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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
i
Dedication
To the soul of my mother
To my dear father, brothers and sisters
To my friends and colleagues
ftÄt{xÄwx|Ç
ii
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.
iii
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
iv
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
v
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(.
1
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
4
(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
6
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
7
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).
8
Fig (1-1) Industrial production of various types of yoghurt (Tamime and Deeth, 1980)
Cool to incubation temperature
9
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).
10
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,
11
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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