 102 10
p. fluorescens 10038 + + + - - -
P.flurescens 9428 + + + - - -
P.flonrsucence 11251 + + + + - -
A. tolerance + + + + + +
Symbol += growth when subculture after heating
- Absence of growth when subculture after heating
22
CHAPTER TWO
MATERIALS AND METHODS
2.1 Collection of samples
2.1.1 Source of samples
A total of sixty pasteurized milk samples were collected from market
in Khartoum State. From four different factories.
2.1.2 Sampling method
Five (5) types of packed pasteurized milk were collected. The
samples were placed into iceboxes then transported directly to the
laboratory. Samples were examined for bacterial isolation and
identification.
2.1.3 Samples for bacteriological examination
The samples were treated as follows:
The milk packets were washed first then wiped with cotton dipped in
70% alcohol, then using sterile syringe to take 93 ml milk sample and
placed in a sterile vial.
2.2 Sterilization
2.2.1 Sterilization of equipments
Sterilization of glass-were such as Petri-dishes test tubes, flasks and
pipette performed in hot air oven at 160°C for 1.5 hours. Other glass-were
as Macerates bijou and universal bottles were sterilized by autoclaving at 15
23
Ib/inch2 at 121°C 15minutes. Instruments such as spatula, forceps and loops
were sterilized by flaming after dipping in alcohol.
2.2.2 Sterilization of culture media and solutions
Culture media such as blood agar base, MacConkey, and Nutrient
agar were sterilized by autoclaving at 15Ib/inch2 pressure at 121°C for
minutes.
Carbohydrates media were sterilized by steaming for 30 minutes on
three successive days.
2.3. Cultural methods
2.3.1 Cultural techniques
Solid media: a large loop (0.4 cm) was sterilized by flame and bijou
vial was opened sterilized by flame and sterile loop was used to steak onto
plates of solid media that was used. For sub-culturing into liquid medium a
colony was picked up with sterile wire loop and was put into the medium.
Inoculation of solid and semi solid media
For further culturing on solid media wither the sample source was
solid or liquid, culturing was done by stabbing or streaking.
2.3.2 Incubation of culture: (Cowan and Steel, 1975)
All culture media, solid, semi-solid or liquid media were incubated
aerobically at 37°Cfor 8-24 hours, except for MR-VP, indole media which
were kept for 48 hours and urease activity for 5 days.
24
2.3.3 Examination of culture
All culture on solid media was examined with the naked eye for
growth, colonial morphology and change in color.
2.3.4 Purification of cultures
Purification was done by the selection of a single discrete colony and
sub-culturing into liquid to solid media until purified. Gram’s staining
checked the purity of new culture.
2.4 Identification of bacteria
The identification from purified isolated colony was used in
accordance to the outline set by the manual of Cowan and Steel, (1975), as
based on the genus criteria, which include:
1. Reaction to gram’s stain
2. Shape of the organisms
3. Presences or absence of spore
4. Motility
5. Catslase acclivity
6. Oxidase production
7. Colonial characteristic on different media and heamolysis on
blood
8. Aerobic or anaerobic growth
9. Oxidation fermentation test
25
2.5 Species differentiation based on biochemical reactions for isolated
bacteria Cowan and Steel, (1975)
The biochemical tests were prepared and performed as instructed in
manual and the following tests were performed accordingly:
2.5.1 Catalase test
The isolated bacteria grown on blood agar and one drop of 3%
hydrogen peroxide solution were placed on he surface. The immediate
evaluation of gas within five minutes indicated. Catalase activity.
2.5.2 Oxidase test
A piece of filter paper was placed into a clean Petri-dish and 2-3
drops of 1% tetramethyl-p-phenyl enediamine dihydrochloride were then
placed on the paper. Then the organism tested was picked, smeared on
impregnated paper, development of dark purple color indicates a positive
result within seconds.
2.5.3 Sugar fermentation
Peptone water medium was used as the basal medium for
carbohydrate fermentation with the addition of Andrade’s or bromocresol
purple as indicator. The cultures were examined daily for seven days be for
they were discarded.
Change of color to red indicates positive reaction in case of
Andrade’s reagent and yellow in case of bromocresol purple.
26
2.5.4 Oxidation fermentation test O.F
Two tubes of Hugh and Leifson, media were inoculated with the
tested culture, one being covered with a layer of sterile soft paraffin to
depth of about 1-2 cm. they were incubated at 37°C and examined daily up
to 14 days. Fermentation indicated by change in both tubes (Barow and
Feltham, 1993).
2.5.5 Citrate utilization
Koser’s citrate medium was indicated with the tested organism and
inoculated with the tested organism and incubated at 37 °C for up to 48
hours. Positive result for citrate was recognized by bluish color due to
ammonia production. Negative tests were examined after further incubation
for up to 14 days.
2.5.6 H2 S production test
A tube of peptone water inoculated, and lead acetate paper was as
inserted between the pulge and the tube. The tubes were examined daily for
7 days for blackening of paper due to H2S production.
2.5.7 Motility test
A small piece of the colony of bacterium need to be tested was picked
by the end of the straight wire and stabbed in the centre of the semi-solid
agar in the tube, this preparation incubated at 37 °C over night motility was
detected by turbidity of the medium in the tube.
27
2.5.8 Urease test
A drop of christensen’s urea medium was heavy loaded with the
tested organism. A positive result was shown by the development of red
color.
2.5.9 Indole production
Peptone water medium was inoculated with the tested organisms and
incubated at 37°Cfor 48 hours. A positive result was indicated by red color,
after addition of 0.5 ml Kovac’s reagent.
2.5.10 Methyl Red (MR) reduction test
Glucose phosphate medium was inoculated and incubated at 37°C
for 2 days. Two drops of methyl red solution were added, shaken well then
examined. Red color indicates positive result where yellow color indicates
negative result.
2.5.11 Voges-proskauer test
Glucose phosphate medium was inoculated at 37°C for 48 hours then
three ml of 5% naphthol solution were added to the culture followed by 1
ml of 40% potassium hydroxide. The medium was shaken well and left.
After 15 minutes and one hour the medium was examined for development
of pink color. That indicates positive reaction due to production of Acetyl
methyl carbinol.
28
2.6 Preparation and formulation of culture media
2.6.1 Solid media: blood agar base (oxoid cm 55) g/l
Formula
Lab lem co beef extract 10
Peptone (oxoid L 37) 10
Sodium chloride 5
Agar 15
PH 7.5 (approximately)
This medium was obtained from oxoid ltd. (England) it was prepared
according to manufactures instructions by dissolving 40 grams of the
dehydrated medium in one liter of distilled water, and then sterilized by
autoclaving at 15Ib /inch2 at 121 °C for 15 minutes. Then the medium was
cooled to 50°C defibrinated sterile sheep blood was added at the rate of 5-8
percent and mixed with gentle rotation. The medium was poured into Petri-
dishes.
2.6.2 MacConkey agar (oxoid cm 7)g/L
Formula
peptone (oxoid 137) 20
lactose 10
bile salts (oxoid L33) 5
sodium chloride 5
neutral red 0.075
agar 12
PH 7.4 (approximately)
29
This medium was prepared according to manufacture’s instructions
by dissolving 45 grams in one liter of distilled water then sterilized by
autoclaving for 15 minutes at 15 Lb/inch2 (121°C) and poured into Petri-
dishes.
2.6.3 Nutrient broth g/L
Formula:
Lab. Lemoco, beef extract 10
Peptone (oxoid L37) 10
Sodium chloride 5
PH 7.5 (approximately)
According to manufactures instructions 25 grams of medium was
dissolved in one liter of distilled water then distributed into universal bottles
and was sterilized by autoclaving at 15Lb/inch2 at 121°C.
2.6.4 Peptone water (oxoid cma)g/L
Formula:
Peptone (oxoid L37) 10
Sodium chloride 5
PH 7.2 (approximately)
15 grams of medium was dissolved in one liter of distilled water
according to intimations then distributed in 5ml amounts. The medium was
sterilized for 15 minutes at 15 Lb/inch2 at 121°C.
30
Peptone water sugar (Barrown and Felthman 1993). This medium
contains 900 ml peptone water 10 of andrades reagent. PH was adjusted to
7.1-7.3 before sugars were added. The medium was sterilized by
autoclaving for 15 minutes.
Specific sugar was added by dissolving 10 grams in 90 ml of distilled
water, sterilized by steaming then the sugar solution was added to peptone
distributed into sterile test tubes 5 ml then sterilized by autoclaving for 15
minutes.
2.6.5 Tryptone water (Oxoid cm 87)
(indole production medium) g/L
Formula:
Tryptone (oxoid L42) 10
Sodium chloride 5
PH 7.5 (approximately)
The medium was prepared according to manufacture. Instructions.
Fifteen grams were dissolved in one liter of distilled water. Then distributed
into test tubes and sterilized by autoclaving at 15Lb/inch2 at 121 °C for 15
minutes.
2.6.6 MR-VP- medium (oxoid cm 45) g/L
Peptone (oxoid L99) 5
Dextrose 5
Phosphate butter 5
PH 7.5 (approximately)
31
The medium was prepared according to manufactures instructions by
dissolving 15 grams one liter of distilled water.
The medium was distributed into tubes and sterilized by autoclaving
at 15Lb/inch2 at 121 °C for 15 minutes.
2.7 Reagents
All reagents obtained from (B.D.H). British Drug House Chemicals,
UK
2.7.1hydrogen peroxide (B.D.H.UK)
Is composed of .04 grams methyl red, 40 ml alcohol and 100ml
distilled water. The methyl red was dissolved in ethanol and then diluted by
addition-distilled water for MR test.
2.7.2 Kovac’s reagent
This reagent contains paradimethylamino benzalehyde in 5 grams
amyl alcohol at 50°C-55 °C, then cooled and the acid was added carefully.
The reagent was stored at 4C° and used for indole.
2.7.3 Voges- proskaur (VP test)
Reagent A: 5% Alpha-naphthol in alcohol
Reagent B: prepared by 40% aqueous. Solution Na OH.
2.7.4 Lugol’s lodine g/L
Iodine
Potassium lodide 10
Distilled water 100ml
32
The Iodine and potassium lodide were dissolved in some of distilled
water and adjusted to 100 ml with distilled water. It was used for grams
stain.
2.7.5 Nitrate test reagents (B DH U K).
a. Solution A: it is prepared by dissolving of 0.33% sulphanitic acid in
one liter of 5 N acetic acid by gentle heating.
b. Solution B: 0.6% dimethyl-alphanaphthol in 5 N. acetic acid
c. Zinc dust: oxidase reagents 1% Alpha-naphthol in 95% ethanol.
2.7.6 Oxidase reagents:
1% alpha-naphthol in 95% ethanol.
2.8 Staining technique:
Smears were made from culture media in clean sterile slides. The smear
were fixed and stained as follows: Gram’s stain (Cowan and Steel, 1975).
This stain was performed according to the manual.
a. Crystal violet solution was applied for 1.5 minutes.
b. Then the slide was washed with distilled water.
c. Lugol’s lodine solution was applied for 0.5 minute
d. Then the lodine solution was tipped off without washing
e. The slide was decolourized with few drops of acetone
f. Then the slide was washed thoroughly with distilled water
33
g. The slide was counter stained with dilute carbol fuchsine for 0.5
minute. The slide was washed with distilled water, drained and
blotted for drying.
Gram positive organisms appear violet where as gram negative
organism appeared red.
34
CHAPTER THREE
RESULTS
Sixty pasteurized milk samples were collected from different shops
in Khartoum State within the period of September-December 2006. The
milk samples were subjected to general bacteriological examinations.
The samples that were positive were shown in Table (3) and Figure (1).
3.1 Bacteriological findings
As shown in Table (3) and Figure (1)21 of the sample (35%) were
positive for bacteria were as 39 samples (65%) were negative for bacterial
growth.
3.2 Species of bacteria isolated from packed pasteurized milk samples
The species of bacteria that were isolated are shown in Table (4)and
Figure (2) The total number of isolates was 21, (35%) the isolates were five
different bacterial species three were gram-negatives while the other two
species were positive bacilli.
3.3 Species of gram-negative bacteria isolated from packed pasteurized
milk samples
Cultural characters and biochemical properties of enterobacter
aerogenes and proteus mirabilis were shown in Table (8).
35
3.4 Gram-positive Bacillus spices isolated from pasteurized milk
samples
Two bacillus gram-positive species were isolated as shown in Table
(9). Bacillus cerus and Actinobacillus were identified.
Table (7) shows identification of pseudomonas species that were
isolated from pasteurized milk sample.
Figure (5), (6) and (7) showing the percentage of bacterial isolate
from different factories.
There was Variation between Deferent factories in the No: of
isolation it was shown in Table (10) and Figure (3)
36
Table (3): Bacteriological screening of packed pasteurized milk
samples from Khartoum State (Sep. Dec. 2006)
Item /test Positive Negative Total
No. of sample 21 39 60
As percent % 35% 65% 100%
37
39
21
NegativePositive
Figure (1): The bacteriological screening of packaged pasteurized milk
samples.
38
Table (4): Bacteria isolated from packed pasteurized milk samples
from shops in Khartoum State
Species No. of isolates Percent%
Enterobacter aerogenes 9 42.85%
Prateus mirabilis 2 9.52%
Pseudomonas aeroginosa 3 14.28%
Bacillus cereus 5 23.28%
Actinobcillus lignersii 2 9.52%
39
No. of Isolate from total milk sample
00.05
0.10.15
0.20.25
0.3
Ent
roba
cter
aero
gene
Bac
ilus
ceru
s
Pro
teus
mira
bilis
Pse
udom
onas
aeru
gino
sa
Act
inob
acill
uslig
nier
sii
No. of Isolate from total milk sample
Figure no. (2): The number of isolate from total milk samples
40
Table (5): The results of biochemical reactions for identification of
gram-negative bacteria at genus level
Genus of
bacteria
Gram
reaction
Motility Growth
on air
Catalase Oxidase O/F Glucose
Enterobac
teria -ve rods + + + - F +
Pseudomo
nas -ve rods + + + + O +
Proteus -ve rods + + + F -
41
Table (6): Identification of enterobacter from different pasteurized
milk samples
Test Result
Shape and gram’s stain Gram –ve rods
Sugar fermentation +
Catalse +
Oxidase -
H2S -
Citrate utilization +
Urease +
MR -
VP +
H2O2 +
Table (7): Classification of pseudomonas species
Species Gram
stain Motility Oxidase Catalase Of Glucose lactose Urease H2O2 Sueroes
Ps. - + + - 0 + - + + -
42
Table (8) Biochemical tests done for identification of entero-bacteria species (Ent-aerogenes and proteus) according to
Cown and Steel, 1975.
Seci
es
Gra
m st
ain
Mot
ility
O.F
Oxi
dase
Cat
alas
e
Gbu
cose
Xyl
ose
Lact
ose
Man
itol
Sucr
ose
Citr
ate
Ure
ase
MR
VP
H2O
H2O
2
Enterobacter
Aeragenes
- + F - - + + + + + + + + + - +
Proteus Mirabili - + F + - - - - - - - - + + - +
43
Table (9): Identification of bacillus species in pasteurized milk samples
B.species Gram
reaction
Motility O/F Oxidase Catalase H2O2 Citrate Indole VP
Bacillus + - F + + + + - +
44
Table (10): Illustrate no. of isolate in different factory
Factory No. of samples No. of isolate
Kenana 20 16
Best 10 4
Taza 10 1
Diama 10 0
Capo 10 0
Total 60 21
45
16
1
4
10 10
4
9
6
0 00
2
4
6
8
10
12
14
16
18
Kenana Taza Best Daima Capo
PositiveNegative
Figure No. (3): The distribution of positive isolation percentage from
different factories
46
0
2
4
6
8
10
12
14
16
18
Kenana Taza Best Daima Capo
PositiveNegative
Figure No. (4): The comparative representation of isolate from
different factories
47
Taza
10%
90%
Positive Negative
Figure No. (5): The percentage of bacterial isolate from Taza Factory
Samples
48
Kenana
80%
20%
Positive Negative
Figure No. (6): The percentage of bacterial isolate from Kenana
Factory Samples
49
Best
40%
60%
Positive Negative
Figure No. (7): The percentage of bacterial isolate from best Factory
Samples.
50
CHAPTER FOUR
DISCUSSION
Successful operations of both control agencies and individual
members of the fluid milk industry depend fundamentally on production,
maintenance and sales to consumers of milk with low bacterial content
(American Public health Association , 1960).
So the heat treatment of milk prior to packaging is an important
critical control point to ensure that spoilage organisms are eliminated or at
least reduced in number for optimum keeping quality more over its use
among infants elders and disable persons makes it’s public health
importance a major issue (IDF, 1994).
This present investigation designed to study possible causes of
packed pasteurized milk contamination that was marketed in Khartoum
State.
The samples, were cultured in blood and MacConkey agar and sub-
cultured in nutrient agar.
According to morphological and biochemical reactions enterobacter,
proteues, pseudomonas, bacillus and actinobacillus species were found and,
studied.
From samples only 35% of the samples were positive for presence of
bacteria.
51
Isolation of bacteria from pasteurized milk samples, can be attributed
to poor hygienic production or ineffective pasteurization of milk (Harding,
1999).
Contamination of samples may occur because of the health and
hygiene conditions of the of the cow, the environment in which the cow is
housed and milked, the procedures used in cleaning and sanitizing the
milking, storage equipments, temperature and length of storage all influence
microbial numbers in raw milk (Murphy and Boor, 2000).
Enterobacter aerogens which was gram negative lactose fermenter
entero-bacteria constitutes the highest percentage (42.85%).
Its presence indicates fecal contamination. It was found only in
Kenana pasteurized milk, and one sample in Taza milk. Other species of
bacteria isolated from packed pasteurized milk were pseudomonas
(14.28%), proteus sp. (9.52%), Bacillus (23.85%), and actiono bacillus
(9.52%).
The presence of pseudomonas aerogenosa species it can be
attributed to the tact that such bacteria grows well in cool temperatures, or it
may entered the product in water contamination with pseudomonas spp. is
often one of the components for contamination of water in resident areas
(Winston, 2003).
The reporting of pseudomonas sp. in this study agreed with the
findings Badria, (2006). Proteus spp. which is an enterobacteria was found
52
at a level of (42%). P.mirablis which is a non lactose fermenter, known by
characteristic swarming on MacConkey, was also reported by James (1986)
and Baderia (2006).These are three types of coliforms which are
enterobacter, proteus and pseudomonas their presence indicate dirty cows
or dirty areas in milking system. Bacillus cerus was isolated at a rate of
(23.85%). Bacillus, which was gram-positive rods, cause spoilage of milk.
Their presence suggested that they survive in milk or buildings in the
system, and develop resistant which can help them to stand pasteurization.
Isolation of Bacillus spp. in this study agreed with the findings of
Nashwa (2004), which reported the highest percentage bacillus over all
isolates.
In some factories no bacteria isolation was reported. This may be
due to small numbers of samples or UHT milk which is essentially sterile.
On the other hands pasteurized milk have living bacteria or bacterial spores
in it. They are heat resistant. This milk thus will spoil as these bacteria
germinate.
Hence the sterile milk has long shelf life and can be kept at room
temperature, pasteurized milk will be affected by refrigeration.
Also a Sudden unexpected spike and return normal, may lead to
cooling problems failure of cleaning system sanitizing failure or milking
wet cows. may lead to contamination and bacterial growth. Coliforms
bacteria are linked to manures.
53
Actinobacillus aerogenos was reported at 9.52º level which may
enter milk from various sources in producing farms, and may attributed to
residues on surfaces of pasteurizing equipment.
The presence of, bacilli or actinobacili, also due to stagnant milk at
pasteurization temperature, and foam round in distribution pan of cabinet
cooler also play a role in this process. (American Public Health association,
1963).
Presence of coilforms, and pseudomonas in pasteurized milk. Also
indicates post pasteurization contamination. The equipments , residues on
improperly sanitized equipments, splashing from floors or equipments,
condensate dipping and similar sources play a role in the contamination of
pasteurized milk.
54
CONCLUSION AND RECOMMENDATION
From results of this study
1. Some of the factories distribute low quality pasteurized milk in
Khartoum State.
2. The presence of physophilic bacteria indicated that both heat
treatment and storage temperature need to be revised
3. Pasteurization at UHT plays an important role in survival and
destruction of different bacterial contaminations
Recommendations
1. Examination of factories should be carried out regularly to ensure
clean milk production and supply
2. Raw milk should be of low bacterial load and free from pathogenic
and spoilage microorganisms and chemical contaminants.
3. Improvement of processing, storage condition and marketing of
dairy products.
4. A well equipped quality control laboratory should be established
beside efficient staff has to be employed
5. The official authorities should implement quality assurance
programs and regular monitoring.
6. Further work in needed to evaluate the conditions of extended shelf
life of dairy products.
55
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