MICROBIAL CONTAMINATION OF YOGHURT

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 ABSTRACT

An assessment of the microbial contamination of selected yoghurt sold in

Onitsha Market was determined in this study using standard microbiological
procedures. The physical parameters of the brands at time of purchase were also
determined. The results showed that the pH values of the samples ranged from
4.29-4.56, while their temperature readings were between 8 and 170c. The mean
total count of samples on Brain Heart Infusion (BHI) and De Mann Rogosa
Sharpe (MRS) agar media ranged from 2.0×107 to 6.0×108 and 1.0×108 to 5.4
× 108 cfu/ml respectively. The yoghurt isolates were identified as Streptococcus
and Lactobacillus species; these isolates were resistant to commonly used
antibiotics and inhibited the growth of Staphylococcus aureus and Pseudomonas
aeruginosa from clinical samples. No viable growth of isolates was observed in
simulated gastric fluid of pH 1.5 to 2.5. Slight decrease in viable count of
Lactobacillus spp. from 4.0×107 to 3.0×107 cfu/ml and Streptococcus spp. from
3.0×108 to 2.0×108 cfu/ml was observed in bile of pH 8.28 to 8.30. The isolates
were recovered from faecal samples two weeks after ingestion with mean count
ranging from no growth (zero) to 5.8×108 cfu/ml on MRS agar media.

The result of this study therefore indicated poor Microbiological standards of

commercial yoghurts sold in Onitsha market at the time of this research. The
isolates were found to exhibit some probiotic potentials and no pathogen was
isolated from samples. It is recommended that strains of microorganisms that
can deliver full probiotic potentials to consumers be used in commercial
yoghurt production. This result underlines the need for improved hygienic
measures in the processing, storage and distribution of these products to avert
public health challenges.

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 TABLE OF CONTENT

ABSTRACT

CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND OF THE STUDY

1.2 STATEMENT OF PROBLEM

1.4 AIMS AND OBJECTIVES

1.5 SIGNIFICANCE OF STUDY
1.5 LIMITATION OF THE STUDY
1.6 RESEARCH QUESTION
1.7 DEFINITION OF TERMS

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 CONCEPTUAL REVIEW

2.2 YOGHURT CULTURE BACTERIA

2.3 YOGHURT

2.4 HEALTH BENEFITS OF YOGHURT

2.5 YOGHURT APPEARANCE

2.6 FERMENTED MILK PRODUCTS

2.7 MILK FERMENTATION AND BIOCHEMICAL CHANGES

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 METHODOLOGY

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3.1.1 STUDY AREA

3.1.2 SAMPLE COLLECTION

3.2 MICROBIOLOGICAL ANALYSIS

3.2.1. PREPARATION OF MATERIALS

3.2.2. PREPARATION OF SERIAL DILUTIONS

3.2.3. ENUMERATION OF TOTAL AEROBIC BACTERIA (TEB)

3.2.4. ENUMERATION OF COLIFORM BACTERIA

3.2.5. ENUMERATION OF YEAST AND MOULDS

3.3. ISOLATION AND IDENTIFICATION OF MICROORGANISMS

3.3.1. GRAM REACTION

3.4. BIOCHEMICAL TEST

3.4.1. CATALASE TEST

3.4.2. INDOLE TEST

3.4.3. OXIDASE TEST

3.4.4. MOTILITY TEST

3.4.5. COAGULASE TEST

3.4.6. CITRATE UTILIZATION TEST

3.4.7. METHYL RED TEST

3.4.8. VOGES PROSKAUER

3.4.9. CARBOHYDRATE FERMENTATION TEST

3.5 DATA ANALYSIS

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CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 RESULTS

4.2 DISCUSSION

CHAPTER FIVE

5.0 SUMMARY AND CONCLUSION

5.1 SUMMARY

5.2 CONCLUSION

REFERENCES

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 CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Yoghurt consumption has become very popular in Nigeria ever since the
production of locally made yoghurt started. According to Durga et al (2016),
Yoghurt in itself is a very nutritious diet for people across all age groups.
Yoghurt quality varies from one producer to another as there is no well-
described standard for its production. Yoghurt is described as a cultured dairy
product produced by the lactic acid fermentation of milk using a combination of
bacteria such as Lactobacillus bulgaricus and Streptococcus thermophilus in the
ratio of 1:1(Hui, 1992). Yoghurt is made from skimmed milk usually from
cows, sometimes from other animals such as goat or sheep (Miller et al., 1964).
Milk from which yoghurt is made is an excellent source of protein, vitamins and
minerals like calcium and some antibacterial substances such as lysozyme and
lactoperoxidase, as well as large amount of lactose sugar, peptone, phosphate
and nitrogen-based enzymes (Alfa-Lawal, 1984).

Although, it is a traditional beverage in the Balkans and Middle East (Ghandge

et al., 2008), yoghurt is consumed by all people of all nations. Yoghurt is
produced by symbiotic actions of two lactic acid bacteria, namely Streptococcus
thermophilus and Lactobacillus bulgaricus which ferment lactose to lactic acid,
which gives it, its sour taste (Steinkraus, 1997; Tamine and Robinson, 2004;
Kumar and Mishra, 2004; WDC, 2014). Yoghurt can serve as food and plays an
important role in human nutrition, health maintaining, therapeutic and dietetic
functions (Younus et al., 2002; Khan et al., 2008).

The nutritional quality of yoghurt has been reported and is known to contain
high-quality protein, calcium and phosphorous. Its carbohydrate can be utilized
easily by those intolerant to lactose (Younus et al., 2002; Alakali et al., 2008;

6
Ghandge et al., 2008). It is also believed that yoghurt has valuable therapeutic
properties and helps in curing gastrointestinal disorders (Athar, 1986; Wolinsky,
2000; Younus et al., 2002; Vasiljevic and Shah, 2008).

Yoghurt has been described as a nutritiously balanced food containing almost

all the nutrients present in milk but in a more assailable form (Anthar, 1986).
Microorganisms present in fermented dairy products stabilize bowl microflora.
The health claims associated with the consumption of yoghurt include
alleviation of lactose intolerance, lowering of serum cholesterol level, and
treatment of diarrhea and possibility of exhibiting anticancer activity
(Nickerson, 1994). Yoghurt can be consumed as both food and thirst quenching
beverage. Yoghurt due to its high nutritive value is susceptible to contamination
by pathogenic microorganisms causing spoilage (Nickerson, and Sinskey,
1972). Yoghurts may provide additional health benefits, for example it may
reduce cholesterol levels (DiRienzo, 2000). Study suggested that certain
diseases with gastrointestinal tract such as lactose intolerance, diarrhea, colon
cancer and other bacterial infection were inhibited through high consumption of
yoghurt (Dave and Shah, 1997). Molds and yeasts are the primary contaminants
in yoghurt produced commercially in Nigeria. They are responsible for off-
flavor, loss of texture quality due to gas production and package swelling and
shrinkage (Suriyarachichi, and Fleet, 1981).

1.2 STATEMENT OF PROBLEM

Food is one of the vehicle involved in the transmission of diseases.

Microorganisms like all other living things need food to grow and reproduce.
As a result of this, contaminating microorganism consume the chemical
components of food and later replace them with metabolic products, which are
capable of altering the texture, tenderness, flavor and color of food, or even
cause loss nutritional value or become unpleasant and harmful to consumers.

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Millions of people die every year as a result of food borne diseases and about
5000 victims die every year (CDC, 2004).

Poor food handling is largely responsible for food contamination with bacteria,
fungi, and protozoa being the major contaminants and their presence in food
causes a wide range of diseases. The effect of these microorganisms has been
mild, severe or fatal depending on the ineffective dose (CDC, 2004).

1.3 AIMS AND OBJECTIVES

This study was conducted with the aim of investigating the microbiological
quality of yoghurt

The objectives are:

1. To investigate the microbiological contamination and qualities of yoghurt

sold in Onitsha roundabout.
2. To investigate the nutritional qualities of locally yoghurt sold in Onitsha
roundabout.
3. To come out with a recommendation that will reduce the microorganisms
load.

1.4 SIGNIFICANCE OF STUDY

The research work will enable us to know the types of microorganisms

associated with yoghurt sold in Onitsha roundabout. The result obtained will be
used to create awareness and educate the public about the possibility of
contamination of yoghurt produced.

This study will help every small and large-scale yoghurt producer to maintain
adequate hygienic condition to make the good quality and healthy yoghurt
which will reduce the microorganisms load.

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1.5 LIMITATION OF THE STUDY

Microorganism can contaminate all foods and drinks we consume daily but this
particular work focus mainly on the microbial contamination of yoghurt.
1.7 RESEARCH QUESTIONS
At the end of this work, student involved shall be able to give answers to the
following questions:
i. What are the 4 types of microorganisms?
ii. What is microorganism?
iii. What type of bacteria is commonly found in yoghurt?
1.8 DEFINITION OF TERMS

i. Microorganism: is an organism that is so small that it is microscopic

(invisible to the naked eye).

ii. Microbiological contamination: refers to the non-intended or accidental

introduction of microbes such as bacteria, yeast, mould, fungi, virus, prions,
protozoa or their toxins and by-products.
1.9 PROJECT ORGANISATION

The work is organized as follows: chapter one discuss the introductory part of
the work, chapter two presents the literature review of the study, chapter three
describes the methods applied, chapter four discusses the results of the work,
chapter five summarizes the research outcomes and the recommendations.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 CONCEPTUAL REVIEW

Yogurt is a food produced by bacterial fermentation of milk (Fredrickson,

2007). The bacteria used to make yogurt are known as yogurt cultures. The
fermentation of lactose by these bacteria produces lactic acid, which acts on
milk protein to give yogurt its texture and characteristic tart flavor (Fredrickson,
2007). Cow's milk is commonly available worldwide and, as such, is the milk
most commonly used to make yogurt. Milk from water buffalo, goats, ewes,
mares, camels, and yaks is also used to produce yogurt where available locally.
The milk used may be homogenized or not, even pasteurized or raw. Each type
of milk produces substantially different results.

Yogurt is produced using a culture of Lactobacillus delbrueckii subsp.

bulgaricus and Streptococcus thermophilus bacteria. In addition, other
lactobacilli and bifidobacteria are sometimes added during or after culturing
yogurt. Some countries require yogurt to contain a certain amount of colony-
forming units (CFU) of bacteria; in China, for example, the requirement for the
number of lactobacillus bacteria is at least 1 million CFU per milliliter
(Bertrand-Harb et al., 2003).

To produce yogurt, milk is first heated, usually to about 85 °C (185 °F), to

denature the milk proteins so that they do not form curds. After heating, the
milk is allowed to cool to about 45 °C (113 °F).[3] The bacterial culture is
mixed in, and that temperature of 45 °C is maintained for 4 to 12 hours to allow
fermentation to occur (Gilliland, 2009).

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2.2 YOGHURT CULTURE BACTERIA

The thermophilic LAB, Streptococcus thermophilus & Lactobacillus delbrueckii

subsp. bulgaricus are used together as important starter microorganisms in the
production of yoghurt and some kind of cheeses. Because both bacteria are able
to grow alone in milk, this indirect positive interaction is called proto-
cooperation (Fredrickson, 2007). This positive relationship often has a
beneficial effect on bacterial growth and on the production of lactic acid and
aromatic compounds. Lactic acid production results in the lowering of pH and
this makes it unsuitable for growth of spoilage or pathogenic microorganisms
(Donkor et al., 2007).

The proteolytic activity of the two yoghurt bacteria is moderate but is very
significant and leads to symbiotic growth of the two organisms, and production
of flavour compounds. L. bulgaricus is known to be the more proteolytic (Rapp,
2006) of the two bacterial strains used for yoghurt production. L. bulgaricus has
the ability to hydrolyse caseins whereas S. thermophilus has only limited
proteinase activity (Tamime and Deeth, 2010).

A B

C D

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Figure 2.1 Lactic acid bacteria: (A) Yoghurt bacteria (Streptococcus
thermophilus & Lactobacillus delbrueckii subsp. bulgaricus), (B) Lactobacillus
acidophilus, (C) Streptococcus thermophilus, (D) Bifidobacterium bifidum

2.3 YOGHURT

Yoghurt is a very popular flavorful and healthful food and one of a family of
cultured dairy products in all over the world. This product is dependent upon the
fact that casein (the major protein of milk) is insoluble at its isoelectric point
(pH 4.6, where the net charge of the casein is 0). Lactic acid bacteria produce
lactic acid which reduces the pH from the natural pH of milk (pH 6.5-6.6) to pH
4.6 and lower. Yoghurt is produced by lactic acid bacteria that grow best at
about 40o C. Since early times it has been an important food item in the Middle
Eastern Mediterranean coast. Commercial

production of yoghurt increased rapidly in Europe after Metchinkoff`s (2001)

findings that consumption of sour milk prolongs life. The typical yoghurt
flavour is caused by lactic acid, which imparts an acidic and refreshing taste,
and a mixture of various carbonyl compounds like acetone, diacetyl, and
acetaldehyde, the latter of which is considered the major flavour component
(Law, 2001; Ott et al., 2009; Tamime and Deeth, 2010).

Yoghurt or yoghurt-like products have also been used as the most popular
vehicle for incorporation of probiotic organisms (Dave & Shah, 2007). The
LAB must survive in the gastrointestinal tract to provide beneficial properties.
When viable LAB are consumed through fermented milk, the dairy constituents
offer excellent buffering capacity. Furthermore since LAB are in yoghurt (pH 4
- 4.5) the cells may be conditioned to low pH environment and survivability
may be high in gastric juice which has low pH.

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2.4 HEALTH BENEFITS OF YOGHURT

Healthy reasons to eat yoghurt are accumulating especially with the continuing
research findings on the consumption of yoghurt and prevention of diseases
formation. These are briefly described in the following:

1) Many people who cannot tolerate milk either because of protein allergy
or lactose intolerance can enjoy yoghurt. The culturing process makes yoghurt
more digestible than milk (Bertrand-Harb et al., 2003).

2) The friendly bacteria in yoghurt reduces the conversion of bile into

carcinogenic bile acids and this seems to deactivate harmful substances (such as
nitrates and nitrites before they are converted to nitrosamines) before they can
become carcinogenic (Commane et al., 2005).

3) Consumption of yoghurt during antibiotic prescription will minimize the

effects of the antibiotic removal of friendly bacteria in the intestines. The live
bacterial cultures in yoghurt can help replenish the intestines with helpful
bacteria before the harmful ones take over (Macfarlane and Cummings, 2009).

4) Yoghurt can decrease yeast infection and it has prevention of growth of

pathogenic bacteria (Gilliland, 2008).

5) Yoghurt is a rich source of calcium (Smith et al., 1985). Because the live-
active cultures in yoghurt increase the absorption of calcium, serving of yoghurt
gets more calcium into the body than the same volume of milk. Smith et al.,
(1985) and Rusoff (1987) verified that bioavailability of calcium in fermented
milk is high and readily absorbed. Daily intake of yoghurt may also either
reduce the risk of osteoporosis because it increases calcium assimilation in body
or help lactase deficient individuals take steps to prevent osteoporosis (Wynckel
et al., 2001).

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6) Yoghurt is an excellent source of protein (McGee, 2005). Besides being a
rich source of proteins, the limited proteolysis of the milk proteins during
fermentation makes these proteins easier to digest. For this reason proteins in
yoghurt are often called “pre- digested protein”and have beneficial uses for
certain people who lack the digestive enzyme due to disease states (Savaiano
and Levitt, 2004).

7) Fermented milk products are excellent dietary minerals, particularly

calcium, phosphorus, magnesium and zinc (Rusof, 2007).

8) Several LAB are capable of synthesising B-vitamins (Nilson et al., 2005)

and their concentration in fermented milk is generally high (Shahani &
Chandan, 2009).

9) There are a few studies that have shown that yoghurt can reduce the
blood cholesterol. This is because the live cultures in yoghurt can assimilate the
cholesterol or because yoghurt binds bile acids (which has also been shown to
lower cholesterol), or both (Liong & Shah, 2006).

10) Yoghurt and various dairy contain LAB are believed to confer a variety
of important nutritional and therapeutical benefits to consumers including
antimutagenic, anticancer and anti-carcinogenic activity (Rao et al., 2006; Rao
et al., 2009; Fernandes et al., 2007).

11) It is well known that whey proteins, especially β-lactoglobulin (BLG) and
to a lesser extent a-lactalbumin (ALAC), are allergenic (Wal, 2008). Hydrolysis
of these proteins by lactic bacteria may decrease this allergenicity.

12) Certain whey peptides are known to have biological activity such as
opioid and bactericidal activity (Schlimme & Meisel, 2005).

13) Several peptides arising from proteolysis of milk proteins have been cited
as exerting biological activity (Meydani and Ha, 2000) and influence calcium

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absorption and have pharmacological effects on the central nervous system,
cardiovascular system, and digestive system including immuno-modulating
properties (Meisel & Schlimme, 2010).

2.5 YOGHURT APPEARANCE

Appearance and physical characteristics are important quality parameters of

yoghurt. Good quality yoghurt should be thick and smooth with no signs of
syneresis. Set yoghurt with a high level of syneresis on the surface may be
regarded as a low quality product, even though this is a natural phenomenon.
Conventionally, syneresis is reduced by increasing the total solids of yoghurt
mix to around 14% (w/w) with dry dairy ingredients (Tamime & Deeth, 2010)
or by using stabilizers. Dry dairy ingredients such as skim milk powder, whey
protein isolate, whey protein concentrate, sodium (Na)-caseinate or calcium
(Ca)-caseinate are commonly used to increase the solids content of the yoghurt
mix. Nevertheless, fortification with these ingredients affects production costs.
The use of stabilizers including gelatine, modified starches, or gums may affect
the consumer perception of yoghurt. The use of stabilizers is also prohibited in
some European countries (De Vuyst & Degeest, 2009).

2.6 FERMENTED MILK PRODUCTS

Fermented food has a long history of safe usage and is found in diets throughout
the world. Fermentation is broadly defined as a biochemical changes in organic
substances that are caused by the action of microorganisms or enzymes to
produce organic acid, alcohol, carbon dioxide and energy in the form of ATP
(adenosine triphosphate). Fermentation is applicable for many purposes, among
others to extend the shelf life by protection and preservation of foods, producing
desirable taste and flavour, enhancement of nutritional value, producing
required physicochemical properties, improvement of food safety and food
security (Caplice & Fitzgerald, 2009).

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The optimum fermentation conditions (temperature, pH, moisture, nutrient and
oxygen) depend on the type of microorganisms used. Familiar fermentation
includes using yeast in bread-making, production of alcoholic beverages and
conversion of corn into fuel ethanol. Lactic acid bacteria (LAB) carry out
fermentations leading to the production of yoghurt, cheese, sausage, sauerkraut,
wine or beer and pickles. They form a major part of the diet of people around
the world. Some composition of milk products are shown in Table 2.2.
Fermented milk products, including yoghurt and cheese, are formed when
bacteria break down lactose to produce lactic acid, which sours the milk.

2.7 MILK FERMENTATION AND BIOCHEMICAL CHANGES

Microbial fermentation in food fermentation involves the breakdown of sugar

and protein which results in the production of a large array of organic
compounds that contribute to the flavour, preservation and outer appearance of
the food product (Hugenholtz et al., 1999). Milk fermentation is initiated by
lactobacilli and streptococci bacteria which use nutrients in milk for their
growth and alter the nutritional composition and physical appearance of milk
(Loones, 2009).

Lactose is used by lactic acid bacteria (LAB) as the principal source of carbon
for growth and energy. It is initially hydrolyzed by lactase into galactose and
glucose (Greenberg & Mahoney, 2002) followed by subsequent glucose
conversion to D- or L- lactic acid via the glycolytic, Embden-Meyerhof-Parnas
pathway (Hemme et al., 2010). The lactic acid fermentation consists of two
major pathways that include homolactic fermentation which produces lactic
acid and heterolactic fermentation which produce equimolar amount of lactic
acid, carbon dioxide and ethanol (Vakil and Shahani, 2010).

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 Whole Chocolate Yoghurt
milk milk (whole whole Chedar Butter
Nutrients Unit 3.25% fat milk) milk 1cup cheese milk
1 cup
1 cup 1 oz. 1 oz.
Energy Kcal 149.92 208.38 136.64 114.13 99
Water G 214.7 205.75 208.81 10.42 220.82
Protein G 8.03 7.93 14.04 7.06 8.11
Fat G 8.15 8.48 0.44 9.4 2.16
Carbohydrate by
difference G 11.37 25.85 18.86 0.36 11.74
Fibre ,total dietary
G 0 2 0 0 0
Ash G 1.76 2 1.89 1.11 2.18
Minerals
Calcium mg 291.34 280.25 295.72 204.49 285.18
Magnesium mg 32.79 32.58 28.3 7.88 26.83
Iron mg 0.12 0.6 0.12 0.19 0.12
Phosphorus mg 227.69 251.25 233.51 145.18 218.54
Potassium mg 369.66 417.25 378.77 27.9 370.69
Vitamins
Vitamin C mg 2.29 2.28 1.3 0 2.4
Thiamine mg 0.09 0.09 0.07 0.01 0.08
Niacin mg 0.21 0.31 0.02 0.18 0.14
Riboflavin mg 0.4 0.41 0.35 0.11 0.38
Vitamin B6 mg 0.1 0.1 0.08 0.02 0.08
Vitamin B12 mg 0.87 0.84 0.91 0.23 0.54
Vitamin A IU 307.44 302.5 301.35 300.23 80.85
Fatty acids
Saturated G 5.07 5.26 5.14 5.98 1.34
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Monosaturated G 2.36 2.48 2.19 2.66 0.05
Polysaturated G 0.3 0.31 0.23 0.27 0.05
Cholesterol mg 33.18 30.5 31.12 29.74 8.58
Amino acids
Tryptophan G 0.11 0.11 0.05 0.09 0.09
Threonine G 0.36 0.36 0.35 0.25 0.39
Leucine G 0.79 0.78 0.86 0.68 0.81
Lysine G 0.64 0.63 0.76 0.59 0.68
Valine G 0.54 0.53 0.7 0.47 0.6
Arginine G 0.29 0.29 0.26 0.27 0.31
Proline G 0.78 0.77 1.01 0.8 0.82
Tyrosine G 0.39 0.38 0.34 0.43 0.34
Serine G 0.44 0.43 0.41 0.53 0.42
Table 2.2 Comparison of nutrient composition in various milk products

Source: Newer Knowledge of Dairy Foods / Appendix (From USDA, ARS,

USDA Nutrient Database for Standard, Reference, Release 12)

Volatile fatty acids, ethanol, acetaldehyde, acetoin and butanone are yielded
through fermentation. The lactic acid, formed in the reduction of pH of milk,
resulted in a pleasant soury taste.

LAB has very limited capacity to synthesize amino acids by using inorganic
nitrogen sources. Therefore they are dependent on preformed amino acids, the
requirement for amino acids differs among the species of these bacteria in the
growth medium as nitrogen source (Williams et al., 2002). Protein is degraded
by proteolysis and increases the peptide and free amino acid content of
fermented milk products (Livia 2002). Lipids are sparingly hydrolysed by LAB
lipases which are more active towards lower but not higher molecular weight
triglycerides (Yvone et al., 2003; Nihal et al., 1986). Although lipases are

18
present in S. thermophilus & L. delbrueckii subsp. bulgaricus, they have little
effect on free fatty acid content of fermented milk products (Fernandes et al.,
2001). LAB require minerals and vitamins for growth (as mineral catalysis and
mediators in the enzymatic reaction respectively) but their requirement is small
and would not significantly alter the total content of fermented milk products
(Fernandes & Shahani, 2009). The bioavailability of some of the minerals may
be changed due to pH changes caused by fermentation.

Numerous scientific papers and review articles (Hughes and Hoover, 2001;
Kurmann and Rasic, 2001; Modler et al., 2010) have reported the health
benefits associated with the consumption of fermented dairy products. Some of
the proposed health benefits are thought to be conferred by live bacteria
contained in the products. For instance the higher free amino acid content in
fermented milk is possibly due to partial hydrolysis of the milk proteins by LAB
(Friend & Shahani, 2004).

Foods containing probiotic bacteria which were originally used as a mean to

enhance storage life in much of the undeveloped countries are currently
categorized as "functional foods". Such products are now gaining widespread
popularity and acceptance throughout the developed world. A number of health
benefits for product containing live probiotic bacteria have been claimed
including alleviation of symptoms of lactose intolerance, treatment of
diarrhoea, anti-carcinogenic properties (Daniel et al.,2005), antimutagenic
activity (Nadathur and Bakalinsky, 2005), reduction in blood cholesterol and
improvement in immunity.

19
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 METHODOLOGY

3.1.1 STUDY AREA

This study will be conducted in Yola North Local Government of Adamawa

State. The region is characterized by a balance dry and rainy seasons. The rainy
days occur between May and October and the dry season occurs between
November and April. The primary occupation of the people is farming, business
and civil servants.
3.1.2 SAMPLE COLLECTION
Yoghurt samples will be purchased randomly from Supermarket and other local
vendors in the market and will be wrapped properly to avoid the contact with
air. All possible efforts will be made to minimize the time lag between
collection and analysis, so that no significant change in yoghurt quality would
occur. Samples will then be transported to the laboratory as soon as possible in
an insulated foam box with ice to maintain temperature ranging from 4°C to
6°C for analysis.
3.2 MICROBIOLOGICAL ANALYSIS

3.2.1. PREPARATION OF MATERIALS

All media will be obtained in dehydrated forms and prepared according to the
manufacturer’s instructions. Glassware such as Petri-dishes, test tubes, pipettes,
flasks, and bottles will be sterilized in a hot oven at 170°C for two hours,
whereas distilled water will be sterilized by autoclaving for 15 min at 121°C.

3.2.2. PREPARATION OF SERIAL DILUTIONS

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This will be done according to APHA in which 1 ml of yoghurt from a
homogenous sample is serially diluted into 9 mL of sterile distilled water to
prepare eightfold dilutions from 10−1 to 10−8. 50 μl of diluted samples will be
spread over prepared dried plates with different media.

3.2.3. ENUMERATION OF TOTAL AEROBIC BACTERIA (TEB)

Nutrient agar will be used to determine the total aerobic bacterial count and
appropriate dilutions will be pour-plated. The cultured plates will be incubated
aerobically at 37°C for 24 hours. TEB will be counted after the colonies are
evaluated.

3.2.4. ENUMERATION OF COLIFORM BACTERIA

MacConkey agar(supplemented with0.5g/l nystatinwill be used to determine the

coliform count. The cultured plates will be incubated aerobically at 37°C for 24
hours after pour plating of the appropriate dilutions. The colonies will be
evaluated and counted at the end of the incubation.

3.2.5. ENUMERATION OF YEAST AND MOULDS

Sabouraud Dextrose agar (supplemented with 0.5 g/l chloramphenicol) will be

used to determine yeast and mould counts. After the pour-plated plates will be
incubated aerobically at 25°C for 3–5 days, the developed colonies will be
evaluated and counted.

3.3. ISOLATION AND IDENTIFICATION OF MICROORGANISMS

The distinguished colonies on the incubated plates will be picked and purified
by repeated sub culturing done by streaking on the appropriate media with a
sterile loop (the strategy consisted of picking 1 colony to represent every visibly
different morphology on each plate) using the streak method. Purified colonies
will be prepared in their respective agar: Nutrient agar for total aerobic bacteria,

21
MacConkey agar for coliforms and Sabouraud Dextrose agar for yeast and
moulds. From these preparations, 0.5 ml of each will be pipetted into 0.5 ml of
glycerol and stored in a freezer at −5°C awaiting identification. Bacterial
isolated from the samples will be characterized based on colonial,
morphological, standard microbiological and biochemical reactions. All the
bacterial cultures were subcultured prior to their use in further experiments and
the obtained fresh cultures were used for biochemical tests.

By microscopic observation of each culture following incubation, the purity of

isolates was confirmed and preliminary identifications were done.

3.3.1. GRAM REACTION

Gram staining reaction has the wide application that is capable of

distinguishing virtually all bacteria into one of two large group which are the
gram positive or gram negative as described by Dr Hans Christian Gram (1884).
Smear of each isolate will be made on the slide and heat fixed. Primary stain
(crystal violet) will be added in drops. Lugol’s iodine will be added for 45
seconds decolorized with acetone and washed with water. It will then be air
dried examined at Xl00 under oil immersion. Positive gram staining appears
purple and negative grams staining appears pink.

3.4. BIOCHEMICAL TEST

3.4.1. CATALASE TEST
1ml of 3% of hydrogen peroxide will be transferred into clean test tubes. A
colony from a 24hours pure culture will be inoculated into the hydrogen
peroxide solution. Positive catalase test shows the presence of gas bubbles while
a negative test reveals absence of gas bubbles. The test is used to identify
Staphylococcus aureus.
3.4.2. INDOLE TEST

22
This test detects the ability of certain bacteria to decompose the amino acid
tryptophan with the release of indole which accumulates in the medium the test
organism is inoculated at 37°C. indole production will be detected by adding
1ml of kovac’s reagent to overnight culture. Indole reagent contains
dimothilaminobenzaldyde. The reaction with the indole gives a pink-red
colouring of surface layer within 10minutes if positive and there is no pink-red
colouring if negative.
3.4.3. OXIDASE TEST
A few drops of kovac's reagent will be added to piece of filter paper on a petri
dish. The bacteria isolates will then be smeared on the filter paper with a glass
rod. The paper will be observed. Positive result gives a dark purple color while
negative result shows no color change. This test will be used to identify
coliforms.
3.4.4. MOTILITY TEST
This test is done to differentiate motile from non-motile organisms. A wire loop
will be used to inoculate a motility medium by making a stab to the bottom of
the tube and incubated afterwards for 24-48 hours. If the organism is motile, the
tube will appear cloudy and the organism will spread out of the stab line, Non-
motile organism will grow along the streak line only and the media will not be
cloudy.
3.4.5. COAGULASE TEST
The use of blood plasma is being introduced in coagulase test. A loop full of
human plasma will be added to culture isolate on a slide. Positive isolate gives
agglutination reagent with plasma. Test will also be carried out at 37°C for 24
hours' positive tubes shows coagulation of the plasma in the tube. This test was
used to identify Staphylococcus aureus.
3.4.6. CITRATE UTILIZATION TEST
This test is based on the ability of the test organism to use citrate as a carbon
source for metabolism with resulting alkalinity and utilization of ammonia as its

23
source of nitrogen. Using a straight wire 2¬-3mls of sterile slanted Simmon
citrate agar will be inoculated with the test organism. The inoculated media will
be incubated at 37oc for 24 hours. Citrate utilization will be shown by change in
colour of the indicator from light green to dark blue.
3.4.7. METHYL RED TEST
This will be used to detect the production of sufficient acid during fermentation
of glucose which will be indicated by change in colour of the methyl red
indicator. Isolates will be inoculated into tube of previously prepared glucose
peptone and incubated at 37oC for 2 days. Then 5 drops of methyl red solution
will be added to each tube and colour change will be observed. Positive result
gives yellow with the indicator as reported by Raima.
3.4.8. VOGES PROSKAUER
Tubes of glucose phosphate peptone water will be inoculated and incubated at
37°C for 2 days.
Ml of 40% KOH and 3 ml of 5% solution of 2-naphrol in absolute ethanol will
be added to each tube. A positive result gives crimson colour in 30 minutes.
3.4.9. CARBOHYDRATE FERMENTATION TEST
It tests for the presence of acid and/or gas produced from carbohydrate
fermentation. Basal medium containing a single carbohydrate source such as
Glucose, Lactose, Sucrose or any other carbohydrate is used for this purpose. A
pH indicator (such as Andrade’s solution, Bromcresol purple (BCP),
Bromothymol blue (BTB) or Phenol red) is also present in the medium; which
will detect the lowering of the pH of the medium due to acid production. Small
inverted tubes called Durham tube is also immersed in the medium to test for
the production of the gas (hydrogen or carbondioxide)
3.5 DATA ANALYSIS

Data obtained will be analyzed using percentage and formula respectively.

Values were tested using Analysis variance (ANOVA).

24
25
 CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

The analysis of the samples and materials are presented in the following
sections.

4.1 RESULTS

The total bacterial count ranges from 2.0 × 107 to 6.0 × 108 on BHI and from
1.0 × 108 to 5.4 × 108 on the MRS (Table 1), only sample E had no growth on
the MRS agar medium after the period of incubation. The different
characteristics of the colonies were observed and represented in the Table 1.

Table 1a: Mean total count and colonial characteristics of isolates on BHI
and MRS agar.

Sampl Mean total Colon Size Shape Elevation Margin Colour Surface appearance
e code count cfu/ml y code (mm)

BHI
medium
YGA 3.0 × 107 A1 5 Regular Low Entire Cream Moist, shiny and mucoid
convex
A2 <1 Regular Low Entire Yellow Moist and shiny
convex
YGB 5.0 × 107 B1 1 Regular Low Entire Cream Moist and shiny
convex
B2 <1 Regular Low Entire Yellow Moist and shiny
convex
YGC 2.0 × 107 C1 3 Regular Low Entire Cream Moist and shiny
convex
C2 <1 Regular Low Entire Yellow Moist and shiny
convex
8
YGD 6.0 × 10 D1 8 Regular Low Entire Cream Moist, shiny and mucoid
convex

26
 D2 2 Regular Low Entire Yellow Moist and shiny
convex
YGE 5.0 × 108 E1 <1 Regular Low Entire Cream Moist and shiny
convex
E2 6 Regular Low Entire Yellow Moist and shiny
convex

MRS
medium
YGA 2.0 × 108 A 4 Regular Flat Entire Cream Mucoid and dry
YGB 1.0 × 108 B 3 Regular Low Entire Cream Moist and shiny
convex
YGC 5.0 × 108 C 5 Regular Flat Entire Cream Moist and shiny
YGD 5.4 × 108 D 3 Regular Low Serrated Cream Moist and shiny
convex
YGE NO - - - - - - -
GROWTH

Key: YGA = HOLLANDIA YGB = HABIB YGC = PINKBERRY YGD =

YOGURBERRY YGE = FARMFRESH

The susceptibility of the yoghurt isolates to commonly used antibiotics is shown

in Table 2. Streptococcus species were resistant to amoxicillin and three other
commonly used antimicrobials, while sensitive to gentamycin and ciproflaxacin.
Lactobacillus species were resistant to gentmycin, streptomyc and amoxicillin.
Table 1b: Physical parameters of the yoghurt samples analysed
Sample No. Average pH values Average
temperature
values (0C)
YGA 4.32 17
YGB 4.29 16
YGC 4.35 8
YGD 4.34 9

27
 YGE 4.56 5
Key: YG = Yoghurt

Table 1b shows the results of the analysis of the physical parameters of the
samples. Temperature readings ranged from 8 to 170C while pH readings were
between 4.29 and 4.56. Also the pH readings of between 4.29 and 4.46 are
somewhat above the high acidity and low pH of between 3.8 and 4.2 expected
for yoghurt storage. At this pH of 3.8 – 4.2, yoghurt is not a hospitable medium
for pathogens which will not grow and will not survive well either. These poor
conditions as reported in this study do not support the delivery of wholesome
yoghurt to consumers as asserted by Nduka (2004).
Table 2: Susceptibility of yoghurt isolates to commonly used antibiotics
Inhibition Inhibition zone S. aureus zone
Antibiotics Conc. (µG)Sensitivity Sensitivity
zone (mm (mm
(mm) ) )
Amoxycillin 25 r 8 r 8 10
Ofloxacin 5 s 20 s 20 15
Streptomycin 10 s 15 r 8 12
Chloramphenicol 30 s 12 r 9 14
Ceftriazone 30 r 6 s 16 9
Gentamycin 10 s 14 r 10 14
Pefloxacin 5 s 12 s 12 12
Cotrimaxozole 25 r 5 s 17 10
Ciproflaxacin 10 s 15 s 20 15
Erythromycin 5 s 14 s 12 11
Key: S = sensitive; R = resistant.
The yoghurt isolates were found to posses varying inhibitory activity against S.
aureus and P. aeruginosa. While the Lactobacillus spp. had inhibitory activity
against S. aureus and P. aeruginosa, Streptococcus spp. had no effect against S.
aureus.

28
 The experiment shows that when yoghurt isolates was exposed to a simulated
gastric fluid at the pH 1.5 to 2.5, all the yoghurt isolates (Lactobacillus spp. and
Streptococcus spp.) after incubation period of 24 h at 37°C, were found to have
no viable count.
Table 3: Mean total count and colonial characteristics of bacteria isolates
from faeces on MRS agar
Sample Yoghurt Mean Colon Size Shape Elevation Margi Colour Surface
code name total count y code n appearance
(mm)
YGA Hollandia No growth - - - - - - -
YGB Habib No growth - - - - - - -
YGC Pinkberry 5.0 × 108 C 5 Regular Low Entir Cream Moist and
convex e shiny
YGD Yogurberry 1.0 × 108 D 7 Regular Low Entir Cream Moist and
convex e shiny
YGE FarmFresh 4.8 × 107 E 3 Regular Low Entir Cream Moist and
convex e shiny
Key: YG = Yoghurt
Exposure of yoghurt isolates to bile at pH 8.29 to 8.30 shows that the isolates
(Lactobacillus spp. and Streptococcus spp.) survived a period of 24 h incubation
at 37°C. The viable counts of the Lactobacillus spp. isolates were, however,
slightly decreased from 4.0 ×107 to 3.0 ×107 cfu/ml. While the Streptococcus
specie decreased from 3.0 × 108 to 2.0 × 108 cfu/ml.
After a period of two weeks, viable growth of Lactobacillus on MRS was
obtained from faecal samples of individuals that consumed yoghurt samples
except for samples A and B (Super Sunnex and GT yoghurt) which had no
growth. The mean total bacteria count ranges from zero to 5.8×108 cfu/ml
(Table 3).
4.2 DISCUSSION

29
The study on safety and probiotic potentials of different yoghurt brands sold in
Onitsha has shown that the yoghurt producing companies provides information
such as batch number, manufacturers address, NAFDAC number, but they do
not give information on microbial composition/contents.

The study has also shown that some yoghurts circulating in Onitsha south–east
Nigeria contains viable microorganisms which are able to survive production
process, preservation temperature and refrigeration temperature of 4°C. The
gram reaction and microscopic examination of the yoghurt isolates reveals that
all the isolates were gram positive and there was no trace of gram negative
bacteria, which might indicate the presence of contamination from coliforms/
enterobacteriaceae. The microbiological and biochemical analysis reveals the
identified organisms were Lactobacillus species and Streptococcus species.

Yoghurt isolates were resistant to some commonly prescribed antibiotics.

Amoxicillin, streptomycin, ceftriazone, chloramphenicol, gentamicin and
cotrimazole were not effective against all yoghurt microflora, while oflaxacin,
pefloxacin, and erythromycin, were very active against the yoghurt microflora.
Antibiotic resistant in bacteria may be intrinsic or acquired. Intrinsic resistance
is a naturally occurring trait and may be considered as specie characteristics,
whereas acquired resistance drives either from genetic mutation or acquisition
of foreign DNA from other bacteria (Teuber et al., 1999; Belletti et al., 2009).
The relationship between antibiotic use and resistance was reviewed by Singer
et al. (2006).

In several fermented foods as in probiotic, Lactobacilli are often a relevant

microbial component and can interact with gut microflora (Ammor,
2007).There is need for further investigation on the antibiotic resistance
implication of LAB because in spite of the large consumption of live
Lactobacilli, the presence of acquired antibiotic resistance is not crucial for their

30
classification as generally recognized as safe by the U.S. Food and Drug
Administration (Belletti et al., 2009). It has been reported that there is no barrier
between pathogenic (for example, streptococci), potentially pathogenic (for
example, enterococci), and commensal (for example, lactobacilli and
lactococci) LAB regarding acquired antibiotic resistance, and identical genes
responsible for resistance are found among these organisms (Mathur and Singh,
2005; Belletti et al., 2009). Horizontal transfer of resistance factor from
Lactobacillus to Enterococcus of a gene involved in the resistance towards
erythromycin and tetracycline has been shown (Jacobsen et al., 2007; Ouoba et
al., 2008).

The study has shown that the Lactobacillus species and Streptococcus species
had inhibitory activity against S. aureus and P. aeruginosa; this means that the
yoghurt isolates have the ability to inhibit the growth of some pathogenic
organisms when found in the same habitat, such as the gastro intestinal tract.
Several researches on effect of probiotics on pathogenic organisms reveals that
regular intake of probiotics can reduce potentially pathogenic bacteria in upper
respiratory tract and GIT (Ulrich and Jan-Olaf, 2003; Hoveyda et al., 2009;
Allen et al., 2010). Beneficial effect of probiotics has been observed in several
models of gastrointestinal infection, clinical trials in colonized human adults
and children show that while probiotics do not completely eradicate pathogens
such as H. pylori, they maintain significantly lower levels of the bacterium and
thus, in combination with antibiotics, increase eradication rates and/or decrease
antibiotics’ adverse effects (Gotteland et al., 2006).

The application of probiotic cocktails (combinations of two or more strains with

potentially different mechanisms of antimicrobial action), have been reported
(Hickson et al., 2007; Sleator, 2010). Using a mixture of two strains of
Lactobacillus murinus and one strain each of Lactobacillus
salivarius subspecies salivarius, Lactobacillus pentosus and

31
Pediococcus pentosaceous (collectively referred to as LIVE5). Casey et al.
(2007) obtained significant reductions in both clinical and microbiological signs
of Salmonella typhimurium infection in a porcine model animals treated with
this cocktail. Hamilton-Miller (2003), Johnson-Henry et al. (2004) and Hoveyda
et al. (2009) noted that a mixture of Lactobacillus strains reduces gastric
inflammation and bacterial colonization in Helicobacter pylori-infection and in
irritable bowel syndrome. Furthermore, probiotics have also been demonstrated
to be effective against enteric viruses, particularly rotavirus (Heyman, 2000).
Besides treating enteric infections, “designer probiotics” have been recruited to
combat HIV (Rao et al., 2005; Lagenaur and Berger, 2005), and reduction in
pregnancy related complications have also been reported (Braga et al., 2011;
Myhre et al., 2011).

Exposure of the yoghurt isolates to simulated gastric fluid of pH 1.5 to 2.5

resulted in no growth. This indicates that the specific pH levels and incubation
time condition affected the survival of the yoghurt isolates. In that case, strains
with high acid tolerance should be used in the production of yoghurt, so as to
enable them survive in the gastro intestinal tract and confer desired health
benefits. Acid stability and intestinal mucosal adhesion properties are among
the criteria used to select probiotic microbes (Salminens et al., 1998; Sheehan et
al., 2006, 2007). Viability is an important factor, but not the only criteria for
quality assurance (Elina et al., 2001). One of the limitations of probiotics in
clinical application is that the most effective probiotic strains often prove to be
fragile during industrial processing such as drying or heating (Sheehan et al.,
2006). Improving the stress tolerance of probiotic strains is thus an important
biological and clinical goal (Sheehan et al., 2007; Sleator and Hill, 2008).

32
 CHAPTER FIVE

5.0 SUMMARY AND CONCLUSION

5.1 SUMMARY

The study showed the trend of tolerance of yoghurt isolates to bile at 8.29 to
8.30. Here, all isolates of Lactobacillus and Streptococcus species survived
treatment with bile at the incubation period of 37°C for 24h. The viable counts
of the Lactobacillus specie and Streptococcus specie, however, decreased from
4.0×107 to 3.0×107 cfu /ml and from 3.0×108 to 2.0×108 cfu /ml respectively.
Other studies on lactic acid bacteria corroborate with these findings (Haller et
al., 2001). Bile production contributes to the antimicrobial arsenal developed by
mammalian host to inhibit microbial colonization and potential infection; this
could contribute to reduction in viable cell count as observed in this study. The
bile resistant traits of intestinal yoghurt isolates are crucial for maintaining
viability during GI transit and are desirable attributes of an orally administered
probiotic (Charteris et al., 2000; Elina et al., 2001; Elkins and Lisa, 2004).

This study also showed that the yoghurt samples after being consumed by
healthy volunteers, two weeks after the gastro intestinal tract sill had a good
number of the yoghurt microflora. It could be explained that yoghurt microflora
were able to adhere and colonize the intestinal mucosa, these characteristics are
necessary for selecting probiotics for commercial use in foods and therapeutic,
to ensure that they are retained inherently. The ability to adhere is also, to some
extent likely to be connected to the ability to stimulate the immune defense
(Ouwehand et al., 2009; Plant and Conway, 2002). However, permanent
colonization by the probiotics is unwanted and remains unsolved whether
colonization is critical for probiotics to have their effect at all (Ouwehand et al.,
2009; Fedorak and Madsen, 2004). That the isolates were found/recovered from

33
stool even though they did not survive the simulated gastric fluid after 24h
incubation, calls for further investigation.

5.2 CONCLUSION

It can be concluded that some yoghurts circulating in Yola North of Adamawa

state Nigeria, contain no contaminating gram negative
microorganisms/coliforms and are thus acceptable. The starter culture used in
the production possesses some desired attributes of a probiotic organism such as
surviving production process, storage temperature, resistance to bile and some
commonly used antibiotics, ability to inhibit the growth of some pathogenic
organisms, and colonization/adherence to intestinal mucosa (recovery from
faecal samples). The use of strains of probiotic organisms better adapted to
resist gastric acid and more of the commonly used antibiotic is advocated,
further work in this regard is also necessary.

Milk can harbor a number of microorganisms and can be a good source of food
borne diseases. In most foods, the total bacterial count is often an indication for
the sanitary quality, safety and utility of foods. It may reflect the conditions
under which the product is manufactured such as contamination of raw
materials and ingredients, the effectiveness of processing and the sanitary
conditions of equipment and utensils at the processing plants. It is possible to
have pathogen-free yoghurts through high and strict preventive measures.

34
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