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

أ

DEDICATION

To my father and mother

To my dear small family wife and son

To all people in my heart

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

1

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).

2

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”.

3

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).

4

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

5

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

6

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).

7

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

8

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

9

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

10

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

11

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).

12

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:-

13

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.

14

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

REFERENCES

Abdalla, W. M. and Al Zubeir I. E. M. (2006). In Microbial hazard associated with fermented milk "Roub and Mish" processing in Sudan. International Journal of Dairy Science, 1(1): 21-26.

Abdel-Malek, Y. (1978). Traditional Egyptian dairy fermentations. Global Impacts of Applied Microbiology, 5:198-208.

Abdel Gadir, W. S., Ahmed, T. K. and Dirar, H. A. (1998). The Ttraditional fermented milk products of the Sudan. Intl.J. Food Microbial, 44: 1-13.

Adams, M. R. (1990). Topical aspects of fermented foods. Trends in Food Science and Technology, 1: 141- 144.

Alfa-laval A. B., Dairy Handbook published by Alfa-Lava, A.B. Dairy Handbook Engineering Division. P.O. Box 1008, S-22103 Lund, Sweden.

Alexander, J. W; Boyce, S. T. and Babcock, G. F. (1990). The process of microbial translocation. Ann. Surg. 212, 496-512.

Ali, M.Y., Islam, M. A.; Alam, M. J. and Islam, M. N. (2002). Quality of yoghurt (Dahi) made in laboratory and available in the market of Mymensingh Town in Bangladesh. Pak.J. Boil. Sci., 5(3): 343-345.

Al-Tahiri, R. (2005). A Comparison on microbial conditions between traditional dairy products sold in Karak and Same Products produced by modern dairies. P. J. Nutr., 4(5): 345-348.

Axelsson, L. (1998). “Lactic Acid Bacteria : “Classification and Physiology” in Lactic Acid Bacteria, Microbiology and Functional Aspects, edited by S. Salminen and A. Von Wright (Marcel Dekker Inc., New York) pp. 1-73.

Barrow, G.I. and Feltham, R. K.A. (1993). Cowan and Steel Manual for the Identification of Medical Bacteria, 3rd ed., Cambridge University Press, London, UK

36

Bentley, R. and Meganathan, R. (1982). "Biosynthesis of vitamin K (menaquinone) in bacteria.   Microbiol. Rev., 46 (3): 241–80.

Beukes, E.M.; Bester, B. H. and Moster, J. F. (2001). The Microbiology of South African traditional fermented milk. In. J. Food Microbiol., 63:189-197.

Bhat, J. V. K. and Fernande, F. (1984). Chemical and microbiological studies on mawa. Indian. J. Dairy Sci. vol. 1: 49-58.

Brigidi, P.; Bolognani, F.; Rossi, M.; Cerre, C. and Matteuzzi, D. (1993). Cloning of the gene for cholesterol oxidase in Bacillus spp., Lactobacillus reuteri and its expression in Escherichia coli. Letters in Applied Microbiology 17: 61-64.

Campbell-Platt, G. (1987). Fermented foods of the world, a dictionary and guide., Butterworths, London. ISBN: 0-407-00313-4.

Carr, F. J.; Chill, D. and Maida, N. (2002). “The lactic acid bacteria: A literature survey”. Critical Rewievs in Microbiology, Vol. 28: 281-370.

De Simone, C. (1986): Microflora, youghurt and the immune system. International Journal of Immunotherapy, Suppl II: 19-23.

DeMan, J. C.; Rogosa, M. and Sharpe, M. E. (1960). A medium for the cultivation of lactobacilli. J. Bacteriol. 23:130.

Dirar, H. A. (1993). The indegenous fermented foods of the Sudan. C. A. B. International, Wallingford.

Duszkiewicz-Reinhard, W.; Gujska, E. and Khan, K. (1994): Reduction of stachyose in legume flours by lactic acid bacteria. Journal of Food Science, 59: 115-117.

El Sadic, G. M.; Naguib, K. and Negm, A. (1972). Acetaldehyde and diacetyl production in "Zabady" and "Zabady' culture. Milchwissen schaft, 27:756.

37

El-Baradei, G.; Delacroix-Buchet, A. and Ogier, J. C. (2008). Bacterial biodiversity of traditional Zabady fermented milk. International Journal of Food Microbiology, 121:295–301.

Feng P., Weagant S. and Grant M. (2002). Enumeration of Escherichia coli and the Coliform Bacteria. Bacteriological Analytical Manual (8th ed.).

Fuller, R. (1989). Probiotica in man and animals. Journal of Applied Bacteriology, 66.

Goldin, B. R. and Gorbach, S. L. (1984). The effect of oral administration on Lactobacillus and antibiotics on intestinal bacterial activity and chemical induction of large bowel tumors. Developments in Industrial Microbiology, 25: 139-150.

Gustafsson, B. E. (1983). Introduction to the ecology of the intestinal microflora and its general characteristics. Nutrition and the Intestinal Flora. B. Hallgren ed. ISBN 91 22 00593 5.

Haberer, P.; Holzapfel, W. H. and Wagner, H. (1997). Moegliche Rolle von Milchsaeurebakterien bei der Cholesterinsenkung im Blutserum. Mitteilungsblatt der Bundesanstalt fuer Fleischforschung, Kulmbach, 36: 202-207.

Harrigan, W. F. and McCance, M. E. (1976). Laboratory Methods in Food and Dairy Microbiology. Academic Press, London.

Harrison, V. C. and Peat, G. (1975). Serum cholesterol and bowel flora in the newborn. American Journal of Clinical nutrition, 28: 1351-1355.

Harun-ur-Rashid MD.; Togo K.; Ueda M. and Miyamoto T. (2007). Probiotic Characteristics of Lactic Acid Bacteria Isolated from Traditional Fermented Milk ‘Dahi’ in Bangladesh. Pakistan Journal of Nutrition 6 (6): 647-652.

Hassan, R. A., EL Zubeir, I. M. E. and Babiker, S. A. (2008). Chemical and microbial measurements of fermented camel milk “Garris” from transhumance and nomadic herds in Sudan. Australian Journal of Basic and Applied Sciences, 2 (4): 800-804.

38

Hepner, G.; Fried, R. R.; Jeor, S. S.; Fuseti, L. and Morin, R. (1979). Hypercholesteremic effect of youghurt and milk. American Journal of Clinical Nutrition, 32:19.

Hesseltine, C. W. and Wang, H. L. (1983). The importance of traditional fermented foods. BioScience, 30: 402-404.

Holt, J. G. (1994). Bergey's Manual of Determinative Bacteriology (9th ed.). Williams and Wilkins. ISBN 0-683-00603-7.

Hosono, A. and Hisamatsu, S. (1995). Binding of amino acid pyrolysates and aflatoxins to autoclaved cells of Enterococcus faecalis FK-23. Bioscience, Biotechnology, and Biochemistry 59: 940-942.

Houghtby, G. A.; Matuin L. .J. and Koeing, E. K. (1992). Microbiological count methods, standard methods, for the microbiological in examination of dairy products 16th edition. Marshall T.R. PHD. Editor, Washington D. C., 20005.

Isolauri, E. (1996). Studies on Lactobacillus GG in food hypersensitivity disorders. Nutrition Today, 31: 28S-31S.

Jokovic, N.; Nikolic, M.; Begovic, J.; Jovcic, B.; Savic, D. and ToPisirovic, L. (2008). A survey of the lactic acid bacteria isolated from Serbian artisanal dairy product Kajmak. Internationl Journal of Food Microbiology, 127: 305- 311.

Kasravi, F. B.; Adawi, D.; Molin, G.; Bengmark, S. and Jeppson, B. (1997). Effect of oral supplementation of lactobicilli on bacteria translocation in acute liver injury induced by D-galactosamine. J. Hepatology, 26: 417-424.

Kosikowski, (1982). Cheese and fermented foods. Edwareds Brothers, Inc. Ann Arbor. Michigan, USA.

Kurmann, J. A. (1994). The production of fermented milk in the world: aspects of the production of fermented milks. Int. Dairy Federation Bull., 179: 16-26.

Lidbeck, A.; Övervik, E.; Rafter, J.; Nord, C. E. and Gustafsson, J. Å. (1992). Effect of Lactobacillus acidophilus supplements on mutagen

39

excretion faeces and urine in humans. Microbial Ecology in Health and Disease, 5: 59-67.

Liljeberg, H.G.M.; Loenner, C. H. and Bjoerck, I. M. E. (1995). Sourdough fermentation or addition of organic acids or corresponding salts to bread improves nutritional properties of starch in healthy humans. Journal of Nutrition, 125: 1503-1511.

Lore, A.T.; Mbugua, K. S.; and Wango, H. J. (2005). Enumeration and identification of microflora in suusac, a Kenyan traditional fermented camel milk product. Lebensm- Wiss.u.- Technol, 38:125-130.

Lovell, R. H. (1990). Dairy microbiology, 2th edition. Robinson, K. R. Editor, London, pp. 250_270.

Madigan, M. and Martinko, J. (2005). Brock Biology of Microorganisms, 11th ed., Prentice Hall, ISBN 0131443291.

Marin, M. L.; Tejada Simon, M. V.; Murtha, J.; Ustunol, Z. and Pestka, J. J. (1997). Effects of Lactobacillus spp. on cytokine production by RAW 264.7 macrophage and EL-4 thymoma cell lines. Journal of Food Protection, 60: 1364-1370.

Marteau, P. and Rambaud, J. C. (1993). Potential of using lactic acid bacteria for therapy and immunomodulation in man. Microbiology Reviews, 12: 207-220.

Mathara, J. M.; Schillinger, U.; Kutima, P. M.; Mbugua, S. K. and Holzapfel, W. H. (2004). Isolation, identification and characterization of dominant microorganisms of kale naoto: the Maasai traditional fermented milk in Kenya. International Journal of Food Microbiology, 64(3): 269-278.

McIntosh, G. H. (1996). Probiotics and colon cancer prevention. Asia Pacific Journal of Clinical Nutrition, 5: 48-52.

Mead, P. S.; Slutsker, L.; Dietz, V.; McCaig, L. F.; Bresee, P. M.; Shaprio, C. and Tauxe, V. (1999). Food-related illness and death in the United States. Emerging Infectous Diseases, 5: 607-625.

40

Mital, B. K. and Garg, S. K. (1995). Anticarcinogenic, hypoholesterolemic and antagonistic activities of Lactobacillus acidophilus. CRC Critic. Rev.\ Microbiol. 21:175-214.

Morgan, D.; Nawman, C.P.; Hutchinson, D.N.; Walkes, A.M.; Rowe, B. and Maijd, F. (1993). Verotoxin-producing Escherichia coli O 157:H7 infections associated with the comsumption of yogurt. Epidemiology and Infection, 111:181-187.

Nord, C. E. and Kager, L. (1984). The normal flora of the intestinal tract. Neth. J. Med, 27: 249-252.

Okuklu, P. (2005). Investigation of chromosomal and plasmid DNA profiles of lactococcus lactis ssp. Lactis. Izmir Institute of Technology. P: 2-5

Perdigon, G.; Alvarez, S.; Rachid, M.; Aguero, G. and Gobbato, N. (1995). Immune system stimulation by probiotics. Journal of Dairy Science 78: 1597-1606.

Poppel, G. V. and Schaafsma, G. (1996). Cholesterol lowering by a functional yoghurt. Food Ingredients Europe, Conference proceedings, 31-32.

Reddy, B. S.; Ekelund, G.; Bohe, M.; Engle, A. and Domellöf, L. (1983). Metabolic epidemiology of colon cancer: Dietary pattern and fecal sterol concentrations of three populations. Nutr Cancer, 5: 34-40.

Reid, G.; Howard, J. and Gan, B.S. (2001). "Can bacterial interference prevent infection?". Trends Microbiol, 9 (9): 424–428.

Robinson, R. K. and Samona, A. (1992). Health aspects of ‘bifidus’ products: a review. International Journal of Food Sciences and Nutrition, 43: 175-180.

Robinson R. K., (1985). Dairy microbiology. Vol 2. The microbiology of milk products. Elsevier Applied Science Publisher; London and New York.

Ryan, K. J. and Ray C. G. (2004). Sherries Medical Microbiology (4th ed.). McGraw Hill. ISBN 0838585299.

41

Saarivirta, (1983). Nutritive value of fermented milk. Handout 4th International training course on fermented milk. Helsinki, 29.7.1983.

Sahlin, P. (1999). Fermentation as a Method of Food Processing. Lund Institute of Technology. Lund University. Department of Applied Nutrition and Food Chemistry, pp: 16 - 17.

Salminen Deighton, M. and Gorbach S. (1993). Lactic acid bacteria in health and disease. Lactic acid bacteria. ed. S. Salminen and A. von Wright, 237-294. New York, USA: Marcel Dekker Inc.

Sato, K.; Saito, H. and Tomioka, H. (1988). Enhancement of host resistance against Listeria infection by Lactobacillus casei: Activation of liver macrophage and peritoneal macrophages by Lactobacillus casei. Microbiol Immunol., 32: 689-698.

Savadogo, C. A. T.; Ouattara1, P. W.; Savadogo A. S.; Ouattara, N. B. and Traore A. S. (2004). Microorganisms involved in Fulani Traditional fermented milk in Burkina Faso. Pakistan Journal.of Nutrition, 3(2): 134-139.

Savage, D. C. (1983). Microbial ecology of the gastrointestinal tract. Nutrition and the Intestinal Flora. B. Hallgren ed. ISBN 91 22 00593 5.

Shahani, K. M. (1983). Nutritional impact of lactobacillic fermented foods. Nutrition and the Intestinal Flora. B. Hallgren ed. ISBN 91 22 00593 5.

Strandhagen, E.; Lia, A.; Lindstrand, S.; Bergstroem, P.; Lundstroem, A.; Fonden, R. and Andersson, H. (1994). Fermented milk (ropy milk) replacing regular milk reduces glycemic response and gastric emptying in healthy subjects. Scandinavian Journal of Nutrition 38: 117-121.

Swank, G. M. and Dietch, E. A. (1996). Role of the gut in multiple organ failure. Bacterial translocation and permeability changes. World J. Surg. 20 : 411-417.

42

Tamai, Y.; Oishi, H.; Nakagawa ,I.; Watanabe, Y.; Shinmoto Tamine, A. Y. and Robinson, R. K. (1988). Fermented milks and their future trends: technological aspects. J. Dairy Res., 55: 281-307.

Tamai, Y.; Oishi, H.; Nakagawa, I.; Watanabe, Y.; Shinmoto, H.; Kuwabara, Y.; Yamato, K. and Nagai, S. (1995). Antimutagenic activity of the milk fermented by mixed-cultured with various lactic acid bacteria and a yeast. Journal of Japanese Society of Food Science and Technology, 42: 383-387.

Taranto, M. P.; Sesma, F.; Pesce de Ruiz Holgado, A. and Valdez, G. F. D. (1997). Bile salts hydrolase plays a key role on cholesterol removal by Lactobacillus reuteri. Biotechnology Letters, 19 : 845-847.

Teuber, M. (1995). “The genus Lactococcus” in The Genera of Lactic Acid Bacteria, edited by Wood, B. J. B. and Holzapfel, W. H. (Blackie Academic and

Thomas, T. D. (1985). Role of lactic acid bacteria and their improvement for production of better fermented animal products. New Zealand J. Dairy Sci. Tec., 20: 1-10.

Thompson, A. (2007). "E. coli Thrives in Beach Sands". Live Science.. Retrieved on 2007-12-03.

Todar, (2007) (Todar's Online Textbook of Bacteriology. University of Wisconsin-Madison, USA.

Tsuji, K.; Ichikawa, T.; Tanabe, N.; Abe, S.; Tarui, S. and Nakagawa, Y. (1992). Effects of two kinds of koji on blood pressure in spontaneously hypertensive rats. Journal of the Agricultural Chemistry Society of Japan 66: 1231-1246.

Uzeh, E. R.; Ohenhem, E. R. and Rojugbokan, K.A. (2006). Microbiological and nutritional quality of dairy products: Nono and Wara. Nature and Science, 4(3).

Van der Waaij, D.; Berghuis-de Vries, J. M. and Wees, L. V. D. (1972). Colonization resistance of the digestive tract and the spread of bacteria to the lymphatic organs in mice. J. Hyg., 70: 335-342.

43

Wells, C. L. (1990).Relationships between intestinal microecology and the translocation of intestinal bacteria. Antoine van Leeuwenhoek, 58: 87-93.

Wilhelm, G. (1993). Studies on the therapeutic properties of acidophilus milk. Symposium of the Swedish Nutrition Foundation.