Food Microbiology Microorganisms in Food Food Preservation Food-borne Illness Fermented Foods.
MICROBIOLOGY OF TRADITIONAL FERMENTED MILK PRODUCT (ROUB) · MICROBIOLOGY OF TRADITIONAL FERMENTED...
Transcript of MICROBIOLOGY OF TRADITIONAL FERMENTED MILK PRODUCT (ROUB) · MICROBIOLOGY OF TRADITIONAL FERMENTED...
MICROBIOLOGY OF TRADITIONAL FERMENTED MILK
PRODUCT (ROUB)
By
SALAH IBRAHIM KHIDER HUSSAIN
B.Sc. (Hon.) Animal Production Sudan University of Science and Technology
)2002(
A Thesis
Submitted in Partial Fulfillment for the Requirements
of the Degree of Master of Science
In Tropical Animal Production
Supervisor:
Dr. Mohamed Osman Mohamed Abdalla
Department of Dairy Production
Faculty of Animal Production
University of Khartoum
October, 2009
ب
ACKNOWLEDGMENT
All grateful to Allah for the assistance, health, patience and will to
accomplish this work.
I would like to express my gratitude and thanks to my supervisor
Dr. Mohamed Osman Mohamed Abdalla for his supervision, help and
proper guidance during all the stages of this research.
Also my thanks are extended to all members of Department of
Dairy Production, Faculty of Animal Production, University of Khartoum
who helped me.
My thanks to everyone who helped me in my work.
ج
LIST OF CONTENTS Title Page DEDICATION i ACKNOWLEDGMENT ii LIST OF CONTENTS iii LIST OF TABLES v ABSTRACT vi المستخلص vii CHAPTER ONE: INTRODUCTION 1CHAPTER TWO:LITERATURE REVIEW 3 2.1 Fermentation and fermented milk products 3 2.1.1 Fermentation: Definition of fermentation 3 2.1.2 Origin of fermented milk 32.1.3 The fermentation of milk 4 2.1.4 Types of fermented milks 5 2.2 Health effects of fermented foods 5 2.2.1 Probiotic effect 5 2.2.2 Flatulence reducing effect 7 2.2.3 Anticholesterolemic effect 7 2.2.4 Effect on transit time, bowel function and glycemic index 7 2.2.5 Anticarcinogenic effect 8 2.2.6 Immunoactive effects 9 2.2.7 Important health properties of fermented milk 10 2.3. Traditional fermented milk products in Sudan 11 2.3.1 "Roub": 11 2.3.2 "Gariss" 11 2.3.3 Other dairy products 122.4.1 Procedures of "Roub" processing 12 2.4.2 Containers used in Roub making 13 2.4.2.1"Bukhsah" preparation 14 2.4.3 Uses of "Roub" 14 2.5 Microbiology of "Roub" 14 2.5.1 Lactic acid bacteria 142.5.2 Yeasts 15 2.5.3 Moulds 16
د
2.5.4 Staphylococcus aureus 16 2.5.5 Escherichia coli 16 2.5.6 Salmonella spp. 17 2.6 Effect of lactic acid on pathogenic organisms 17 2.7 Microbiological properties of some fermented dairy products 18 CHAPTER THREE: MATERIALS AND METHOD 20 3.1.1 Source of samples 20 3.1.2 Preparation of media 20 3.1.2.1 Plat count agar medium (scharlawu 01-161) 20
3.1.2.2 MRS agar medium 20 3.1.2.3 Mannitol saly agar medium 21 3.1.2.4 SS agar medium 21 3.1.2.5 MacConky agar medium 21 3.1.2.6 Malt extract agar medium 213.1.3 Preparation of samples dilution 22 3.2 Examination of culture 22 3.2.1 Total viable bacterial count 22 3.2.2 Lactic acid bacteria count 22 3.2.3 Staphylococcus aureus count 23 3.2.4 Salmonella spp. count 23 3.2.5 E. coli count 233.2.6 Yeasts and Moulds count 23 3.3 Biochemical test 23 3.3.1 Gram stain 23 3.3.2 Catalase test 243.3.3 Oxidase test 24 3.3.4 Indol test 24 3.4 Statistical Analyses 24 CHAPTER FOUR: RESULTS 25 CHAPTER FIVE: DISCUSSION 32 CHAPTER SIX : CONCLUSION AND RECOMMENDATIONS 34 REFERENCES 35
ه
LIST OF TABLES
Table Title Page
Table (1) Microbiological profile of “Roub” from Al-Obeid area, North Kordofan State (Log10 cfu/ml) 27
Table (2) Microbiological profile of “Roub” from Nyala area, South Darfur Stat (Log10 cfu/ml) 28
Table (3) Microbiological profile of “Roub” from Abu-Naama area, Blue Nile State (Log10 cfu/ml) 29
Table (4) Microbiological profile of “Roub” from three areas under study (mean ±SD) 30
Table (5) Percentage of positive samples from the areas under study 31
و
MICROBIOLOGY OF TRADITIONAL FERMENTED MILK PRODUCT
(ROUB)
Salah Ibrahim Khider Hussain
Degree of Master of Science in Tropical Animal Production
ABSTRACT
This investigation was carried out to evaluate the microbial load of a Sudanese
fermented dairy product, locally fermented (Roub) from different areas in Sudan.
Roub samples were collected from Nyala (South Darfur State ) , El Obied
(North Kordofan State) and Abu Naama (Blue Nile State) and transported to the
Faculty of Animal Production in sterile disposable plastic containers in ice box at
4oC. The samples were subjected to microbiological examination (total viable
bacteria, Staphylococcus aureus, Salmonella, lactic acid bacteria and yeasts and
moulds).
The results indicated the present E. coli, Staphylococcus aureus, lactic acid
bacteria, yeasts and moulds in the Roub sample from Nyala, El Obied and Abu
Naama, while Salmonella sp. was not detected in all samples tested. E. coli was not
detected in all samples collected from Abu Naama area. Total viable bacteria were
detected in all samples (100%), while E. coli was detected in 30% of the samples
from El Obied, 40% from Nyala and was not detected in samples from Abu Naama
area.
Staphylococcus aureus was detected in 40%, 60% and 40% of samples from El
Obied, Nyala and Abu Naama, respectively while lactic acid bacteria were detected
in all samples tested. Yeasts and moulds were detected in 100%, 90% and 90% of
samples from El Obied, Nyala and Abu Naama areas, respectively.
ز
)الروب( المتخمر تقليدياَ الدقيقه فى منتج االلباناالحياء
صالح ابراهيم خضر حسين
درجة ماجستير العلوم فى االنتاج الحيوانى
المستخلص
جمعت عينات . أجرى هذا البحث لتقييم المحتوى الميكروبى للروب من مناطق مختلفة بالسودان
واليـة النيـل (وأبو نعامـة ) والية شمال كردفان( األبيض و ) والية جنوب دارفور ( الروب من نياال
فى درجـه إلى معمل قسم االلبان كلية االنتاج الحيوانى في أواني من البالستيك المعقمه و نقلت )األزرق
البكتريـا ،بكتريا القولون ،الحيه العدد الكلى للبكتريا(العينات للفُحص الميكروبي خضعت. م4oحراره
).الفطريات و االعفان ،بكتريا حامض الالكتيك ،السالمونيال ،العقدية
مـن الفطريـات واألعفـان و بكتريا حامض الالكتيك والبكتريا العقدية و بكتريا القولونعزلت
لم تكتشف بكتريا السالمونيال فى كـل العينـات األبيض وأبو نعامة ، بينما ونياال التى جمعت من عيناتال
عزلت البكتريـا .أبو نعامةمنطقه لم تكتشف بكتريا القولون فى العينات التى جمعت منكما ، التى جمعت
% 40و % 30الحيه من كل العينات التى جمعت من المناطق الثالث فى حين عزلت بكتريا القولون مـن
ـ ة من العينات التى جمعت من منطقتى االبيض و نياال بينما لم تعزل من العينات التى جمعـت مـن منطق
من العينات التى جمعت من االبيض و نياال % 40و % 60و % 40عزلت البكتريا العقديه في .ابونعامه
فى حين ان بكتريا حامض الالكتيك عزلت فى كل العينات و عزلت الفطريات و ، و ابونعامه على التوالى
.بونعامه على التوالىمن العينات التى جمعت من االبيض و نياال و ا% 90و % 90و % 100الخمائر فى
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CHAPTER ONE
INTRODUCTION
Preservation of food is one of the oldest methods known to mankind.
A typical example is the lactic acid fermentation which is used in the
manufacture of fermented dairy products, like cheese and fermented milks
(Robinson, 1985).
The nature of fermented products is different from one region to
another. Thus is depending on the local indigenous microflora, which in turn
reflected the climatic conditions of the area. Thus traditional fermented milk
in regions with cold climate contained mesophilic bacteria such as
Leuconostoc spp., whilst thermophilic bacteria, which include mostly
Lactobacillus and Streptococcus, prevailed in regions with a hot, subtropical
or tropical climate (Thomas, 1985; Tamai et al., 1988 and Kurmann, 1994).
"Roub" is the most important indigenous fermented milk product of
considerable economic and dietary importance of the people of Sudan
especially in the west and the east of Sudan (Abdel Gadir et al., 1998).
In Sudan favorite sour milk "Roub" is made from fresh raw cow's
milk and to some extent goat's and sheep's. However, camel's milk is still the
milk of choice for some people in Sudan. Since the existence of the art of the
traditional manufacture of "Roub" in Sudan until present, the whole process
depends entirely on natural fermentation of milk. This kind of fermentation
process is the result of the presence of microorganisms and their enzymes in
milk. In the dairy industry these microbes are known as "starter cultures".
Attentions are still going to be focused on the need of the dairy industry for
new isolates of lactic acid bacteria as yet unused for dairy fermentations
(Abdel Gadir et al., 1998).
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Environmental conditions in each country affect the properties of the
predominant native microflora and this limits the use of some universal
starters. A rational solution for this problem might be to select starter
cultures among the native flora that could be used successfully in the dairy
industry.
The objectives of this study are:
1) To determine the bacterial load of traditionally made Roub
collected from different areas.
2) To isolate some lactic acid bacteria from “Roub”.
3) Detect of some pathogenic bacteria from “Roub”.
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CHAPTER TWO
LITERATURE REVIEW
2.1 Fermentation and fermented milk products:
2.1.1 Fermentation:
Definition of fermentation:
Campbell-Platt (1987) defined fermented foods as those foods which
have been subjected to the action of microorganisms or their enzymes so that
desirable biochemical changes cause significant modification to the food.
However, to the microbiologist, the term “fermentation” describes a form of
energy-yielding microbial metabolism in which an organic substrate, usually
a carbohydrate, is incompletely oxidised, and an organic carbohydrate acts
as the electron acceptor (Adams, 1990). This definition means that processes
involving ethanol production by yeasts or organic acids by lactic acid
bacteria are considered as fermentations, but not the production of fish
sauces in Southeast Asia, that still has not been shown to have a significant
role for microorganisms, and not the tempe production since the metabolism
of the fungi is not fermentative (Adams, 1990). Whichever definition is
used, foods submitted to the influence of lactic acid producing
microorganisms are considered fermented foods.
2.1.2 Origin of fermented milk:
Fermented milk was originated in the Near-East, perhaps before the
Phoenician era, and spread through central and Eastern Europe. The earliest
example of fermented milk was warm, raw milk from cow’s, sheep's, goat’s,
camel’s or horse’s of the nomads roaming the area which was turned into
clabber, or curd by bacteria and their end products (Abdel Gadir et al.,
1998).
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2.1.3 The fermentation of milk:
The term was first applied when the only known reaction of this kind
was the production of wine. The early fermentation concepts stated that
fermentation was strictly a chemical aspect of the process that the meaning
was changed to signify the breakdown of sugars into ethanol and CO2
(Abdel Gadir et al., 1998). Between 1856 and 1875, Louis Pasteur showed
conclusively that fermentation was a result of microbial action and each type
of fermentation was accompanied by the growth of a particular type of
micro-organism (Kosikowski, 1982). Louis Pasteur also marked the birth of
chemical microbiology with his association of microbes with the
fermentation in 1857. The term "fermentation" thus became associated with
the idea of cells, gas production and production of organic by-products
(Abdel Gadir et al., 1998).
The evolution of gas and presence of whole cells were invalidated as
criteria for defining fermentation when it was discovered that in some
fermentation such as the production of lactic acid no gas is liberated, while
the fermentation processes could be obtained with cell-free extracts,
indicating that the whole cell may not be necessary (Abdel Gadir et al.,
1998) . Fermentation is clearly needed to be redefined when the situation
was further complicated by the discovery that the ancient process of vinegar
production, which yielded considerable quantities of organic by-products
was a strictly aerobic process. Fermentation came to be regarded as the
anaerobic decomposition of organic compounds to products which could not
be further metabolized by the enzyme systems of cells without intervention
of oxygen.
The fermentation products differed with different microorganisms,
being mainly governed by the enzyme complex of cells and the
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environmental conditions. The economic values of these by-products lead to
the development of industrial microbiology (Abdel Gadir et al., 1998).
2.1.4 Types of fermented milks:
A characteristic common feature to all milks is the presence of lactic
acid, for example cultured buttermilk, yoghurt, acidophilus and Bulgarian
milks. Other fermented milks are alcoholic in addition to acidic, for
example, Kefir and Koumiss. The term "cultured" is frequently substituted
for "fermented" in buttermilk, yoghurt and sour cream because,
commercially, these foods were among the first to be produced on a large
scale by pure bacterial cultures (Kosikowski, 1982).
2.2 Health aspects of fermented foods:
2.2.1 Probiotic effect:
One of the reasons for the increasing interest in fermented foods is its
ability to promote the functions of the human digestive system in a number
of positive ways (Sahlin, 1999). This particular contribution is called
probiotic effect. Already early in 1900, Metchnikoff pointed out the use of
fermented milks in the diet for prevention of certain diseases of the
gastrointestinal tract and promotion of healthy day to day life. Since then a
number of studies have now shown that the fermented food products have a
positive effect on health status in many ways. The human intestinal
microbial flora is estimated to weigh about 1000 grams and may contain
1016–1017 colony forming units (cfu) representing more than 500 strains. For
physiological purposes, it can be considered to be a specialized organ of the
body with a wide variety of functions in nutrition, immunology and
metabolism (Gustafsson, 1983). Studies on mice have shown that the
indigenous microorganisms in the stomach are Lactobacillus, Streptococcus
and Torulopsis, while in the small intestine, ceacum and colon several
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different species (Bacteroides, Fusobacterium, Eubacterium, Clostridium,
etc.) coexist (Savage, 1983). The gastrointestinal microflora in humans is
also known to contain hundreds of species. Even though there is a wide
variation among individuals, the number of species and size of the
population are usually kept stable in normal healthy subjects. There is a
constant struggle in maintaining the desirable balance and a dynamic
equilibrium between microbial populations within the intestinal flora
(Robinson and Samona, 1992). The anaerobic organisms, which outnumber
the Gram negative enteric bacteria by about 10000:1, are associated with the
intestinal epithelium limiting adherence of potential pathogens by effective
colonization (Van der Waaij et al., 1972; Nord and Kager, 1984; Swank and
Dietch, 1996). The stability of the intestinal microflora is affected by many
factors including dietary habits. Decrease in the number of anerobic bacteria
is associated with increase in the number of Gram negative pathogens in the
intestinal tract and their translocation to extraintestinal tissues. Under normal
conditions the intestinal wall prevents translocation of organisms both dead
and live as well as microbial products like toxins from the gut to the blood.
However, in patients with systemic insult like starvation, shock, injury and
infection or specific insult of the gastrointestinal canal through
inflammation, chemotherapy or radiation, the gut mucosal permeability will
be increased leading to translocation of microbes (Alexander et al., 1990;
Wells, 1990 and Kasravi et al., 1997). A fermented food product or live
microbial food supplement which has beneficial effects on the host by
improving intestinal microbial balance is generally understood to have a
probiotic effect (Fuller, 1989).
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2.2.2 Flatulence reducing effect:
During fermentation of the beans for preparation of tempe, the trypsin
inhibitor is inactivated, and the amount of several oligosacharides which
usually cause flatulence are significantly reduced (Hesseltine and Wang,
1983). Bean flour inoculated with Lactobacillus and fermented with 20%
moisture content, showed a reduction of the stachyose content
(Duszkiewicz-Reinhard et al., 1994).
2.2.3 Anticholesterolemic effect:
Hepner et al. (1979) reported hypercholesteremic effect of yoghurt in
human subjects receiving a one-week dietary supplement. Studies on
supplementation of infant formula with Lactobacillus. acidophilus showed
that the serum cholesterol in infants was reduced from 147 mg/ml to 119
mg/100 ml (Harrison and Peat, 1975). In an in vitro study, the ability of 23
strains of lactic acid bacteria isolated from various fermented milk products
to bind cholesterol was investigated, and no cholesterol was found inside the
cells (Taranto et al., 1997). Poppel and Schaafsma (1996) have also reported
the ability of yoghurt to lower the cholesterol in serum by controlled human
trials. Possible role of lactic acid bactera in lowering cholesterol
concentration and various mechanisms by which it may be possible has been
discussed by Haberer et al. (1997). Brigidi et al (1993) have cloned a gene
encoding cholesterol oxidase from Streptomycin lividans into Bacillus,
Lactobacillus and E .coli.
2.2.4 Effect on transit time, bowel function and glycemic index:
The transit time for 50% (t50) of the gastric content was significantly
reduced for regular unfermented milk (42±10 min) in comparison with a
fermented milk product indigenous to Sweden called "långfil" or ropy milk
(62±14 min). Another study reported increase in transport time and
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improved bowel function in patients with habitual constipation (Wilhelm,
1993). The number of defeacations per week increased from three during
control period to seven using conventional fermented milk and fifteen when
acidophilus milk was served. Regular unfermented milk also gave
significantly higher increase in glycemic index curve than fermented milk
product called långfil (Strandhagen et al., 1994). Liljeberg et al., (1995)
showed that the presence of acid, specially acetic or lactic, lowered the
glycemic index in breads to a significant level. Koji which is prepared from
Aspergillus oryzae and beni-koji made from Monascus pilosus were found to
express rises in blood pressure (Tsuji et al., 1992).
2.2.5 Anticarcinogenic effect:
There are interesting data on anticarcinogenic effect of fermented
foods showing potential role of lactobacilli in reducing or eliminating
procarcinogens and carcinogens in the alimentary canal (Reddy et al., 1983;
Shahani, 1983; Mital and Garg, 1995). The enzymes ß-glucuronidase,
azoreductase and nitroreductase “which are present in the intestinal canal”
are known to convert procarcinogens to carcinogens (Goldin and Gorbach,
1984). Oral administration of Lb. rhamnosus GG was shown to lower the
faecal concentration of ß-glucuronidase in humans (Salminen and Gorbach,
1993) implying a decrease in the conversion of procarcinogens to
cancinogens. Fermented milk containing Lactobacillus acidophilus given
together with fried meat patties significantly lowered the excretion of
mutagenic substances compared to ordinary fermented milk with
Lactococcus fed together with fried meat patties (Lidbeck et al., 1992). The
processes of fermentation of foods are also reported to reduce the
mutagenicity of foods by degrading the mutagenic substances during the
process. Lactic acid bacteria isolated from Dahi (traditional Indonesian
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fermented milk), were found to be able to bind mutagens and inhibit
mutagenic nitrosamines (Harun-ur-Rashid et al., 2007). Milk fermented with
Lactobacillus acidophilus LA-2 was demonstrated to suppress faecal
mutagenicity in the human intestine (Sahlin, 1999). Studies on
antimutagenic activity of milk fermented with mixed-cultures of various
lactic acid bacteria and yeasts showed that, the fermented milk products with
mixed cultures of lactic acid bacteria had a wider range of activity against
mutagens than those produced with a single strain of lactic acid bacteria
(Tamai et al., 1995). However, a review by McIntosh (1996) concluded that
there is only limited data to support the hypothesis that probiotic bacteria are
effective in cancer prevention. On the other hand, a study by Hosono and
Hisamatsu (1995) on the ability of the probiotic bacteria to bind
carcinogenic substances have reported that E. feacalis was able to bind
aflatoxins B1, B2, G1 and G2 as well as some pyrolytic products of
tryptophan.
2.2.6 Immunoactive effects:
Some lactic acid bacteria present in fermented milk products, are
found to play an important role in the immune system of the host after
colonization in the gut (De Simone, 1986). Oral administration of
Lactobacillus casei caused an improvement of the function of the peritoneal
macrophages and increased the production of IgA (Sato et al., 1988). The
mechanism of this effect is not clearly known, but it is speculated that the
lactobacilli, their enzymes or the metabolic products present in the
fermented food product may act as antigens, activating production of
antibodies. Marin et al. (1997) studied the influence of lactobacilli used in
fermented dairy products on the production of cytokines by macrophages.
The results indicated that for most strains, direct interaction with
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macrophages caused a concentration dependent increase in tumour necrosis
factor and interleukin. A study by Perdigon et al. (1995) showed that,
Lactobacillus casei could prevent enteric infections and stimulate the
secretion of IgA in malnourished animals but also translocate bacteria, while
yoghurt could inhibit growth of intestinal carcinoma through increased
activity of IgA, T- cells and macrophages. In a review by Marteau and
Rambaud (1993), the authors concluded that there is a potential for using
lactic acid bacteria for therapy and immunomodulation in mucosal diseases,
especially in the gastrointestinal tract. Isolauri (1996) presented a study
suggesting that Lactobacillus spp. strain GG could be used in the prevention
of food allergy. It is suggested that dietary antigens induce
immunoinflamatory response that impairs the intestine’s barrier function and
that probiotic organisms could be a mean of introducing a tool to reinforce
the barrier effect of the gut.
2.2.7 Important health properties of fermented milk:
Sometime between 1898 and 1908 a good deal of controversy existed
over the special health-giving properties of fermented milk foods. Extremists
claimed a larger life expectancy for the consumers where these foods are
staple. They pointed to high percentage of centenarians in regions where
fermented milks are consumed. Other saw nothing more in fermented milks
than good basic food (Kosikowski, 1982). However souring of milk inhibits
the growth of many pathogenic bacteria, so that outbreaks of intestinal
diseases are much less likely than with fresh milks especially in hot
countries. It is most likely that the healthfulness of fermented milks depends
on their contents of protein, lactose, fat, calcium and other important
minerals and vitamin B complex (Saarivirta, 1983). It has been claimed that
the biological value of proteins of fermented milks is better than the ordinary
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milk. There is, however, no evidence showing that simple protein fragments
together with the unchanged protein have better nutritive value than the
original proteins (Saarivirta, 1983). Since fermented milks have usually less
fat, they contain less fat soluble vitamins than the ordinary milk. They are,
however, excellent sources of B-vitamins; the amount of any single vitamin
depends on the culture condition and the type of fermented milk in quest
(Saarivirta, 1983).
It has been demonstrated that individual with limited ability to digest
lactose (lactose intolerance) can usually consume nutritionally useful
quantities of fermented milk (Saarivirta, 1983).
2.3. Traditional fermented milk products in Sudan:
2.3.1 "Roub":
It is the most common fermented milk product of Sudan. Milk is
fermented overnight, and the resulting sour milk is churned to give butter;
the remaining buttermilk is "Roub". The principal aim behind "Roub"
production is the need to facilitate the extraction of butter from the milk. The
butter (furssah) is later boiled to give butter oil or ghee, which can be stored
for use in the lean season. "Roub" production is in the hands of animal-
owning nomadic tribes, and the bulk of it is produced during the rainy
season from July to October (Dirar, 1993).
The traditional manufacturing procedures of "Roub" in Sudan are not
different with different regions despite the disparities in climate and culture.
However, differences are still there in the whole process. These differences
are mainly confined to the containers used and their names and to some
extent to the use of "Roub" and "Roub" products (Abdel Gadir et al., 1998).
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2.3.2 "Gariss":
Camel's milk is produced in certain areas of Sudan, under nomadic
conditions, amounting to about 33,000 tons/year, because camels in Sudan
are reared under unstable nomadic roaming, their milk is not always
available to urban or village residents. The camel's milk being abundant in
remote localities, the herders have to prepare Gariss, a fermented product, on
which they sustain living for several months as the sole source of various
nutrients (Dirar, 1993). Gariss from camel's milk is made by a semi-
continuous fermentation process where the fermentation is carried out in two
leather bags of tanned goat skin embedded in green or wet grass carried on
the back of camels and subjected to continuous shaking by the jerky walk
inherent to camels. Whenever part of the product is withdrawn for
consumption, a portion of fresh camel's milk is added to make up volume
and this continues for months (Dirar, 1993).
2.3.3 Other dairy products:
Biruni, also called leben-gadim, which is fermented unchurned milk
ripened for up to 10 years. A related product, but not ripened, is mish, which
is made by prolonged fermentation to the extent that maggots thrive in it.
The product is consumed whole, with the maggots included. These two
products are closely related to Egyptian mish (Abdel-Malek, 1978).
Dairy products that have entered the Sudan from Egypt within the last
century are jibna beida (white cheese), zabadi (yogurt), and black cumin
flavored mish. These products are strictly confined to urban communities,
where the Egyptian influence is more strongly felt (Dirar, 1993).
2.4.1 Procedures of "Roub" processing:
In areas where the survey was carried out, the practice of "Roub"
making was almost the same and can be outlined as follows:-
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Source of milk is normally cow's milk (in Kordofan) and in addition,
goat's, sheep's and camel's milk can be used. Usually milk for "Roub"
making is used as raw i.e. not boiled. However, there are still some people
who prefer heating milk (boiling) before processing. It is clear that it is just a
matter of taste and liking, people who prefer boiling of milk say that the
resultant "Roub" is better in taste and they called it "Roub barid" (cold Roub
i.e. moderately acidic).
They usually (unless the container is new and not used before) do not
add any of previous "Roub" (as starter culture) to the raw milk under
processing, however, still there are some producers who prefer adding part
of previous "Roub" as starter culture. "Roub" has a short lifetime i.e.
deteriorates rapidly; in addition they all agree that there is a need to add
"Roub" so as to expedite the fermentation.
Milk for "Roub" making generally is left for 12 hours after which the
fermentation is complete and some people leave it for 24 hours; but they all
agree that the fermentation period can go up or down according to the
ambient temperature.
Coagulation of milk (i.e. complete fermentation) is usually noticed by
experience. There is no specific way of judging the milk other than normally
by sight and sense.
Milk is shaken (churned) after being curdled. The shaking time is
usually 10 minutes after which a separation of butter is obtained. Addition of
water to ease the collection of butter is common to all Roub-producer. Water
is added just at the breakpoint (of butter and butter milk) which is detected
by experience.
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2.4.2 Containers used in Roub making:
The common traditional container is called "Bukhsah" but for those who
tend to boil the raw milk they use a container called "Burma" (which is made
from clay). After milk is heated, it is transferred to the "Bukhsah" where
fermentation takes place; it is also called "Berkaba" (Abdel Gadir et al.,
1998).
2.4.2.1"Bukhsah" preparation:
"Bukhsah" is some kind of gourd, which in its unshaped form is wholly
closed object. They open it from one side (usually the tapering end) and then
spin the opening with some kind of tree-fiber called "Saaf" until the opening
becomes narraw and considered as the mouth of "Bukhsah". Usually from
the same "Saaf" they make a mouth-lid as a cover which tightly closes the
mouth. They then spin a handle for "Bukhsah" (usually made from skin of
cattle), this handle is for hanging the "Bukhsah" during shaking process.
"Bukhsah" is usually rinsed at least after each two "Roub" makings, and then
exposed to the sun for drying (Abdel Gadir et al., 1998).
2.4.3 Uses of "Roub":
"Roub" is usually used as a drink and is considered a good nourishing
food, and the nomads depend on it.
Sometimes people add what is called "waika" (a kind of sun-dried
okra) to process "Mullah Roub" (kind of stew) and sometimes "Roub" is left
to get sharply soured and then the curd is collected leaving the whey which
is sweetened with table sugar and drunk ( as in Bija tribes).
2.5 Microbiology of "Roub":
2.5.1 Lactic acid bacteria:
The lactic acid bacteria consisting of a number of diverse genera are
grouped as either homofermenters or heterofermenters on the basis of
15
fermentation end products (Carr et al., 2002). Homolactic fermentation is
known as Embden-Meyerhof-Parnas pathway for glycolysis (Teuber, 1995).
The homofermenters produce only lactic acid as the major end-product by
the fermentation of carbohydrates (Axelsson 1998 and Carr et al., 2002)
The homofermentative group of lactic acid bacteria includes
Streptococcus, Pediococcus, Lactococcus, and Lactobacillus species. The
heterofermenters on the other hand produce a number of other products such
as carbon dioxide, acetic acid, ethanol besides lactic acid (Axelsson 1998
and Carr et al., 2002). Homofermentation and heterofermentation are
differentiated by the production of aldolase which is the key enzyme for
glycolysis. The homofermenters possess aldolase which ferments glucose to
lactic acid. The heterofermenters do not produce this enzyme and follow
pentose-monophosphate pathway instead. They cannot breakdown fructose
1, 6 diphosphate to triose phosphates, and only oxidize glucose-6- phosphate
to 6-gluconate by phosphoketolase (Okuklu, 2005).
2.5.2 Yeasts:
Debaryces hansenii has been isolated from cheese and other foods.
Cells appear spherical to short oval and are arranged singly, in pairs or in
short chains and propagate by multipolar budding. On slide cultures
pseudomycelium is absent or, if present, is very primitive. There are unable
to ferment lactose (Lovell, 1990).
Yeasts can break down protein and fat and therefore cause quality
defects in cheese and butter. Yeasts grow best in acid environment and
therefore usually found in acid products as cultured milk. Yeasts develop
most vigorously at about 30oC in the presence of air. Yeasts are generally
undesirable organisms from the dairy point of view because they can cause
16
fermentation in milk and dairy products, adversely effecting both flavor and
smell (Alfa-laval Dairy Hand book).
2.5.3 Moulds:
Moulds are commonly found in dairy products. On mould extract
agar, colonies are white in colour and appear yeast-like and butyrous,
particularly when older. The commonest species of moulds in milk and milk
products, do not in practice survive high temperature short time
pasteurization (HTST). The presence of mould in heat treated product is
therefore sign of recontamination. Many different families of moulds exist;
those which are of the greatest importance in the dairy industry are
Penicillium (Alfa-laval Dairy Hand book).
2.5.4 Staphylococcus aureus :
Staphylococcus is a genus of Gram-positive bacteria. Under the
microscope they appear round (cocci), and form in grape-like clusters (Ryan
et al., 2004).
The Staphylococcus genus includes just thirty-three species (Holt,
1994). Most are harmless and reside normally on the skin and mucous
membranes of humans and other organisms. Found worldwide, they are a
small component of soil microbial flora (Madigan and Martinko, 2005).
2.5.5 Escherichia coli:
Escherichia coli is a Gram negative bacterium that is commonly
found in the lower intestine of warm-blooded organisms (endotherms). Most
E. coli strains are harmless, but some, such as serotype O157:H7, can cause
serious food poisoning in humans, and are occasionally responsible for
costly product recalls. The harmless strains are part of the normal flora of
the gut, and can benefit their hosts by producing vitamin K2 (Bentley and
17
Meganathan, 1982) or by preventing the establishment of pathogenic
bacteria within the intestine (Reid et al., 2001).
E. coli is not always confined to the intestine, and its ability to survive
for brief periods outside the body makes it an ideal indicator organism to test
environmental samples for fecal contamination (Thompson, 2007). The
bacterium can also be grown easily and its genetics are comparatively simple
and easily-manipulated or duplicated through a process of metagenics,
making it one of the best-studied prokaryotic model organisms, and an
important species in biotechnology and microbiology (Feng, 2002).
2.5.6 Salmonella sp.:
Salmonella is a Gram-negative facultative rod-shaped bacterium in the
same proteobacterial family as Escherichia coli, the family
Enterobacteriaceae, trivially known as "enteric" bacteria. Salmonella is
nearly as well-studied as E. coli from a structural, biochemical and
molecular point of view, and as poorly understood as E. coli from an
ecological point of view. Salmonellae live in the intestinal tracts of warm
and cold blooded animals. Some species are ubiquitous and other species are
specifically adapted to a particular host. In humans, Salmonella is the cause
of two diseases called salmonellosis: enteric fever (typhoid), resulting from
bacterial invasion of the blood stream, and acute gastroenteritis, resulting
from a food borne infection/intoxication (Todar, 2007).
2.6 Effect of lactic acid on pathogenic organisms
Lactic acid inhibits many pathogenic bacteria, and the undissociated
form of the acid is considered to be the active component (Robinson and
Samona, 1992). The amount of undissociated lactic acid depends on both the
concentration of lactic acid and the pH. The pKa of lactic acid is 3.86, giving
that at pH 4.8 only 10% while at pH=3.86 half of the lactic acid is in the
18
undissociated form. In contrast to the lactate ion, the uncharged
undissociated form of lactic acid can penetrate the cell membrane of the
bacteria. The cytoplasm of the bacteria has a much higher pH than the
surrounding, which provokes the dissociation of the lactic acid molecule
inside the bacteria, thus liberating H+. This will lower the pH of the
cytoplasm. The amount of undissociated lactic acid passing through the cell
membrane at a point near pH 4 is too high for the mechanisms in the cell
dealing with the regulation of pH in the cytoplasm. The pH inside the
bacteria will drop and eventually cause the destruction of the pathogen
(Abdel Gadir et al., 1998).
2.7 Microbiological properties of some fermented dairy products:
El Sadic et al (1972) studied the microbiological properties of 50
samples of "Zabady" from Cairo market. They found that the total microbial
count averaged 1.2×109 cfu/ml, the dominant species being streptococci
(mainly Strep thermophilus) followed by micrococci Lactobacillus count
averaged 7×108 cfu/ml, with Lactobacillus bulgaricus predomination. Yeasts
and moulds count averaged 7×105 cfu/ml and they mainly Candida spp.
Yeasts were selected in 84% of samples. Colifirm count ranged from 0.0 to
2.4×106 cfu/ml with an average 15×104 cfu/ml, these organisms being
detected in 58% of samples.
Bhat and Fernande (1984) examined the microbial flora of "Mawa".
From 5 representative "150" isolates were obtained. These isolates were
mainly: (8) species of each of Micrococcus, Staph. citoreous and
Microbacterum lacticum, (6) species of Bacillus, Clostridium perfringens
(the only anaerobic Bacillus) and Actinomyces rutger sensii and (3) species
of moulds (Asperigillus, Penicilluim and Pachytrichum). The average colony
19
count/gm of "Mawa" on 2% agar (same samples) was: 33,664,000 (after 24
hrs), 385,621,020 (after 72 hrs) and 1,643,018,200 (after 144 hrs).
Savadogo et al. (2004) found that total plate count agar (aerobic
mesophilic bacteria) ranges of counts were 8.12×105 - 3.6×108 and mean
counts for all samples 6.71×107, Rogosa agar (lactobacilli) ranges of counts
(cfu ml-1) 0.30x106-1×108 and mean counts (cfu ml-1) for all samples
24.44×106 and yeast and moulds ranges of counts (cfu ml-1) 1.83×103-
3.7×106 n=25 and mean counts (cfu ml-1) for all samples.
Al-Tahiri (2005) studied the microbiological conditions between
traditional dairy products sold in Karak in the south of Jordan and same
products produced by modern dairies. He found the microbiology of
unpasteurized milk, yoghurt, lebnah (concentrated yoghurt) and white soft
cheese produced by farmers as total count (5×105- 3×103, 6×102 – 2×104), S.
aureus count (3×102- 4×10, less than 10 – 5×103) and yeast and moulds
count (15×103- 15×10, 4×10 – 5×105), but in products produced by modern
dairies he found the total count (zero- 6×106,8×105 – 5×10), yeasts and
moulds count ( zero- 10) and no Staph. aureus were detected in all samples.
Abdalla and El Zubeir (2006) found high count of E. coli,
Staphylococcus aureus, and Salmonella spp. in yogurt and their mixture and
reported that these organisms are potential for food spoilage. Escherichia
coli, Listeria monocytogenues and Yersinia enterocolitica are three of the
most important food-borne bacterial pathogens and can lead to food-borne
diseases through consumption of contaminated milk and fermented milk
products (Morgan et al., 1993; Mead et al., 1999).
20
CHAPTER THREE
MATERIALS AND METHODS
3.1.1 Source of samples:
A total of 30 "Roub" samples were obtained from three regions in
Sudan ("Nyala", "Al Obied" and "Abu Namma"). "Roub" samples were
collected from the market in sterile "50 ml" plastic flasks and transported in
cooled containers (4oC) to the laboratory of the Department of Dairy
Production, Faculty of Animal Production, University of Khartoum. The
samples were stored at the same temperature (4oC) till the microbiological
examination was carried out during 48 hours.
3.1.2 Preparation of media:
3.1.2.1 Plat Count agar medium (Scharlawu 01-161):
The medium consisted of 5 gm casein peptone, 2.5 gm yeast extract,
1.0 gm dextrose and 15 gm agar. The medium was prepared by suspending
28 gm of the powder in one liter of distilled water, then boiled until
dissolved completely and sterilized by autoclaving at 121oC for 15 minutes.
3.1.2.2 MRS agar medium (7543) :
The medium consisted of 10 gm casein peptone tryptic digest, 10 gm
meat extract, 5 gm yeast extract, 20 gm glucose, 5 gm sodium acetate, 1 gm
polysorbate “80”, 2 gm potassium phosphate, 2 gm ammonium citrate, 0.1
gm magnesium sulfate, 0.05 gm manganese sulfate and 15 gm agar .
The medium was prepared by suspending 70 gm of the powder in one
liter of distilled water, then boiled until dissolved completely and sterilized
by autoclaving at 121oC for 15 minutes.
21
3.1.2.3 Mannitol salt agar medium (7143A):
The medium consisted of 10 gm proteose peptone, 1 gm beef extract,
75 gm sodium chloride, 10 gm D- mannitol, 0.025 gm phenol red and 15 gm
agar.
The medium was prepared by suspending 111 gm of the powder in
one liter of distilled water, then boiled until dissolved completely and
sterilized by autoclaving at 121oC for 15 minutes.
3.1.2.4 Salmonella Shigella (SS) agar medium (7152A):
The medium consisted of 5 gm beef extract, 2.5 gm enzymatic digest
of casein, 2.5 gm enzymatic digest of animal tissue, 10 gm lactose, 8.5 gm
bile salts, 8.5 gm sodium citrate, 8.5 gm sodium thiosulfate, 1 gm ferric
citrate, 0.00033 gm brilliant green, 0.025 gm neutral red and 13.5 gm agar.
The medium was prepared by suspending 63 gm of the powder in one
liter of distilled water, boiled until dissolved completely.
3.1.2.5 MacConky agar medium:
The medium consisted of 17 gm pancreatic digest of gelatin, 3 gm
peptones (meat and casein), 10 gm lactose monohydrate, 5 gm NaCl, 1.5 gm
dehydrated bile, 1 gm crystal red, 30 gm neutral red and 13.5 gm agar.
The medium was prepared by suspending 51.5 gm of the powder in
one liter of distilled water, then boiled until dissolved completely and
sterilized by autoclaving at 121oC for 15 minutes.
3.1.2.6 Malt extract agar medium:
The medium consisted of 12.75 gm maltose, 2.75 gm dextrin, 2.35 gm
glycerol, 0.78 gm peptone and 15 gm agar.
The medium was prepared by suspending 50 gm of the powder in
one liter of distilled water, boiled until dissolved completely and sterilized
by autoclaving at 121oC for 15 minutes.
22
3.1.3 Preparation of serial dilutions:
Eleven milliliter of "Roub" were dissolved in 99 milliliter of sterile
distilled water to make 10-1 dilution, then one ml from the above mentioned
dilution was specially transferred to 9 milliliter sterile distilled water. This
procedure was repeated to make serial dilutions of 10-2, 10-3, 10-4, 10-5 and
10-6. From the last dilution, 1 ml was transferred to Petridish in duplicate
(Houghtby et al., 1992).
3.2 Examination of culture:
Growth on solid media was examined visually with naked eyes for
colonies appearance and change in media.
3.2.1 Total viable bacterial count
Plate count agar was used for the enumeration of total bacterial count,
1 ml quantities of each sample decimal dilutions, 106 was streaked in dried
plate with plate count agar and incubated at 32 ±1oC for 48 ±3 h for
enumeration of total plate count. They were counted by colony counter
(Houghtby et al., 1992).
3.2.2 Lactic acid bacteria count:
MRS agar was used for the enumeration of Lactobacilli, 1 ml
quantities of each sample decimal dilutions, 106 was streaked in dried plat of
MRS agar. The culture was incubated at 35oC for 48 hours (DeMan et al.,
1960). They were counted by colony counter.
3.2.3 Staphylococcus aureus count:
Mannitol salt agar was used for the enumeration of staphylococci, 1
ml quantities of each sample decimal dilutions, 106 was streaked in dried
plate of mannitol salt agar. The culture was incubated at 37oC for 48 hours
(Harrigan and McCance, 1976). They were counted by colony counter.
23
3.2.4 Salmonella spp. count:
Salmonella Shigella agar (SS agar) was used for the enumeration of
Salmonella spp , 1 ml quantities of each sample decimal dilutions, 106 was
streaked in dried plate of SS agar. The culture was incubated at 35oC for 48
hours. They were counted by colony counter
3.2.5 E. coli count:
MacConky agar was used for the enumeration of E. coli, 1 ml
quantities of each sample decimal dilutions, 106 was streaked in dried plate
of MacConky agar. The culture was incubated at 35oC for 48 hours. They
were counted by colony counter.
3.2.6 Yeasts and moulds counts:
This was determined by plating suitable dilution of sample in malt
extract agar plate which was acidified to pH 3.5 using 10% acid as indicated
in Harrigan and McCance (1976). Plates were incubated at 30oC ± 2oC for
up to 5 days.
3.3 Biochemical tests:
Biochemical tests were performed according to Barrow and Feltham
(1993) as follows:
3.3.1 Gram stain
A discrete colony was carefully picked with a sterile wire loop. The
colony from fresh culture (18-24 hr) was emulsified in a drop of saline and
spread evenly on clean slide to make a thin film. The slide was allowed to
dry and fixed by slight flaming and stained. The smear was stained with
crystal violet solution for seconds, then rinsed rapidly with water and
Gram’s iodine solution was added and left to dry for seconds. The iodine
was poured off and the slide was washed with 95% ethanol for 5-15 seconds.
24
The smear was then washed with tap water and stained with safranin
solution for 20 seconds, again washed with water and allowed to dry. On
microscopic examination, the Gram positive organisms appeared purple
while Gram negative ones appeared pink (Harrigan and Mc Cance, 1976).
3.3.2 Catalase test:
The organisms to be tested were put in sterile slides. A drop of 3%
hydrogen peroxide (H2O2) was added to the colony and emulsified.
Production of gas immediately or after 5 minutes indicated a positive result.
3.3.3 Oxidase test:
The oxidase test was performed using oxidase test paper. The colonies
to be tested were removed by a platinum wire or glass rod and smeared
across the surface the oxidase test paper. The positive reaction was showed
by the development of a dark purple color within 10 seconds.
3.3.4 Indole test:
Indole test was performed to detect the ability of an organism to break
down tryptophan to indole. The colonies to be tested were inoculated into
tube containing tryptophan water and incubated at 370C for 48 hrs then
added 1ml of Kovac,s reagent was added and read immediately. The result
was indicated by the appearance of bright pink color in the top layer.
3.4 Statistical analyses:
The samples were analyzed for total viable bacterial count, count,
Staphylococcus aureus count, Salmonella spp. count, E. coli count and
yeasts and moulds count. Means were separated using Duncan multiple
range test with P≤0.05.
25
CHAPTER FOUR
RESULTS
The result in tables 1, 2 and 3 indicate that the mean total viable
bacterial count in EL-Obeid, Nyala and Abu Naama areas was 8.14, 7.56 and
8.07 Log10 cfu/ml respectively. These values showed significant different
(P<0.05) between EL-Obeid and Nyala samples but no significant different
(P>0.05) between El-Obeid and Abu Naama samples and between Nyala and
Abu Naama samples were observed (Table 4). Out of the number of samples
tested, it was found that all samples were positive (100%) for total viable
bacterial count (Table 5).
Means of Staphylococcus aureus count were 6.15, 6.18 and 5.30 Log10
cfu/ml for EL-Obeid, Nyala and Abu Naama areas respectively (Tables 1, 2
and 3), and these means were not significant different (P>0.05) between the
three areas under study (Table 4). The percentage of positive samples for
Staphylococcus aureus were 40% in El-Obeid, 60% in Nyala and 20% in
Abu Naama areas (Table 5).
Escherichia coli showed a mean value of 5.60 Log10 cfu/ml in El-
Obeid area (range: <1 est - 6.30 Log10 cfu/ml), 5.70 Log10 cfu/ml in Nyala
(range: <1 est – 6.60 Log10 cfu/ml), while in Abu Naama area the organism
was not detected in samples tested (Tables 1, 2, 3 and 4). There were no
significant different (P>0.05) between El-Obeid and Nyala areas (Table 4).
Thirty percent of the samples tested were positive in El-Obeid and 40% in
Nyala area, while all samples tested in Abu Naama area were negative.
Salmonella sp was not detected in all samples tested in the three areas
under study (Tables 1, 2, 3 and 4).
26
The mean values for yeasts and moulds were 5.53, 4.64 and 5.50
Log10 cfu/ml for EL-Obeid, Nyala and Abu Naama areas respectively and
these means were not significant different (P>0.05) between the three areas
under study (Table 4). All samples tested were positive (100%) for yeasts
and moulds in El-Obeid area and 90% of samples were positive in Nyala and
Abu Naama areas (Table 5).
The mean count of lactic acid bacteria was 7.80, 7.09 and 7.51 Log10
cfu/ml in EL-Obeid, Nyala and Abu Naama areas respectively (Table 4).
These values showed significant different (P<0.05) between El-Obeid and
Nyala samples but no significant different (P>0.05) between El-Obeid and
Abu Naama samples and between Nyala and Abu Naama samples were
observed (Table 4). All samples tested for lactic acid bacteria were positive
(100%) in the areas under study (Table 5).
27
Table (1). Microbiological profile of “Roub” from El-Obeid area, North
Kordofan State (Log10 cfu/ml)
Organism Range Mean
Total viable bacterial count 7.53±0.32 - 8.47±0.32 8.14±0.40
Escherichia coli < 1 est - 6.30±2.95 5.60±2.62
Staphylococcus aureus <1 est - 6.90±3.30 6.15±3.15
Salmonella sp. ND ND
Lactic acid bacteria 7.04±0.42 - 8.29±0.42 7.80±0.57
Yeasts and moulds 4.00±0.62 - 5.97±0.62 5.53±1.41
ND = Not detected
est = Estimated
28
Table (2). Microbiological profile of “Roub” from Nyala area, South Darfur
State (Log10 cfu/ml)
Organism Range Mean
Total viable bacterial count 7.34±0.12 – 7.72±0.12 7.56±0.40
Escherichia coli < 1 est - 6.60±3.14 5.70±2.62
Staphylococcus aureus <1 est - 6.60±3.28 6.18±3.15
Salmonella sp. ND ND
Lactic acid bacteria 6.78±0.16 – 7.28±0.16 7.09±0.57
Yeasts and moulds <1 est – 4.90±1.48 4.64±1.41
ND = Not detected
est = Estimated
29
Table (3). Microbiological profile of “Roub” from Abu Naama area, Blue
Nile State (Log10 cfu/ml)
Organism Range Mean
Total viable bacterial count 7.24±0.51 – 8.68±0.51 8.07±0.40
Escherichia coli ND ND
Staphylococcus aureus < 1 est – 6.00±2.53 5.30±3.15
Salmonella sp. ND ND
Lactic acid bacteria 5.70 ±0.77 – 8.21±0.77 7.51±0.57
Yeasts and moulds < 1 est – 6.22±1.78 5.50±1.41
ND = Not detected
est = Estimated
30
Table (4). Microbiological profile of “Roub” from three areas under study
(mean ±SD)
Organism El-Obeid Nyala Abu Namma
SL Mean Mean Mean Total viable bacterial count 8.14±0.40 a 7.56±0.40 b 8.07a±0.40 b *
Escherichia coli 5.60±2.62 a 5.70±2.62 a ND NS
Staphylococcus aureus 6.15±3.15 a 6.18±3.15 a 5.30±3.15 a NS
Salmonella sp. ND ND ND NS
Lactic acid bacteria 7.80±0.57 a 7.09±0.57 b 7.51±0.57 ab *
Yeasts and moulds 5.53±1.41 a 4.64±1.41 a 5.50±1.41 a NS
Means within each row bearing the same superscripts are not significantly
different (P<0.05).
* = P<0.05
ND = Not detected
SL = Significant Level
NS = Not Significant
SD = Standard Deviation
31
Table (5). Percentage of positive samples from the areas under study
Organism El-Obeid Nyala Abu Naama
Total viable bacteria count 100 100 100
E. coli 30 40 0
S. aureus 40 60 20
Salmonella spp. 0 0 0
Lactic acid bacteria 100 100 100
Yeasts and moulds 100 90 90
32
CHAPTER FIVE
DISCUSSION
Due to absence of heat treatment of milk prior to fermentation in
addition to utilizing natural fermentation, it is expected that total bacterial
count is high in all areas sampled.
The results of microbiological examination indicate that this product
is highly contaminated with microorganisms of public health concern. The
high number of total bacterial count, Staphylococcus aureus and E. coli
indicates unhygienic conditions during production of milk and further
processing into “Roub” without heat treatment (Uzeh et al., 2006). Similar
results of total bacterial count were reported for different traditional dairy
products (Beukes et al., 2001; Mathara et al., 2004; Savadogo et al., 2004;
Lore et al., 2005; Al-Tahiri, 2005 and Hassan et al., 2008 ). The detection
of E. coli and Staphylococcus aureus in high number is a public health
concern since it indicates faecal contamination during production or
processing of this product. These organisms were isolated by different
researches in other fermented dairy products (Savadogo et al., 2004; Al-
Tahiri, 2005, Lore et al., 2005; Uzeh et al., 2006).
The results of Lactic acid bacteria count show that fermentation is
mainly carried out by lactic acid bacteria in uncontrolled conditions of
fermentation. Similar results were reported by Beukes et al., (2001),
Mathara et al. (2004), Savadogo et al. (2004), El-Baradei et al. (2008),
Hassan et al. (2008) and Jokovic et al. (2008).
33
Salmonella spp. was not detected in all samples tested. This result was
in disagreement with the results reported by Abdalla and El-Zubeir (2006) in
mish and roub.
“Roub” samples were highly contaminated with yeasts and moulds.
This might be possibl due to poor processing conditions and / or
uncontrolled fermentation which lead to contamination with yeasts and
moulds, and this is obvious by alcoholic fermentation resulting in alcohol
production in addition to lactic acid. Ali et al. (2002), Mathara et al. (2004),
Savadogo et al. (2004), Al-Tahiri (2005), Lore et al. (2005) and Uzeh et al.
(2006) detected yeasts and moulds in different traditional fermented dairy
products.
34
CHAPTER SIX
CONCLUSION AND RECOMMENDATIONS
6.1 Conclusion:
Total viable bacteria were counted in all samples in the three areas
under study, while half of samples were tested positive for Staphylococcus
aureus in two areas and in the third area about 20% of samples were
positive. E. coli was fawned in 35% of the samples under test two areas
while there is no detection in the third area. Salmonella spp. was not
detected in all samples tested, while yeasts and moulds were detected
(100%) in all samples of El Obeid area and 90% of the samples from Nyala
and Abu Naama were infected with yeasts and moulds. Lactic acid bacteria
were detected in all samples under study.
6.2 Recommendations
1. More study of “Roub” microbiology is needed on the following:
a. Isolation and identification of lactic acid bacteria responsible for
Roub fermentation from different areas of Sudan.
b. Production of Roub using improved method to meet the standards
c. More research is needed to improve the quality of the product using
selective starter culture.
d. Isolation and identification of pathogenic bacteria involved in Roub
contamination.
35
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