Chemical and Microbiological evaluation of Set   yoghurt during shelf life 

By 

 SalahEldein Hussein Mohammed  

B.Sc. (Honour) Natural Resources and Environmental Studies University of Kordofan  

(2005) 

 

A thesis Submitted in Partial Fullilment for the Requirements of the Degree of M.Sc in Dairy Production and Technology 

 

Supervisor  

  Dr. Mohamed Osman Mohamed Abdalla  

 

Department of Dairy Production 

 Faculty of Animal Production 

 University of Khartoum 

November 2008 

 

 

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Dedication 

 

To the soul of my mother  

To my dear father, brothers and sisters 

To my friends and colleagues  

ftÄt{xÄwx|Ç

 

 

 

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ACKNOWLEDGMENTS 

  Praise be to Allah 

I wish  to  express my  sincere  thanks  and  gratitude  to Dr. Mohamed Osman  Mohamed  Abdalla  for  his  helpful  supervision, constructive  criticism  and  valuable  guidance  throughout  this work.  

My special thanks are due to Dr. Ibtisam Elyas Mohammed Elzubeeir, head  Department  of  the  Dairy  Production  for  her  continuous support, encouragement and kindness. 

My  appreciation  is  due  to  Dr.  Mohammed  Khair  Abdalla  for  his cooperation  in  choosing  the  suitable  experimental  design  and analysis of the data and unlimited help freely offered     to me. 

My thanks are due to Mr. Moawia Albirer and Best factory family for their help during collection of samples. 

Also my  thanks  are  extended  to my  colleagues  in Dairy  Production and  Technology,  Course.  Special  thanks  are  due  my  friends, Mortada Mohammed, Ibrahim Homeda and Rania Abd el‐Gader, I wish to express special thanks to my family.  

 

 

 

 

 

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LIST OF CONTENTS 

  PageDETECTION  i ACKNOWLEDGMENT  ii LIST OF CONTENTS  iii LIST OFTABLES  v ABSTRACT  vi ARABIC ABSTRACT  vii CHAPTER ONE: Introduction                                                               1 CHAPTER TWO: LITRETURE REVIEW                                           3 2.1 Fermentation and fermented milk products                                    3 2.2 Starter cultures   4 2.3 Types of yoghurt  5 2.4 Manufacture of yoghurt  7 2.5 Preparation of the basic mix  9 2.6 Homogenization   9 2.7 Heat treatment of milk   9 2.8 Production of set yoghurt      10 2.9 Cooling of yoghurt  10 2.10 Packaging of yoghurt                        11 2.11 Yoghurt properties  11 2.12 Fermentation and microbiological aspects  13 2.13 Shelf life of yoghurt  13 2.14 Defects of yoghurt  14 2.15 Nutritional value of yoghurt   15 CHAPTER THREEMATERIALS AND METHODS  16 3.1 Collection of samples   16 3.2 Chemical analysis of yoghurt  16       3.2.1 Determination of total solids content   16       3.2.2 Determination of titratable acidity   16 

 

 

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      3.2.3 Determination of fat content                 17       3.2.4 Determination of protein content  17 3.3  Determination of wheying‐off  18 3.4  Microbiological examination  18        3.4.1 Sterilization of equipment   18        3.4.2 Preparation of media  18               3.4.2.1 Violet Red Bile Agar   18               3.4.2.2 Lactobacillus MRS (Man Rogosa and Sharpe) Agar  19               3.4.2.3 Acidified potato dextrose agar (P.D.A)  19 3.5 Culture Methods  19 3.6 Statistical analysis   20 CHAPTER FOUR RESULTS AND DISCUSSION   21 4.1 Chemical composition of yoghurt  21 4.2 microbiological properties of set yoghurt  22 Table (1) fat contents and protein contents of set yoghurt samples during shelf life.  23 Table (2) Total solids contents and Acidity percentage of set yoghurt samples during shelf life.  24 Table (3) Wheying‐off percentage separated during shelf life  25 Table (4) Coliform count of Set yoghurt samples during shelf life  27 Table (5) Yeasts and Molds count of set yoghurt samples during    shelf life  28 Table (6) Lactobacillus bulgaricus set yoghurt samples during shelf life  29 CHAPTER SIX CONCLUSION AND COMMENDATIONS  30 Conclusion  30 Recommendations                    30 REFERENCES  31 

                             

  

 

 

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LIST OF TABLES

 

 

 

LIST OF TABLES

 

 

 

 

Table                                                                     

page  page1  Fat and protein contents of set yoghurt samples during  shelf life .                      23 2  Total solids contents and Acidity percentage of samples during  shelf life .        24 3  Wheying‐off percentage separated during  shelf life                                               25 4  Coliform count of Set yoghurt samples during  shelf life  27 5  Yeasts and Molds count of set yoghurt samples during  shelf life .                       28 6  Lactobacillus bulgaricus set yoghurt samples during  shelf life                                 29 

 

 

vi

 

ABSTRACT 

 

A study was performed to determine the effect of shelf life on chemical and microbiological characteristics of set yoghurt manufactured in Khartoum State. Samples were collected and kept in the laboratory at refrigerator temperature.

The chemical analysis was carried out to determine the contents protein, fat and total solids contents. The physiochemical analysis included titratable of acidity and wheying-off. Whereas should be analyzed and microbiological examination (coliform, lactobacillus bulgaricus and yeasts and molds counts) were performed at 1, 3, 6 and 9 day intervals.

Results revealed that protein, fat, total solids and acidity and whey-off did not show any significant difference (P>0.05) effects throughout the storage period.

Results of microbiological examination showed non significant variation (P>0.05) in the count of coliform bacteria, yeasts and molds and Lactobacillus bulgaricus throughout the shelf life of 9 days.

 

 

 

 

vii

 

المستخلص

ت ة هذه أجري يم  الدراس ي  لتق ائي والميكروب أثير الكيمي الت

ادي رةللزب ك الخث صالحية المتماس رة ال الل فت ادي . خ ات الزب ت عين جمع

وم ة الخرط ه بوالي بن ومنتجات صنيع الل صنع لت ن م ى ت و حفظ ،م ة عل درج

.حرارة الثالجة

ائى ل الكيم ى التحلي ة عل تملت الدراس ة أش سبة ( لمعرف روتينن ،الب

دهن ة و ال د الكلي ائي .)الجوام ل الفيزي د و التحلي ه لتحدي سبة الحموض ن

شرش سبة ال م ،ون ا ت ل آم ة تحلي ات لمعرف أثيرالعين وجي الت الميكروبيول

ى د الكل طة الع ل بواس ن لك ا ال م ورم بكتيري لسء،كلوروف الآتوباس

).عشرة أيام (الصالحية فترة خالل والفطريات، الخمائرو بلقاريكس

ائج وأ حت النت سبةأن ض روتين، ن دهن، الب د  ال ة الجوام ، الكلي

ة شرش الحموض سبة ال م ون أثر ل ا تت ال معنوي رة لخ صالحية فت )P>0.05 (ال

ا حتو أ آم ائج ض دد أن النت ى الع ا الكل ا للبكتري ة والبكتري ض المنتج لحم

ك ائر الالآتي ات والخم م والفطري أثر ل ا تت الل معنوي رة خ صالحية فت  ال

)P>0.05(.  

 

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CHAPTER ONE

Introduction

Yoghurt is a fermented milk product obtained by fermentation of lactose

to lactic acid by the action of two types of lactic acid bacteria Streptococcus

thermophilus (S. salivarius-ssp. thremophilus) and Lactobacillus bulgaricus

(L. delbrueckii ssp. bulgaricus) and these two microorganisms must live in

large numbers in the final product (Tayfour, 1994).

Yoghurt is an extremely popular fermented milk food in Europe, Asia

and Africa. It is known by quite different names in different parts of the

world: Leben or Raib (Egypt- Saudia Arabia) Madzoon or Matzoon

(Armania), Naja (Bulgaria) and Dalia (India) (Kosikowski, 1966).

In many developing countries of Asia and Africa, yoghurt is more likely

to be produced as naturally soured milk and consumed by the adult people

more than fresh whole milk. It is generally considered a safe product and its

unique flavor appeals to so many people. That consideration is incorporate in

expensive nutrients make it an almost complete food in these areas .In

particular, consumers in the Americans are now more aware of the fine

properties of yoghurt, and its consumption is increasing particularly in large

cities (Kosikowski,1966).

It is produced in different forms such as whole milk yoghurt, skim milk

yoghurt, cream yoghurt, fruit yoghurt and liquid yoghurt (Balasubramanyam

and Kulkarnis, 1991).

In 1973, Food and Agriculture Organization (FAO) and World Health

Organization (WHO) of the United Nations put certain standards for types of

yoghurt according to their fat content, and hence, yoghurt was classified into

whole fat (above 3.0%), moderate fat (3.0-0.5%) and low fat yoghurt

2

(<0.5%), set yoghurt and liquid yoghurt (of low viscosity) (Tamime and

Deeth, 1980) and these may be plain (natural) or they may contain additives

like fruit or flavors, or it may be coloured (Tayfour, 1994). A major

concern of yoghurt industry is the production and maintenance of a product

with optimum consistency and stability. The factors known to improve

consistency are increasing total solids, manipulation of processing variables

and characteristics of starter culture.

The objectives of study are:

1- Evaluation of chemical characteristics of set yoghurt during shelf life.

2- Evaluation of microbiological quality of set yoghurt during shelf life.

3

CHAPTER TWO

LITRETURE REVIEW

2.1 Fermentation and fermented milk products

Fermentation of milk was originated in the Near East and through

Central and Eastern Europe. Fermentation is defined as any modification of

chemical or physical properties of milk or dairy products, resulting from the

activity of microorganisms or their enzymes which cause the main marked

changes (Frank and Marth, 1988). Moreover, the earliest example of

fermented milk was warm, raw milk from cows, sheep, goats, camels or

horses of the nomads roaming the area (Elmardi, 1988). Fermentation was

first used around ten thousand years ago when humans made the transition

from food collectors to food producers and was a common method to extend

the longevity of dairy products. Today, this process remains the same except

that Streptococcus themophilus and Lactoobucillus bulgaricus can be used in

the fermentation of yoghurt (Tamime and Robinson, 1991).

Fermented milk products are cultured dairy products made from skim,

whole or slightly concentrated milk that require specific lactic acid bacteria to

develop their characteristic flavor and texture (Thapa, 2000). Fortunately, the

dominant bacteria were lactic acid Streptococci and lactobacilli, which

generally suppress the spoilage and pathogenic organisms very effectively

(Kosikowski, 1982). Moreover, desired alternation of food microorganisms

are referred to as fermentation regardless of the type of metabolism (Banwart,

1981). Similarly, fermentation is defined as anaerobic breakdown of an

organic substance by enzyme in which the final hydrogen acceptor is an

organic compound. These products include cultured butter milk, sour cream,

yoghurt, acidophilus milk, kefir and concentrated fermented milk products

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(Hargrove and Alford, 1972). Moreover, production of good fermented milk

required a source of good flavored, low bacteria in milk, heat treatment of the

milk, an active properly functioning appropriate starter, quick chilling of

fermented product and high standard sanitation (Kosikowski, 1982).

2.2 Starter cultures

The starter culture, Lactobacillus bulgaricus and Streptococcus

thermophilus, are required for the fermentation in yoghurt production

(Vedamuthu, 1991).

The classical yoghurt starter culture is a mixture of Streptococcus

thermophilus and Lactobacillus delbruechkii ssp. bulgaricus, with a cocci-

rods ratio of usually 1:1 (Hasan and Frank, 2001; Hutkins, 2001).

L. bulgaricus is a lacitic acid bacterium that has been found to be

relatively sensitive to low temperatures. According to Vedamuthu (1991) this

microorganism is usually seen as rod shaped and although typically slender,

may be curved in pairs or chains.

As a homofermentative thermophile, L. bulgaricus is known to be

relatively tolerant to heat, with an optimum growth temperature of

approximately 45—50ºC (Rasmussen, 1981; Rybka and Kailasapathy, 1995;

Vedamuthu, 1991).

L. bulgaricus is of strong tolerance for oxygen, therefore it is

considered to be a relatively slow growing organism, due to lack of oxygen.

As a result of this, until the oxygen levels are reduced during fermentation,

this microorganism will not grow rapidly (Marth and Steele, 1998).

The other starter microoganism involved in yoghurt production, S.

thermophilus is a spherical-shaped, typically found in pairs or long chains and

is noted for the ability to withstand higher temperatures contributing to the

classification of homofermentative thermophile (Vedamuthu, 1991). The

thermal resistance is demonstrated in the evidence of S. thermophilus survival

5

during heating at 60ºC for 30 minutes and characterized by the used of these

bacteria in various high temperature fermentations (Wilkins et al., 1986).

Despite being able to survive at higher temperatures, S. thermophilus fails to

grow at 10ºC, separating this microorganism from other streptococci that

grow at lower temperature (Marranzini, 1987). Other apparent attributes aside

from optimum, minimum and maximum growth temperature, include the

inability of S. thermophilus to grow in high salt concentrations and the

inability to produce ammonia from arginine (Marth and Steele, 1998). These

attributes are important for identification, since S. thermophilus does not

possess a necessary antigen for serological identification and therefore,

physiological techniques must be utilized (Marranzini, 1987).

S. thermophilus and L. bulgaricus exist in a complex cooperative

relationship in yoghurt in which one bacterium produces stimulatory agents

for the other (Tamime and Deeth, 1980; Wilkins et al., 1986; Vedamuthu,

1991). L. bulgaricus has been shown to produce certain amino acids such as

valine, leucine and histidine, which are essential for S. thermophilus to grow.

These amino acids are the result of proteolysis of casein by L. bulgaricus

(Marranzini, 1989; Abu-tarboush, 1996).

S. thermophilus in turn encourages the growth of L. bulgaricus by

producing formic acid and carbon dioxide (Matalon and Sandine, 1986;

Rajagopal and Sandine, 1990).

2.3 Types of yoghurt The various types of yoghurt differ according to their chemical

composition, their method of production, their flavour and the nature of post-

incubation processing. The legal or proposed standards for the chemical

composition of yoghurt in various countries are based on three possible types

of yoghurt classified according to fat content (full, medium or low)

(FAO/WHO, 1973). Such classification is used in composition standards to

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facilitate standardization of product and protect the consumer. However, there

are two main types of yoghurt, set and stirred, based on the method of

production and on the physical structure of the coagulum. Set yoghurt is the

product formed when fermentation/ coagulation of milk is carried out in the

retail container, and the yoghurt produced is in continuous semi-solid mass,

while, stirred yoghurt results when the coagulum is produced in bulk and the

gel structure is broken before cooling and packaging (FAO/WHO, 1973).

Fluid yoghurt may be considered as stirred yoghurt of low viscosity, since

viscosity is mainly governed by the level of solids in the basic mix, fluid

yoghurt can be produced by making stirred yoghurt from a mix with a low

level of total solids, e.g. 11% (Rousseau, 1974). To make a good quality

product, raw milk used must be low of bacterial count free from antibiotics,

sanitizing chemicals, mastitis milk and colostrum and the milk also should be

free from contamination by bacteriophages (Thapa, 2000). Traditionally, fluid

yoghurt is manufactured by mixing equal quantities of yoghurt and water (Fig

1:1) and one of the main characteristics of such a product is the separation of

the solid and the whey phases, hence, it is customary to shake the product

before consumption (Tamime and Deeth, 1980). Falvouring of yoghurt is

another method often used to differentiate various types of yoghurt.

Flavoured yoghurts are basically divided into three categories (plain or

natural, fruit and flavored yoghurt). The first type, plain or natural is the

traditional yoghurt. Sometimes the sharp acidic taste of the natural product is

masked by addition of sugar (Tamime and Deeth, 1980). Fruit yoghurts are

made by addition of fruit, usually in the form of fruit preserves, puree or jam

(Tamime and Deeth, 1980). However they added that falvoured yoghurt is

prepared by adding sugar or sweetening agents and synthetic flavorings and

colourings to plain or natural yoghurt. The post- incubation processing of

yoghurt may lead to many different types of yoghurt. The emergence of new

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yoghurt products has been largely due to recent developments in this field.

Typical examples of these are pasteurized/ UHT yoghurt, concentrated

yoghurt, frozen and dried yoghurt. These products may vary considerably in

chemical composition, physical characteristics, and heat-treatment after

incubation a process which leads to destruction of yoghurt starter bacteria and

reduction in the level of volatile compounds associated with the flavour of

yoghurt (Tamime and Deeth, 1980). Partial separation of the liquid phase

from yoghurt leads to production of concentrated/condensed yoghurt of

around 24% total solids (Tamime and Robison 1988; Robinson, 1977).

Frozen yoghurt which can be either soft or hard, is a product whose physical

state resembles ice-cream rather than yoghurt, while dried yoghurt can be

produced by sun-drying, spray-drying or freeze-drying (Tamime and Robison

1988).

2.4 Manufacture of yoghurt

The basic stages of production are common to all systems, and these

stages are preparation of a basic process milk with 12-14% milk solids-non-

fat (MSNF), heating milk to 85-95oC for 10-20 minutes, inoculating the milk

with a culture in which Lactobacillus bulgaricus and Streptococcus

thermophilus are principal organisms present, incubating the inoculated milk

at 42oC until a smooth coagulum has formed together with the desired level

of acidity and flavour, cooling the finished product and, unless the milk has

been incubated in retail size (set yoghurt ), mixing with fruit or other

ingredients and packaging the stirred yoghurt in containers prior to dispatch

under chilled conditions (Robinson,1986).

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Fig (1-1) Industrial production of various types of yoghurt (Tamime and Deeth, 1980)

Cool to incubation temperature

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2.5 Preparation of the basic mix

The preparation of the basic mix involves the fortification and /or

standardization of milk to achieve the desired rheological properties of the

manufactured product. The most popular method of fortification is the

addition of milk powder to liquid milk (Robinson, 1983). The use of ultra

filtration to concentrate the solids in skim milk is being considered as a

feasible alternative (Bundgaard et al., 1972). The aim of total solids is the

consistency improvement imparted to the yoghurt coagulum (Robinson,

1983). To make a good quality product, raw milk used must be of low

bacterial count, free from antibiotics and sanitizing chemicals, free from

mastatic milk and colostrums and free from contamination by bacteriophages

(Thapa, 2000).

2.6 Homogenization

Is the stage that follows fortification and this treatment affects the

lipid fraction and these changes tend to impact a rather smooth texture to the

coagulum (Banks et al., 1981). It is also reported that homogenization can

reduce the incidence of ‘pips’ in yoghurt (white flacks that appear in some

fruit varieties) (Davis, 1967; Robinson, 1981).

2.7 Heat treatment of milk

Milk is heat treated by three different methods: vat treatment (85°C for

10-14 min), high temperature short time treatment (HTST) (98°C for 0.5 –

1.87 min) and ultra high temperature treatment (UHT) (140 °C for 2-8 sec).

The physical and sensory properties of yoghurt were substantially affected by

method of heat teartment and exposure time. The most viable heating process

investigated was HTST with a residence time of 1.87 min (Parnell-Clunies et

al., 1986).

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Hong and Goh (1979) found that yoghurt from milk heated at 85°Cwas

harder than that from milk heated at 75 °C or 95 °C, and it received highest

subjective scores for appearance, aroma and flavour. Cultured yoghurt from

UHT processed whole milk (149°C for 3 sec) was lower in gel hardness and

apparent viscosity but higher in spreadability and fluidity than yoghurt

processed by conventional vat system (82°C for 30 min). UHT processing of

milk indicated a potential method for commercial manufacture of yoghurt

with consistency or low curd firmness (Labropoulos et al., 1984).

Grgurek (1999) found that post acidity action was slightly higher in

yoghurts prepared with lower amounts of inoculums and he concluded that

the incubation temperature should be properly maintained to achieve

maximum viability.

2.8 Production of set yoghurt

In set yoghurt the coagulation of milk takes place in the retail

containers, and hence the process involves cooling the heated milk to

incubation temperature, adding the starter culture and appropriate flavoring

and coloring ingredients, and filling into the retail containers. For sundae

style yoghurt, the fruit is placed in the container before the milk, followed by

incubation of the product to achieve the desired acidity (Robinson, 1986).

2.9 Cooling of yoghurt

When yoghurt reaches the required acidity (i.e. 0.8-1.0% lactic acid)

cooling of the coagulum commences, and the intention is to reduce the

temperature of the coagulum to below 20ºC within an acceptable time span.

Thus below 20ºC the metabolic activity of the starter organisms is sufficiently

reduced to prevent yoghurt from becoming unpalatable due to excessive

acidity and hence initiation of cooling depends on. The level of lactic acid

required in the end product is usually between 1.2% and 1.4% lactic acid,

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and the rate of cooling that can be achieved with the available equipment, and

in amanner that does not damage the texture of yoghurt (Robinson, 1981).

2.10 Packaging of yoghurt

It is an important step during production the purpose of which can be

summarized as follows: protection of the product against dirt microorganisms

and the environment (e.g. gases) and light, providing relevant information to

the consumer (e.g. name and origin of the product ingredient instructions

expiry date). The packaging material must be non-toxic, and no chemical

reactions should take place between the material and the product (Robinson,

1986).

2.11 Yoghurt properties

Yoghurt properties are influenced by the level of total solids in the

milk. This is important for both the consistency and aroma of the

manufactured yoghurt. In general, an increase in total solids will enhance

these properties (Emmon and Tuckey, 1967). The level of total solids in milk

varies from 9% in skim milk yoghurt to over 20% in other types of yoghurt

(Tamime and Deeth, 1980). Kozhev et al. (1972) concluded that the best

yoghurt is made from milk containing 15.0-16.0% total solids. Although the

composition of commercial yoghurt varies considerably, most low fat yoghurt

falls within the range of 14-15 % total solids. The level of total solids also

affects the titratable acidity of the milk mix due to the buffering action of

proteins, phosphates, citrates and other miscellaneous milk constituents

(Tamime and Deeth, 1980). An increase in total solids results in an increase

in the titratable acidity and reduction in the coagulation time (Humphreys and

Plunkett, 1969).

The viscosity of yoghurt is almost wholly dependent on protein

content of milk. Hence a high protein concentration is essential for the

12

production of viscous yoghurt. However, Tamime and Deeth (1980)

concluded that the addition of milk powder raised the protein level of milk.

Atamer et al. (1996), showed that titratable acidity as lactic acid was

higher in yoghurt samples with high total solids. If a full fat (> 3% fat) set

yoghurt is made then homogenization of the mix is essential to prevent

separation of cream line during incubation, but even for low fat varieties,

homogenization is reported to offer certain advantages, since it reduces the

average diameter of the fat globules to < 2µm, the viscosity of yoghurt is

improved, the syneresis is reduced and the process ensures uniform mixing of

any dry particles added to milk (Robinson, 1986).

Alexiou et al. (1988) found that after homogenization of milk both

consistency and organoleptic properties of yoghurt were significantly

improved.

The optimum conditions for heat treatment are 80-85ºC with a holding

time of 30 min (Galesloot, 1969). The effect of heat treatment can be

summarized as follows: there is denaturation of whey proteins (albumins and

globulins) and an aggregation of casein molecules into a three dimensional

network. This network traps the whey proteins and the yoghurt coagulum

produced subsequently is rendered more viscous (Janness and Patton, 1959).

The bacterial load in milk is reduced, and hence the starter culture has less

competition from adventitious organisms (Robinson, 1983). There is

reduction in the amount of oxygen in the milk (Ling, 1963) and as the normal

yoghurt cultures are microaerophilic, the lowered oxygen tension encourages

their growth (Robinson, 1983). Some limited damage to the milk proteins

may occur during heating and the breakdown products can stimulate starter

activity (Greence, 1957).

The quantity of metabolic compounds produced by combined

cultures was higher than that produced by single strain (Aslim, 1998).

13

After the incubation period, yoghurt is cooled in order to control the

level of lactic acid in the final product. The rate of cooling can affect the

structure of the coagulum. Thus, very rapid cooling can lead to whey

separation due to rapid contraction of the protein filaments which, in turn,

affects their hydrophilic properties (Rasic and Kurmann, 1978).

2.12 Fermentation and microbiological aspects

The classification of lactic acid bacteria by Orla Jensen (1931) was

still recognized as the standard method of classifying these organisms. In the

7th edition of Bergey,s Manual (1957) all these bacteria were grouped in the

family Lactobacillacaeae, which was subdivided into two tribes,

Streptococcaeae (coccal shaped) and Lactobacillaceae (rod shaped).

However, in the 8th edition of Bergey,s manual (1974) , lactic acid bacteria

were reclassified into two families the Streptococcaceae and

Lactobacillaceae. The yoghurt starter bacteria, S. thermophilus and L.

bulgaricus are thermophilic, homofermentative lactic acid bacteria. Moreover

S. thermophilus is distinguished from other members of the genus

Streptococci by its growth at 45oC and failure to grow at 10oC. Unlike most

Streptococci, this species lacks a recognized group antigen for serological

identification and has to be identified by physiological procedures (Bergey,s

Manual, 1974). Classification of S. thermophilus is well documented and

clear cut, in contrast, the classification of L. bulgaricus and it is relationship

to other Lactobacillus species have been the subject of some controversy

(Davis, 1975).

2.13 Shelf life of yoghurt

The end of shelf life of food is ultimately assessed by sensory failure.

The determination in sensory quality is quantified by monitoring changes in

specific attributes or re-education in over all quality acceptability using

different tests that utilize different scales of measurement (Gacula, 1975).

14

These differences in the logistics of measurement, coupled with proportion of

sensory quality loss perceived to be tolerated by consumers (Piga et al.,

2000).

Shah and Ravula (2002). found that increased sugar lead to an

increase of fermentation time and decrease in yoghurt shelf life.

The shelf life of yoghurt can be prolonged based on the type of storage

container used in packaging (Filardo, 2005).

2.14 Defects of yoghurt

The main yoghurt defects can be classified into three categories,

flavour, appearance and texture defects (Tayfour, 1994)

∗ Flavour defects: bitterness caused by keeping for long time that leads

to excess activity of culture,

∗ Hydrolysis of protein by proteolytic bacteria and yeasts, lack of taste

and flavour caused by bad activity of culture (imbalance), incubation for short

time at low temperature or low level of total solids content. Low activity

level caused by activity of culture (low incubation level insufficient

incubation or, at low temperature presence of inhibitors bacteriophages in

milk, excess acidity caused by bad fermentation process. Long incubation

period or at high temperature, low or insufficient cooling, keeping at high

temperature and harsh acid flavour caused by bad culture.

Appearance defects: Whey separation caused by excess acidity due to٭

excess incubation or high temperature in storage, extra keeping period- low

cooling-extra mixing (stirred yoghurt-use of extra pump-bad fruit mixing-

agitation of set yoghurt – low level of total solids and gas production which

are caused by infection by yeasts or coliforms.

Texture defects: curd separation caused by agitation during transportation٭

immediately after bad cooling in a cold room (set yoghurt), low curd

15

consistency caused by low level of incubation (short time or low

temperature), agitation before complete coagulation, very liquid yoghurt

caused by strong mixing, bad incubation (short- time), low level of total

solids (low viscosity production - unconcentrated flavours or fruits) (Tayfour,

1994).

2.15 Nutritional value of yoghurt

In Sudan it is believed that yoghurt (zabadi) is useful for the

treatment of stomach disturbances, and the individuals with such complaints

are advised to eat yoghurt (Dirar, 1993).

Children suffering from infantile diarrhea recovered more rapidly

when fed yoghurt than those given neomycin-kaopectate (Shahani and

Chardan., 1979). Lactic acid is biologically active and capable of suppressing

harmful microorganism's especially putrefactive ones and so has a favourable

effect on human vital activities (Oberman, 1985) and lactic acid was the only

antimicrobial agent active against pathogens (Deeth and Tamime, 1981).

Kilara and Shahani (1976) studied lactose or lactase activity of

cultured and acidified dairy products including yoghurt and concluded that

yoghurt culture released the enzyme lactase which converts lactase into lactic

acid, and lactose intolerant people are adviced to consume there products with

no subsequent problems.

16

CHAPTER THREE

MATERIALS AND METHODS

3.1 Collection of samples

Seventy five samples of yoghurt were obtained from one factory in

Khartoum State during period from June to July 2007, and kept in the

refrigerator (8 ± 2Cْ) under sterile conditions for 9 days and analyzed for

chemical and microbiological properties.

3.2 Chemical analysis of yoghurt

3.2.1 Determination of total solids content

Total solids content was determined according to AOAC (1990).

Three grams of the sample were weighed into dry clean flat bottomed

aluminum dish, and heated on a steam bath for 10 min. The dish was then

cooled in a desiccator weighed and heating, cooling and weighing were

repeated until the difference between two readings was < 0.1 mg. The total

solids content was calculated from the successive equation:

T.S = 1001 ×WW

Where:

W1 = Weight of sample after drying.

W = Weight of original sample

3.2.2 Determination of titratable acidity

The acidity of yoghurt was determined according to AOAC (1990).

Ten grams of sample were placed in a white porcelain dish and 5 drops of

phenolphthalein indicator were added. Titration was carried out using 0.1N

NaOH until a faint pink colour appeared. The titration figure was divided by

10 to get the percentage of lactic acid.

17

3.2.3 Determination of fat content

Fat content was determined by Gerber method according to AOAC

(1990) as follows:

In a clean dry Gerber tube, 10 ml of sulphuric acid (density 1.815 gm/ ml at

20ºC) were poured and then 11 gm of a well mixed yoghurt smple was gently

added. One ml of amyl alcohol (density 0.815 gm/ml at 20ºC) was added to

the mixture, the contents were then thoroughly mixed till no white particles

could be seen. Gerber tubes were centrifuged at 1100 revolutions per minutes

(rpm) for 4 minutes and the tubes were then transferred to a water bath at

65ºC for 3 minutes. The fat percent was then read out directly from the

fatcolumn.

3.2.4 Determination of protein content

The protein content was determined according to Kjeldahal method

(AOAC, 1990).

In a dry clean Kjeldahal flask, 11 gms of yoghurt were added, then 25

ml of concentrated H2SO4 were added follwed by addition of two Kjeldahal

tablets (CuSO4). The mixture was then digested on a heater until a clean

solution was obtained (3 hours) and the flasks were removed and left to cool.

The digested samples were poured into a volumetric flask (100 ml) and

diluted to 100 ml with distilled water. The distillate was received in a conical

flask containing 25 ml of 4% boric acid plus three drops of indicator

(bromocresol green plus methyl red). The distillation was continued until the

volume in the flask was 75 ml. The flasks were then removed from the

distillator, and the distillates were then titrated against 0.1N HCl until the end

point was obtained (red color).

The protein content was calculated as follows:

18

Nitrogen (%) = T × 0.1 × 0.014 × 20 × 100

Weight of sample

Protein (%) = Nitrogen X 6.38

Where:

T: Titration figure (ml).

0.1: Normality of HCl.

0.014: Atomic weight of nitrogen/ 1000.

20: Dilution factor.

3.3 Determination of wheying-off

Wheying-off was determined by sucking the water on the surface of the

curd and pouring in a graduated cylinder. Whey collected was then measured.

3.4 Microbiological examination

3.4.1 Sterilization of equipment

Glassware such as flasks, test tubes, Petri-dishes, pipettes and bottles

were sterilized in a hot oven at 170°C for two hours, whereas distilled water

was sterilized by autoclaving at121°C for 15 minutes (Marshall,1992).

3.4.2 Preparation of media

All media were obtained in a dehydrated form stored in a

hygroscopic environment in a cool dry place, away from light and prepared

according to the manufactures instructions.

3.4.2.1 Violet Red Bile Agar

This medium was used to determine the coliform count (Harrigan and

McCance, 1976). The medium (41.53gm) was dissolved in 1L distilled water

heated to boiling and then sterilized by autoclaving at 121°C for 15 minutes

(15 lb pressure), cooled to 45 ± 2°C and immediately poured into sterile Petri

dishes containing the dilution.

19

3.4.2.2 Lactobacillus MRS (Man Rogosa and Sharpe) Agar

This medium was used to determine Lactobacillus bulgaricus counts.

It was obtained in a dehydrated form (Hi Media Laboratories pvt.Ltd). The

medium (55.15 gram) was dissolved in 1 litre distilled water, heated to boilng

till dissolved completely and sterilized by autoclaving at 121°C for 15

minutes, cooled to 45±1°C

3.4.2.3 Acidified potato dextrose agar (P.D.A)

The medium consisted of 200 gms potatoes infusion form, 20 gms

dextrose and 15 gms agar.

The medium was prepared by dissolvind 39 gm of powder in one litre

of distilled water, followed by boiling to dissolve the medium completely and

it was sterilized by autoclaving at 121ºC for 15 minutes and cooled to 46ºC,

then enough sterile 10% tartaric acid was added

3.5 Preparation of dilutions

Eleven grams from a homogenous yoghurt sample were added to 99

ml of sterile distilled water in a clean sterile flask, then shaked until a

homogenous dispersion was obtained to make 10-1 dilution. One ml from the

above-mentioned dilution (10-1) was aseptically transferred to 9 ml sterile

distilled water. This procedure was repeated to make serial dilutions of 10-1,

10-2, 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8. Cultring method from each dilution, 1

ml was transferred to duplicate Petri-dishes and the culture medium was

poured aseptically into each Petri-dish, mixed gently, left to solidify and

incubated in an inverted position. The typical colonies in each Petri-dish were

counted using a colony counter (Houghtby et al., 1992).

20

3.6 Statistical analysis

All data were analyzed using one way ANOVA by means of statistical

computer software (SPSS-13). Duncan Multiple Range test was used to

determine the significance at P ≤ 0.05.

21

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 Chemical composition of yoghurt

Table (1) presents the effect of storage period on yoghurt during shelf

life. The fat content started at high value of 3.73±0.04 at the beginning of

shelf life, then decreased to 3.61±0.04 at day 3 and stayed at this level

throughout the shelf life of 9 days (P>0.05). The mean values of fat

percentage of yoghurt samples throughout shelf life showed non significant

difference between means, and this is due to the manufacturing of sample

from standardized of milk to fat content of 3.5%. This is supported by

Soomro et al. (2003).

The protein content did non significantly change (P>0.05)

throughout the storage period, although the highest content was at day 3, 3.24

± 0.035, then gradually decreased towards the end (3.12±0.035).

The results of protein content were similar to those of Tarakci and Kücükoner

(2003).

Total solids content gradually increased from 13.87 ± 0.14 at the

beginning to a maximum at day 6, (14.59±0.14) and slightly decreased at the

end, although the variation was not significant (P>0.05) Table (2). These

results are in line with the findings of Younus et al. (2002), there was hardly

any variation in total solids content in different samples of yoghurt, due to

balanced standardization of milk and quality control measures taken to ensure

consistency of end product.

The acidity of yoghurt steadily increased as shelf life of yoghurt

progressed (P > 0.05). The acidity was 0.94±0.011 at day one, and gradually

22

increased to 0.99 ± 0.011 at day 3, 1.13±0.011 at day 6 and 1.22±0.011 at day

9 of storage. The results of acidity in this study are in accordance with the

findings of Younus et al. (2002).

Table (3) presents the amount of whey collected from yoghurt during

shelf life Wheying-off of yoghurt was at minimum and did not significantly

increase. The value was 0.76±0.16 at day one, decreased to 0.60±0.16 at day

6, then increase to 0.69±0.16 at day 9. Shukla et al. (1988) found that

wehying-off is a major defect in yoghurt therefore stabilizers and additives of

milk powder are usually used to check wheying-off in yoghurt.

4.2 Microbiological properties of set yoghurt

Table (4) shows the effect of storage period on the count of coliform.

The count was not significantly affected (P>0.05) by storage of yoghurt

although coliform bacteria started high 2.77±0.07 at day one, decreased to

minimum 2.38±0.07 at day 3, then increased to 2.55±0.07 at the end of

storage period. Coliform examination for yoghurt samples collected revealed

no significant variation between different samples through shelf life of

product. These results were higher than what reported by Al-tahir (2005) and

Younus et al. (2002).

In most of yoghurt samples coliform bacteria were present which might

be due to improper pasteurization of pre-mix prior to its incubation and some

samples of yoghurt contained less count of coliform. It might be probably due

to contamination at product. Similar results have been reported by Younus et

al. (2002), who reported low number of coliform in yoghurt samples.

23

Table (1) fat contents and protein contents of set yoghurt samples during Shelf life.

Fat content Protein content Shelf

life(days) Mean ±SE Maximum Minimum Mean ±SE Maximum Minimum

1 3.73a ± 0.040 5.00 3.11 3.15a ± 0.035 3.56 2.67

3 3.61a ± 0.040 4.10 3.00 3.24a ± 0.035 3.70 2.65

6 3.61a ± 0.040 4.00 3.10 3.12a ± 0.035 3.56 2.61

9 3.61a ± 0.040 4.00 3.00 3.12a ± 0.035 3.84 2.57

S.L N.S N.S

Means in the same column followed by the same letter (s) are not significantly different at (P

= 0.05)

N.S = Not Significance

S.L = Significance Level

SE = Standard Error

24

Table (2) Total solids contents and Acidity percentage of set yoghurt samples during Shelf life.

Total solids Acidity (% lactic acid) Shelf

life(days) Mean ±SE Maximum Minimum Mean ±SE Maximum Minimum

1 13.87a ± 0.144 16.24 12.63 0.94a ± 0.011 1.47 0.87

3 14.05a ± 0.144 16.06 12.34 0.99a ± 0.011 1.85 0.73

6 14.59a ± 0.144 17.00 12.90 1.13a ± 0.011 1.37 0.91

9 14.29a ± 0.144 16.85 12.68 1.22a ± 0.011 1.43 0.89

S.L N.S N.S

Means in the same column followed by the same letter (s) are not significantly different

at (P =0.05).

N.S = Not Significance

S.L = Significance Level

SE = Standard Error

25

Table (3) Wheying-off percentage separated during Shelf life. Shelf life(days) Mean ± SE

1

3

6

9

0.76a ± 0.16

0.68a ± 0.16

0.60a ± 0.16

0.69a ± 0.16

S.L N.S

Means in the same column followed by the same letter (s) are not significantly

different at (P = 0.05).

N.S = Not Significance

S.L = Significance Level

SE = Standard Error

26

Yeasts and molds count did not significantly change (P > 0.05) during

storage period. However the count decreased to a minimum of 1.17 ± 0.14 at

day 6, then increased to 1.28 ± 0.14 at the end of storage period (Table 5).

The results revealed higher mean yeasts and molds count, and this result does

not conform to the Sudanese Standards, which stated that yeasts and molds

count should be less than Log10 1.0 cuf/gm.

Lactobacillus bulgaricus count increased from 7.24 ± 0.8 at the

beginning to 7.88 ± 0.8 at day 6, then slightly decreased to 7.82 ± 0.8 towards

the end of shelf life of yoghurt (Table 6). These results are in line with the

findings of Mohammed et al. (2007) who found that in Japan the fermented

milk and lactic beverage association has established a standard that requires

the presence of >107 viable bacteria/ml dairy product. Moreove These results

are in accordance with findings of Ministerio de La presidencia de Espana

(2003) who found that the Swiss food required that such products contain

≥106 cfu/g and the Spanish Yoghurt Quality Standard requires 107 cfu/ml.

27

Table (4) Coliform count of Set yoghurt samples during Shelf life.

Shelf life

(days) Mean ± SE Maximum Minimum

1 2.77 a ± 0.065 2.89 2.64

3 2.38 a ± 0.065 2.50 2.25

6 2.69 a ± 0.065 2.87 2.56

9 2.55 a ± 0.065 2.68 2.42

S.L N.S

Means in the same column followed by the same letter (s) are not significantly

different at (P=0.05).

N.S = Not Significance

S.L = Significance Level

SE= Standard Error

28

Table (5) Yeasts and molds count of set yoghurt samples during Shelf life.

Shelf life (days)

Mean ± SE Maximum Minimum

1 1.30 a ± 0.14 2.54 0.00

3 1.36 a ± 0.14 2.15 0.00

6 1.17a ± 0.14 2.23 0.00

9 1.28 a ± 0.14 2.20 0.00

S.L N.S Means in the same column followed by the same letter (s) are not significantly different

at (P=0.05).

N.S = Not Significance

S.L = Significance Level

SE= Standard Error

29

Table (6) Lactobacillus bulgaricus set yoghurt samples during Shelf life.

Shelf life

(days) Mean ± SE Maximum Minimum

1 7.24 a ±0.8 7.99 0.00

3 7.85 a ±0.8 8.38 7.46

6 7.88 a ±0.8 8.23 7.64

9 7.82 a ±0.8 8.11 7.45

S.L N.S

Means in the same column followed by the same letter (s) are not significantly different at (P = 0.05).

N.S = Not Significance

S.L = Significance Level

SE= Standard Error

30

CHAPTER SIX

CONCLUSION AND RECOMMENDATIONS

Conclusion

The results obtained during this investigation indicated that during

shelf life of yoghurt the chemical composition and microbial quality did not

significant difference.

Recommendations:

1. Establishment of efficient programs for assurance of high quality product.

2. Quality and safety are needed to ensure marketing of products and

consumer taste.

3. Application of hygienic control using system product.

4. Adjustment of yoghurt mix should approach the standard of the yoghurt

package.

31

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