Mohammed Hassan Mohammed Haj Dr. Osman Ali Osman

ASSESSMENT OF CHEMICAL AND
MICROBIOLOGICAL
QUALITY OF STIRRED YOGHURT
IN KHARTOUM STATE  SUDAN
By
Mohammed Hassan Mohammed Haj
B.Sc. (Honours) Animal Production
University of Gezira
1999
A thesis submitted in partial fulfillment of the requirements for the
Degree
of Master of Dairy Production and Technology
Supervisor
Dr. Osman Ali Osman
Department of Dairy Production
Faculty of Animal Production
University of Khartoum
October - 2006
DEDICATION
To the soul of my friend Zaki Eldein
To my mother and father Hassan Badri
To my brother Khalid and sisters.
With love and respect
Mohammed
In the name of Allah, Most Gracious, Most Merciful.
ACKNOWLEDGEMENT
Praise be to Allah.
I wish to express my sincere thanks and gratitude to Dr. Osman
Ali Osman Elowni for his helpful supervision, constructive criticism,
and valuable guidance throughout this work.
My special thanks are due to Dr. Ibtisam Elyas Mohammed
Alzubeir, the coordinator of the Dairy Production and Technology,
M.Sc courses, for her continued support, encouragement and kindness.
My appreciations is due to Dr. Abdelwahab, for his cooperation
in choosing the suitable experimental design and analysis of the data
and unlimited help freely offered to me.
Iam indebted to the members of the Department of Dairy
Production. Also my thanks are extended to the staff of Dairy
Production laboratory, Faculty of Animal Production.
My thanks are due to my colleagues in the Dairy Production
and Technology, M.Sc. course Special thanks are due my friends,
Nazar, Sami Elbushra, and Mohammed Abdullah.
My gratitude and thanks are extended to Ibn Idris and
Abdelbasit for their patience and unlimited help.
My thanks extended to Mr. Zaki Eldein Ali Omer who started
typing of this thesis and Mr. Abdelhamed Abdelrahim who completed
the typing of this thesis, Finally, I wish to express special thanks to
my family.
ABSTRACT
This study was carried out on stirred yoghurt samples purchased
from the market. The stirred yoghurt was produced by Blue Nile Dairy
Company (CAPO).
Sixty samples were transported to the Faculty of Animal
Production, laboratory to assess the chemical, microbiological content
and shelf life of stirred yoghurt.
Chemical and microbiological analysis were carried out on the
first day of manufacturing and after 2, 4, 6, 8 and 10 days. Ten
samples from six batches were examined.
The investigations involved the analysis of fat, protein, lactose,
ash, total solids, solids-non-fat and measurement of pH, acidity and
viscosity.
Microbiological determinations were conducted for the
identification and classification of lactic acid bacteria and enumeration
of total bacterial count.
The chemical analysis for stirred yoghurt results showed that
the fat ranges were 1.60–4.90%, protein were 1.61–5.36%, lactose
were 6.75–10.92%, ash were 0.54–0.99% and total solids were 14.02–
17.74%, solids-non-fat were 11.34–14.52%, acidity were 0.80–1.50%,
pH were 3.52– 4.72% and viscosity were 38.30–82.70%.
The corresponding averages were 3.12%, 3.32%, 8.80%,
0.83%, 16.06%, 12.93%, 4.08%, 1.00% and 63.60%, while the
microbiological isolation results showed Streptococcus thermophilus
and Lactobacillus bulgaricus. The total viable bacterial count log of
these organism ranged 7.00–7.78 and the average 7.33, 7.00–7.78 and
the average 7.35, respectively.
The log of total bacterial count (cfu) ranged from 7.00 to 8.78
and the average was 7.47.
The results indicated that the storage period had significant
(P< 0.001) effect on the chemical composition except on the
total solids and viscosity. Also there were significant (P< 0.01) effect
of the storage period on the microbiological evaluation of stirred
yoghurt.
‫ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ‬
‫ﺨﻼﺼﺔ ﺍﻷﻁﺭﻭﺤﺔ‬
‫أﺟﺮﻳ ﺖ ه ﺬﻩ اﻟﺪراﺳ ﺔ ﻋﻠ ﻰ اﻟﺰﺑ ﺎدى ﻣﻔﻜ ﻚ اﻟﺨﺜ ﺮة أﺣ ﺪ ﻣﻨﺘﺠ ﺎت ﺷ ﺮآﺔ اﻟﻨﻴ ﻞ‬
‫اﻷزرق ﻟﻸﻟﺒﺎن )آﺎﺑﻮ( ‪ ،‬وﻗﺪ ﺗﻢ ﺷﺮاء اﻟﻌﻴﻨﺎت ﻣ ﻦ اﻟ ﺴﻮق وﺣﻤﻠﻬ ﺎ ﻟﻤﻌﻤ ﻞ آﻠﻴ ﺔ اﻹﻧﺘ ﺎج‬
‫اﻟﺤﻴﻮاﻧﻲ ﺟﺎﻣﻌﺔ اﻟﺨﺮﻃﻮم ﻟﻠﺘﺤﻠﻴﻞ وﻟﺘﻘﻴﻴﻢ ﺟﻮدة اﻟﻤﻨﺘﺞ اﻟﻜﻴﻤﻴﺎﺋﻴﺔ واﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻴﻪ‪.‬‬
‫إﺷﺘﻤﻠﺖ اﻟﺪراﺳﺔ ﻋﻠﻰ اﻟﺘﺤﻠﻴﻞ اﻟﻜﻴﻤﻴﺎﺋﻲ )ﻧﺴﺒﺔ اﻟﺪهﻦ و ﻧﺴﺒﺔ اﻟﺒﺮوﺗﻴﻦ و ﻧ ﺴﺒﺔ‬
‫اﻟﻼآﺘﻮز وﻧﺴﺒﺔ اﻟﺮﻣﺎد و ﻧﺴﺒﺔ اﻟﺠﻮاﻣﺪ اﻟﻜﻠﻴﺔ وﻧﺴﺒﺔ اﻟﺠﻮاﻣﺪ ﻏﻴﺮ اﻟﺪهﻨﻴﺔ و ﺗﺤﺪﻳﺪ ﻧ ﺴﺒﺔ‬
‫اﻟﺤﻤﻮﺿﺔ و ﻗﻴﺎس اﻷس اﻟﻬﻴﺪروﺟﻴﻨﻲ وﻗﻴﺎس اﻟﻠﺰوﺟﺔ(‪.‬‬
‫وإﺷﺘﻤﻠﺖ اﻟﺘﺤﺎﻟﻴﻞ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟﻮﺟﻴﻪ ﻋﻠﻰ اﻟﺘﻌﺮف ﻋﻠﻰ أﻧﻮاع وﻣﺴﻤﻴﺎت اﻟﺒﻜﺘﺮﻳﺎ‬
‫اﻟﻤﺴﺘﺨﺪﻣﺔ ﻓﻲ ﺻﻨﺎﻋﺔ اﻟﺰﺑﺎدي وآﺬﻟﻚ ﺣﺴﺎب اﻟﻌﺪد اﻟﻜﻠﻲ ﻟﻠﺒﻜﺘﺮﻳﺎ‪.‬‬
‫أﺟ ﺮي اﻟﺘﺤﻠﻴ ﻞ اﻟﻜﻴﻤﻴ ﺎﺋﻲ و اﻟﻤﻴﻜﺮوﺑﻴﻮﻟ ﻮﺟﻲ ﻟﻠﺰﺑ ﺎدي ﻣﻔﻜ ﻚ اﻟﺨﺜ ﺮة ﻓ ﻲ اﻟﻴ ﻮم‬
‫اﻷول ﻣﻦ اﻟﺘﺼﻨﻴﻊ وﺑﻌﺪ إﺛﻨﻴﻦ ‪ ،‬أرﺑﻌﺔ ‪ ،‬ﺳﺘﺔ ‪ ،‬ﺛﻤﺎﻧﻴﺔ وﻋﺸﺮة أﻳ ﺎم ‪ .‬ﻋ ﺸﺮة ﻋﻴﻨ ﺎت ﻟﻜ ﻞ‬
‫ﻳﻮم ﺗﻢ ﺷﺮاﺋﻬﺎ ﻋﻠﻰ اﻟﺘﻮاﻟﻲ ﻣﻦ اﻟﺴﻮق‪.‬‬
‫أﻇﻬ ﺮت اﻟﻨﺘ ﺎﺋﺞ اﻟﻜﻴﻤﻴﺎﺋﻴ ﺔ ﻟﻠﺒﺤ ﺚ أن ﻣﺤﺘ ﻮى اﻟ ﺪهﻦ ﻳﺘ ﺮاوح ﺑ ﻴﻦ‬
‫‪ %4.90 –1.60‬وﻧ ﺴﺒﺔ اﻟﺒ ﺮوﺗﻴﻦ ‪ %5.36 –1.61‬وﻧ ﺴﺒـــﺔ اﻟﻼآﺘــ ـﻮز‬
‫‪–6.75‬‬
‫‪ %10.92‬وﻧﺴﺒﺔ اﻟﺮﻣﺎد ‪ %0.99 – 0.54‬و ﻧ ﺴﺒﺔ اﻟﺠﻮاﻣ ﺪ اﻟﻜﻠﻴ ﺔ ‪%17.74 –14.02‬‬
‫و ﻧ ﺴﺒﺔ اﻟﺠﻮاﻣ ﺪ ﻏﻴ ﺮ اﻟﺪهﻨﻴ ﺔ ‪ %14.52 –11.34‬و ﻧ ﺴﺒﺔ اﻟﺤﻤﻮﺿ ﺔ ﺗﺘ ﺮاوح ﺑ ﻴﻦ‬
‫‪%1.50 –0.80‬‬
‫واﻷس اﻟﻬﻴ ﺪروﺟﻴﻨﻲ ‪ %4.72–3.52‬وﻗﻴ ﺎس اﻟﻠﺰوﺟ ﺔ ‪–38.30‬‬
‫‪ .%82.70‬و ﺑﻠﻐ ﺖ اﻟﻤﺘﻮﺳ ﻄﺎت ﻋﻠ ﻰ اﻟﺘ ﻮاﻟﻲ ‪ %3.12‬و‪ %3.32‬و‪ %8.80‬و‪3‬‬
‫‪ %0.8‬و ‪16.06‬و ‪ %12.93‬و‪ %4.08‬و ‪ %1.00‬و ‪.%63.60‬‬
‫ﺑﻴﻨﻤ ﺎ ﺗﻮﺻ ﻠﺖ ﻧﺘ ﺎﺋﺞ اﻟﺘﺤﻠﻴ ﻞ اﻟﻤﻴﻜﺮوﺑﻴﻮﻟ ﻮﺟﻲ ﻋﻠ ﻰ اﻟﺘﻌ ﺮف ﻋﻠ ﻰ‬
‫‪ L.bulgaricus‬و‪S.thermophilus‬‬
‫‪ ،‬وﻗ ﺪ آـــ ـﺎن اﻟـﻌـ ـﺪ اﻟﺒﻜﺘﻴـ ـﺮي ﻟﻜ ﻞ ﻣﻨﻬ ﺎ‬
‫ﺑ ﺎﻟﻠﻮﻏﺮﻳﺜﻢ آ ﺎﻵﺗﻲ‪ 7.78 –7.00 :‬واﻟﻤﺘﻮﺳ ﻂ ‪ 7.33‬و ‪ 7.78 – 7.00‬واﻟﻤﺘﻮﺳ ﻂ‬
‫‪ 7.35‬ﻋﻠﻰ اﻟﺘﻮاﻟﻲ‪ .‬أﻣﺎ ﺑﺎﻟﻨ ﺴﺒﺔ ﻟﻠﻌ ﺪ اﻟﻜﻠ ﻲ ﻟﻠﺒﻜﺘﺮﻳ ﺎ ﺑ ﺎﻟﻠﻮﻏﺮﻳﺜﻢ ﻓﻘ ﺪ ﺑﻠ ﻎ ‪8.78 – 7.00‬‬
‫واﻟﻤﺘﻮﺳﻂ ‪ .7.47‬أﻇﻬﺮت اﻟﻨﺘﺎﺋﺞ أن ﻓﺘﺮة اﻟﺘﺨﺰﻳﻦ ﻟﻬﺎ اﺛﺮ ﻣﻌﻨﻮي )‪ ( P< 0.001‬ﻋﻠ ﻰ‬
‫اﻟﺘﺮآﻴ ﺐ اﻟﻜﻴﻤﻴ ﺎﺋﻲ ﻟﻤﻨ ﺘﺞ اﻟﺰﺑ ﺎدي ﻣﻔﻜ ﻚ اﻟﺨﺜ ﺮﻩ ﻣﺎﻋ ﺪا اﻟﻤ ﻮاد اﻟﺠﺎﻣ ﺪﻩ اﻟﻜﻠﻴ ﻪ وﻗﻴ ﺎس‬
‫اﻟﻠﺰوﺟﻪ‪.‬‬
‫آﺬﻟﻚ هﻨﺎﻟﻚ ﺗ ﺄﺛﻴﺮ ﻣﻌﻨ ﻮي ) ‪ ( P< 0.01‬ﻟﻠﺘﺨ ﺰﻳﻦ ﻋﻠ ﻰ اﻟﻤﺤﺘ ﻮي اﻟﻤﻴﻜﺮوﺑ ﻲ‬
‫ﻟﻤﻨﺘﺞ اﻟﺰﺑﺎدي ﻣﻔﻜﻚ اﻟﺨﺜﺮﻩ‪.‬‬
LIST OF CONTENTS
Page
Dedication……………………………………………………………………………………………...
i
Acknowledgement …………………………………………….………………………………...
ii
Abstract ………………………………………………………..……………………………………...
iii
Arabic Abstract …………………………………………………………………………………...
v
List of Contents ………………………………..………………………………………………...
vii
List of Tables……………………………………..………………………………………………...
xi
List of Figures …………………………………..………………………………………………...
xii
CHAPTER ONE: INTRODUCTION……………………………………………...
1
CHAPTER TWO: LITERATURE REVIEW………………..………………
3
2.1 Fermented dairy products……………..………………………….………..……………
3
2.2 Processing of yoghurt……………..………………………………………………..……
6
2.3 Types of yoghurt…………………………………………………………..…..……………
9
2.4 Composition of Yoghurt…………………………………………………..……………
12
2.5 Factors affecting quality of yoghurt………………………..………..……………
15
2.5.1 Starter cultures……………..…………………………………….…………..……………
15
2.5.2 Heat treatment……………..……………………………………...…………..……………
16
2.5.3 Storage period……………..………………………………………...………..……………
17
2.6 Additives in yoghurt……………..…………………………………………..……………
19
2.6.1 Flavorings and sweeteners……………….…………………………..……………
19
2.6.2 Stabilizers……………..……………………….………………………………..……………
21
2.7 The nutritional value of yoghurt……………..…………………………..…………
21
2.8 Fermentation and microbiological aspects………………..……..……………
22
2.9 Ratio of cocci: rods in starter culture……………………………….……………
23
CHAPTER THREE: MATERIALS AND METHODS……..…………
27
3.1 Source of milk samples……………..…………….………………………..……………
27
3.2 Chemical analysis……………..……………………………..………………..……………
27
3.2.1 Fat content……………….……………………………………………….……………..……
27
3.2.2 Total solids……….……………………………………………………………………..……
28
3.2.3 Ash content…………………………………………….………...……………………..……
28
3.2.4 Protein content……………….…………………..…………….……………………..……
29
3.2.5 Lactose content……………………………………..………………………………..……
30
3.2.6 Solid not fat (SNF) ……………………………….………………………………..……
30
3.2.7 Titratable acidity (T.A) ………..………………….……………………………..……
30
3.2.8 pH – values………………………….…………………….…………..………………..……
31
3.2.9 Viscosity……..…………………………………...……….……………………………..……
31
3.3 Microbiological examination………….………………………………………..……
31
3.3.1 Sterilization…………………………………………………………...………………..……
31
3.3.2 Preparation of serial dilution ………………………………………………..……
31
3.3.3. Type of cultured media used for microbial examination…………
32
3.3.3.1 Plate count agar…………………………………………...……………………..……
32
3.3.3.2 M – 17 medium…………………..……………………………………………..……
32
3.3.3.3 MRS broth………………………………………………………..………………..……
32
3.3.3.4 Nutrient agar…………………………………..…………………..………………..……
32
3.4 Enumeration of microorganisms……………………………..………..……………
33
3.4.1 Streptococcus thermophilus……………..…………………………..……………
33
3.4.2 Lactobacillus bulgaricus…………….……………..……..…………….……………
33
3.4.3 Bacterial count……………..……………………..…………………………..……………
33
3.5 Purification of bacterial isolates……………………………………...……………
33
3.6. Identification of organisms……………………………………………...……………
34
3.6.1 Primary tests……………..……………………………………...……………..……………
34
3.6.1.1 Morphological appearance……………..…………………………..……………
34
3.6.1.2 Shape of cell……………..…………………………..…………………………..………
35
3.6.1.3 Catalase test……………..……………………………………..…………..……………
35
3.6.1.4 Oxidase test……………..…………….……………..…………………………..………
35
3.6.1.5 Motility test……………..…………………………………………………..……………
36
3.6.1.6 Oxidation fermentation test (OF) ………………………..……..……………
36
3.6.2 Confirmatory tests……………..…………………………………………..……………
36
3.7 Statistical analysis……………..……………………..………………………..……………
37
CHAPTER FOUR: RESULTS AND DISCUSSION ……………………
38
4.1 Chemical composition of stirred yoghurt………………….……..……………
38
4.1.1 Fat……………..…………………………..………………………..………………..……………
38
4.1.2 Protein……………..…………………………..…………………………………..……………
40
4.1.3 Lactose……………..……………………………………………………………..……………
40
4.1.4 Ash……………..…………………………..………………………………………..……………
41
4.1.5 Total solids (TS) ……………..……………………………….……………..……………
42
4.1.6 Solids – non–fat (SNF) ……………………...…………………………..……………
44
4.1.7 pH……………..………………………………………...…………………………..……………
45
4.1.8 Acidity……………..……………………..…………..…………………………..……………
47
4.1.9 Viscosity……………..…………………………..………………..……………..……………
48
4.2 Microbiological results of stirred yoghurt………….……..……..……………
50
4.2.1 Standard plate count……………..………………………………….……..……………
50
4.2.2 Identification of isolated LAB……………..…………………………..…………
53
4.2.3 Streptococcus thermophilus……………………………………..…..……………
53
4.2.4 Lactobacillus bulgaricus………………………………………………..……………
56
CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS..…
61
5.1. Conclusion………………………………………………….……………………………..……
61
5.2. Recommendations…………………………...………………………………………..……
62
REFERENCES…………………………………………………………………………………...
63
LIST OF TABLES
Table Title
No.
2.1
Yoghurt and yoghurt – like products known worldwide…
5
2.2
Some typical values of major constituents of milk and
yoghurt……………..………………………………………………….…..……………
25
2.3
Selected characteristics of yoghurt starter culture…………..…
26
1
Fat and protein content of stirred yoghurt samples during
storage….…..…….…..…….…..…….…..…….…..…….……....…….…..…….…..…
39
Lactose and Ash content of stirred yoghurt samples
during storage….…..…….…..………….…..…….…..…….……....…….…..……
43
Total solids (T.S) and Solids–non–fat (SNF) of stirred
yoghurt samples during storage.…..………………….…..…….…..…….
46
pH and acidity development of stirred yoghurt samples
during storage.…..………….…..………………………………………...…..…….
49
5
Viscosity of stirred yoghurt samples during storage….....…..
51
6
Chemical composition of stirred yoghurt using one way
ANOVA analysis.…………………….…..………………………………………
52
Total bacteria count of stirred yoghurt samples during
storage.………….……………………………...………………………………………
54
Microbiology analysis of stirred yoghurt samples during
storage.………….…..……………………………..……………………………………
58
Microbiology analysis of stirred yoghurt using one way
ANOVA analysis.………….……………..………………………………………
59
Identification of lactic acid bacteria.………….…..…………………
60
2
3
4
7
8
9
10
LIST OF FIGURES
Fig.
Title
No.
2.1
Traditional yoghurt production in the Middle East……….…
8
2.2
Industrial production of various types of yoghurt…………..…
13
CHAPTER ONE
INTRODUCTION
Under unrefrigerated storage milk becomes sour in a few hours;
this is due to the action of natural lactic acid bacteria on the milk
sugar, turning it to lactic acid which causes the milk protein to
coagulate (Lucey and Singh, 1997). Traditionally, most communities
in Africa and in Sudan in particular have used this natural
fermentation to produce a variety of fermented milks for home
consumption (Ahmed and Ismail, 1978). Under normal dairy
processing industry, selected lactic starter cultures are used to ferment
milk during preparation of variety of cultured dairy products. The
quality of those products vary from average or below average quality
to high quality products (Tamime and Robinson, 1999).
Yoghurt is a semi–solid fermented milk product which
originated centuries ago in Bulgaria, it’s popularity has grown and is
currently consumed in most parts of the world (Tamime and Deeth,
1980).
The accepted home land of yoghurt is the Balkans peninsula
and the Middle East region, and to communities living in these parts
of the world this type of fermented milk product is identified and
known as natural/plain unsweetened yoghurt. The per capita annual
consumption is high. In Bulgaria, in particular, is 31.5 kg/head/ year
(IDF, 1977). It is evident, therefore, that yoghurt plays an important
role in the diets of these communities. Furthermore, it is customary for
yoghurt to be consumed not only as a refreshing drink, but also as a
main ingredient (Tamime and Deeth, 1980).
There are three types of plain yoghurt: stirred yoghurt, set
yoghurt and liquid yoghurt (with low viscosity). In the set yoghurt, the
product is packaged immediately after inoculation with the starter
culture and is incubated in the packages, for stirred yoghurt the milk is
inoculated by culture, incubated in tank and packaged after cooling
(Chandan, 1999). In many cases of lactose intolerance, cultured milk
products where lactose has been partially broken down can be
acceptable to sufferers of lactose intolerance (Harmann and Marth,
1984). Children suffering from infantile diarrhea recovered more
rapidly when fed yoghurt than those given neomycin – kaopectate
(Shahani and Chandan, 1979).
The objectives of the present study are:
1- Evaluation of the chemical quality of market stirred yoghurt.
2- Evaluation of microbiological quality of market stirred
yoghurt.
3- Isolation and identification of lactic acid bacteria in stirred
yoghurt. Measurement of yoghurt ingredient and the
microbial activity of some bacteria are taken as main
parameter, to reach the objectives mentioned.
CHAPTER TWO
LITERATURE REVIEW
2.1 Fermented dairy products:
Acidification of milk by fermentation is one of the oldest
methods of preserving milk and imparting to it specific favorable
organoleptic qualities. There are different methods of carrying out this
fermentation in various parts of the world and these give rise to a wide
range of fermented milk products, including kumiss, kefir, acidophilus
milk and yoghurt (Thapa, 2000). These products vary considerably in
composition, flavor and texture, according to the nature of fermenting
organisms, the type of milk and the manufacturing process used
(Chandan et al., 1969; Kosikowski, 1977 and Berlin, 1962).
Fermentation is defined as any modification of the chemical or
physical properties of milk or dairy products, resulting from the
activity of microorganism or their enzymes which cause the main
marked changes (Frank and Marth, 1988). Fermentation affects the
carbohydrates, proteins and vitamins as well as producing flavor
compounds (particularly acetaldehyde), some enzymes and of course
bacterial mass (Deeth, 1984). Cultured milks therefore have a change
in composition, as well as an accumulation of the products of bacterial
fermentation and anti–microbial compounds emitted by culture
organisms in order to inhibit the growth of other organisms (Harding
1999). Fermented milk products are widely used in the Balkan for
medicinal purposes against diseases such as pneumonia, dysentery and
less serious complains such as sore throat and laryngitis (Peterson,
1981). There are claims that the digestibility of milk proteins is
improved by fermentation (Marshal, 1986 and Deeth and Tamime,
1981). Lactic acid fermentation products have traditionally been used
by Africans for treating some human ailments and for other ends
(Dirar, 1997). There are also claims that fermented milks contain
some chemical factors that reduces the level of serum cholesterol in
human beings (Mann and Spoery, 1974; Mann, 1977; Richardson,
1978; Grunewald, 1982 and Pulusani and Rao, 1983). Kilara and
Shahani (1976) studied the lactase activity of cultured acidified dairy
products including yoghurt. They concluded that the yoghurt culture
released the enzyme lactase, which break down the lactose contained
in the dairy product, allowing lactose intolerant people to consume
these products with no subsequent problems. The word “yoghurt” is
derived from the Turkish word “jugurt” and Table (2.1) shows the
variety and names by which this product is known in different
countries. Yoghurt is a traditional food and beverage in the Bulkans
and the Middle East. However, it’s popularity has now spread to
Europe and many other parts of the world (Rasic and Kurmann, 1978).
Table (2.1): Yoghurt and yoghurt–like products known worldwide
Traditional name
Country
Jugurt/Eyran
Turkey
Busa
Turkestan
Kissel Mleka
Balkans
Urgotnic
Balkan mountains
Leben/leban
Lebanon and some Arab countries
Zbady
Egypt and Sudan
Mast/Dough
Iran and Afghanistan
Roba
Iraq
Dahi/Dadhi/Dahee
India
Mazum/Matzoo
Armenia
Katyk
Transcaucasia
Tiaourti
Greece
Cieddu
Italy
Mezzoradu
Sicily
Gioddu
Sardinia
Tarho
Hungary
Fiili
Finland
Filmjolk / Fillbunke / Filbunk / Surmelk / Scandinavia
Tettemelk / Taettemjolk
Island
Yoghurt/Yogurt / Yoart / Yourt / Yaourti
Rest of the world “Y” is replaced by
Yahourth/Yogur/Yaghourt
‘J’ in some instances
After: Orla–Jensen (1931), Hammer and Babel (1957), Davidson and
Passmore (1969), Pederson (1971), Nilson (1973); Kon (1972); El –
Sadek et al. (1972).
Although some controversy still exists regarding the exact definition
of yoghurt in terms of its chemical composition and the product
resulting from milk by fermentation with a mixed starter culture
consisting of only Streptococcus thermophilus and Lactobacillus
bulgaricus. The justification for using such an approach has been
discussed (Tamime and Robinson, 1976). Similar definitions has been
used in formulating existing or proposed standards for yoghurt in
many countries (Dehove, 1960 and Danish standards 1968). Yoghurt
is a popular fermented milk product consumed in many parts of the
world. It is produced in different forms such as whole milk yoghurt,
skim milk yoghurt, cream yoghurt, fruit yoghurt and liquid yoghurt
(Balasubraman yam and Kulkarnis, 1991). Yoghurt is one of the most
unique, yet universal dairy products. The uniqueness of yoghurt is
attributable to one symbiotic fermentation involved in its manufacture.
It may be defined as the solid, custard- like fermented milk product
made from fortified high–solids milk using a symbiotic mixture of
Streptococcus thermophilus and Lactobacillus bulgaricus as starter
(Ebenezer and Vedamuth, 1991).
2.2 Processing of yoghurt:
Traditionally, yoghurt has been manufactured from boiled milk
which was concentrated to two thirds of the original volume
(Cronshaw, 1947 and Elliker, 1949). The traditional procedure, which
is illustrated in Figure 2.1, has several draw backs. First, successive
inoculations of the starter cultures tend to up set the balance between
the S. thermophilus and L. bulgaricus; second the low incubation
temperature (blood temperature as compared with the optimum
temperature of (40-45°C) results in slow acidification of milk and can
promote undesirable side effects, e.g. whey syneresis, and adversely
affect the quality of yoghurt, and third, such a method provides no
control over the level of lactic acid produced during manufacture
(Cronshaw, 1947 and Elliker, 1949). The starter, which is obtained
from commercial starter manufacturers or starter banks, is propagated
in rotation and inoculated into the milk under aseptic conditions, the
temperature is accurately controlled and the yoghurt is cooled very
quickly when the desired acidity is reached. Hence, the process can be
well controlled and the quality of yoghurt is standardized (Cronshaw,
1947 and Elliker, 1949). The work on the manufacture of yoghurt has
been reviewed by several authors (Humphreys and Plunkett, 1969;
Tamime and Robinson, 1976; Puhan, 1976 and Bottazzi, 1977).
Figure (2.1): Traditional yoghurt production in the Middle East:
(Tamime
TtTt and Deeth 1980)
Boil milk to cause partial concentration
Cool to incubation temperature, i.e. blood temperature
Starter (previous day yoghurt)
Inoculate with starter
Incubate in bulk until firm coagulate is produced or incubate over night at room
temperature
Cool
Dispatch
Legal standards of yoghurt are mainly based on the chemical
composition of the product i.e. percentage of fat, solids-non-fat (SNF)
or total solids (Tamime and Robinson, 1976). The aim of using high
total solids is consistency improvement the yoghurt coagulum
(Robinson, 1983). Very thin liquid yoghurt caused by strong mixing,
incubation (short time), low level of total solids, bad culture and low
viscosity production (Tayfour, 1994).
2.3 Types of yoghurt:
The industrial manufacture of different types of yoghurt is
shown schematically in Fig 2.2. The various types 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) fat content (FAO/WHO, 1973). They
added that such classification is used in composition standards to
facilitate standardization of product and to 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 a 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 of low 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 2.2) 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). Flavouring 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, pure or jam (Tamime and Deeth, 1980) However
they added that flavoured yoghurt are prepared by adding sugar or
sweetening agents and synthetic flavorings and colorings to plain or
natural yoghurt. The post – incubation processing of yoghurt may lead
to many different types of yoghurt. The emergence of new yoghurt
products has been largely due to recent developments in this field as
they stated. Typical examples of these are pasteurized/UHT yoghurt,
concentrated yoghurt, frozen and dried yoghurt. These products may
vary considerably in chemical composition, physical characteristics,
heat – treatment after incubation a process which leads to destruction
of the yoghurt starter bacteria and reduction in the level of volatile
compounds which are 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 a round
24% total solid (Tamime and Robinson 1978, 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 Robinson, 1978 and Robinson, 1977).
Sun dried yoghurt is produced in many rural areas in the Middle East
where the surplus of summer milk is made into yoghurt and conserved
by this method for winter consumption (Tamime and Deeth, 1980).
Industrial production of dried yoghurt is achieved by either spray
drying or freeze – drying (Tamime and Robinson, 1976).
2.4 Composition of Yoghurt:
Yoghurt is highly nutritious and easily digestible diet
due to the predigested nutrients by bacterial starters, it is perishable in
view of its unused lactose content which can be utilized for the growth
of undesirable microorganisms responsible for spoilage (Durga et al.,
1986). Musa (1997) examined yoghurt prepared from fresh cow’s
milk, he reported for fat content, protein content and total solids
3.2%, 4.5% and 19.39%, respectively.
In terms of over all composition, yoghurt is similar to milk.
However, there are many aspects in which the composition of milk
and yoghurt differs. These differences are either from deliberate
addition of solids to milk or yoghurt, or from changes brought about
by bacterial fermentation (Deeth and Tamime, 1981). The
composition of yoghurt is approximately the same as that of whole
milk; it provides an excellent protein source particularly when it is
fortified with dried milk (Vieseyre, 1964).
Fig. (2.2): Industrial production of various types of yoghurt (Tamime and Deeth, 1980)
Preliminary treatment of milk (standardization/fortification/lactose hydrolysis)
Homogenization
Heat treatment
Starter propagation
Cool to incubation temperature
Inoculation with starter
Incubate
Inoculate in bulk
Cool
Cool
Concentrate
Dispatch
SET
YOGHURT
Freeze
Dispatch
SOFT OR
DEEP
FROZEN
YOGHURT
Mix with fruit
Heat treatment
Cool
Pack
Dispatch
PASTEURIZED
OR UHT
YOGHURT
Pack
Dispatch
STIRRED
YUGHURT
Heat treatment
Pack
Pack
Dry
Mix with equal
quantity of water
Pack
Freeze
Cool
Dispatch
PASTEURIZED
OR UHT
YOGHURT
Cool
Dispatch
Dispatch COCENTRATED Pack
YUGHURT
SOFT OR
Dispatch
DEEP
FROZEN
DRIED YUGHURT
YUGHURT
Dispatch
FLUID
YUGHURT
Deeth and Tamime (1981) concluded that, with all methods of
fortification, the percentage of protein is increased, thus yoghurt will
have an invariably high protein content than milk. Dalles and
Kechagias (1984) found that the acidity of commercial types of
yoghurt in Greece ranged from 1.02 to 2.15% (lactic acid). The major
change resulted from fermentation is the production of lactic acid
from lactose. However, several other important changes also occur. In
many developing countries, “Zabadi” is produced as naturally sour
milk, and makes an important contribution to the diet as a source of
protein, calories and some vitamins (Ahmed and Ismail, 1978).
Kim et al. (1998) reported a titratable acidity of 0.95- 0.99%
for yam– yoghurt which was prepared by addition of yam to skim
milk substrate and cultured with a mixed culture. Collado et al. (1994)
found that the yoghurt drink and yoghurt like products had 0.56% and
0.58% total titratable acidity. Gregurek (1999) found that post acidity
cation was slightly higher in yoghurt prepared with lower amount of
inoculation
and
incubation
temperature
should
be
properly
maintained, to achieve maximum bacterial viability.
Djordjevic et al. (1988) found that there was a lowering of dry
matter during the incubation and during storage and this lowering
takes place in dry fat matter, portion dry matter and is due to the
disappearance of volatile compounds formed during the process of
yoghurt. Uraltas and Nazli (1998) when studied Turkish fruit yoghurt,
found that dry matter content ranged from 22.2 to 23.5% and values
for fat ranged from 2.2 to 2.8% and SNF values ranged from 19.4 to
23.5% for 50 samples. Agaoglu et al. (1997) examined 25 samples of
cokelek (concentrated yoghurt). They found that the average dry
matter content was 18.15%, fat content 1.2%, protein content 4.08%
and mineral content 0.94%.
2.5 Factors affecting quality of yoghurt:
Many factors affect the quality of yoghurt such as type of starter
culture, heat treatment and storage condition.
2.5.1 Starter cultures:
The culture may be of one strain microorganism which called a
single – strain culture, or a number of strains and/or species called a
multi – strain or mixed – strain culture (Kosikowski, 1982).
Yoghurt is produced with a mixed culture of S. salivarius sub –
sp. thermophilus and L. delbrueckii, sub – sp. bulgaricus in a 1:1 ratio.
The coccus grows faster than the rod and it's primarily responsible for
acid production, while the rod adds flavor and aroma (Kosikowski,
1982). The associative growth of the two organisms results in lactic
acid production at a rate greater than that produced by either when
growing alone, and more acetaldehyde (the chief volatile flavour
component of yoghurt) is also produced (Jay, 1986).
The symbiotic relationship between S. thermophilus and L.
bulgaricus, the thermoduric, homofermentative lactic acid bacteria,
has long been utilized in the manufacture of yoghurt, maintenance of
the proper balance between these organisms is considered important,
especially in manufacture of high quality yoghurt (Lee et al. 1974).
Radke–Mitchell and Sandine (1984) assumed a symbiotic
relationship between the strains. The Lactobacilli liberate amino acids
from the protein, and these amino acids encourage the development of
the Streptococcus that stimulates the growth of Lactobacilli by
increasing the initial acidity and also by removing residual oxygen.
Finally the acidity produced by Lactobacilli inhibits the growth of
Streptococci (Hamill, 1968).
2.5.2 Heat treatment:
Milk was processed by three different heating systems, 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
(UHT 140° C for 2-8 sec). The physical and sensory properties of
yoghurt were substantially affected by type of heat processing and
exposure time. The most viable heating process investigated was
HTST with a residence time of 1.87 min (Parnell – clunies et al.
1986).
Hong and Goh (1979) found that yoghurt from milk heated at
85° C was harder than yoghurt from milk heated at 95° C or 75° 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 spread– ability and fluidity than yoghurt processed by conventional
vat system (82° C for 30 min). The UHT processing of milk indicated
a potential method for commercial manufacture of yoghurt with low
curd firmness (Labropoulos et al. 1984). Homogenization can reduce
the incidence of pipes in yoghurt i.e. white flecks that appear in some
fruit varieties (Davis, 1967; Nielsen, 1972 and Robinson, 1977).
2.5.3 Storage period:
Yoghurt is not as stable as one would like, due to proteolytic
activity of L. bulgaricus, yoghurt develops a bitter and a stringent
taste during cold storage. Souring is also continued during storage; by
these processes the quality is limited to 14 days at 7° C (Driessen,
1981). Besides this yoghurt can be spoiled (natural deterioration) after
any contamination and growth of yeasts and molds. This deterioration
is strongly dependent on the type of packaging material used.
However, Driessen (1981) suggested several methods for prolonging
the storage life of yoghurt which include aseptic manufacture,
preservation by chemical agents, preservation by heat- treatment of
milk and culture manipulation.
Temperature control (4-5° C) is most important since higher
temperatures can lead to defects such as bitterness, low temperature
can induce ice–crystal formation and slow freezing is detrimental to
yoghurt, but rabidly frozen yoghurt will keep for several months at 27° C (Humphreys and Plunkett, 1969). They added that cold storage
and careful stock rotation play an important role in ensuring that the
consumer always receives uniform first quality yoghurt.
Keeping quality is essentially determined by the rate of
production of flavour. As such, it is related only to the bacterial
quality of milk as a bacterial strain which produces more flavours will
obviously cause spoilage at a lower count (Urbach and Milne, 1987).
International standards are in general agreement in that,
coliform count should not exceed 10 CFU/ml in yoghurt. However,
because of the different methods of controlling the air borne and
ubiquitous moulds and yeasts particularly the yeast, a tolerance of 100
CFU/ml has been given to good quality yoghurt (Salji et al., 1987).
Toba et al. (1983) concluded that oligosaccharides content of
yoghurt progressively increased during storage. Decline in pH of
yoghurt during cold storage has been reported by Tramer (1973) who
related this effect to the ability of the starter culture organisms to carry
out metabolic processes at storage temperatures.
Most vitamins decrease during storage so the level of vitamins
will largely depend on the age of yoghurt, vitamin B12 appears to be
more stable during storage in yoghurt than in milk (Deeth and
Tamime, 1981).
Reddy et al. (1971) found that storage at 5° C caused a decrease
in the level of vitamins, folic acid and vitamin B12 showed the greatest
loss, while the loss of biotin, niacin and pantothenic acid was small.
2.6 Additives in yoghurt:
Yoghurt may contain variable amounts of nutrient additives that
are classified into flavourings, colourings, sweeteners and stabilizers.
2.6.1 Flavoruings and sweeteners:
Wightwick (1970) mentioned that flavors are used for different
reasons which include:
1. To provide a flavour when little or no initial flavour as in is
carbonated beverages.
2. To mask an existing unpleasant flavour e.g. medicines.
3. To boost a flavour which is too weak e.g. ice cream.
4. To give a twist to a naturally occurring flavour e.g. yoghurt.
The flavour used to achieve the above objectives, fall into three
main categories (Wightwick, 1970).
a) Natural flavours: Which are prepared by macerating vegetables,
spices, herbs, fruits etc… with a suitable solvent.
b) Synthetic flavours: Consist of esters, aldehydes, ketones,
alcohols, lactones, etc…
c) A blend of natural and synthetic flavours. The addition of fruit
juice, concentrated fruit juice, fruit pieces and fruit pastes to milk
depends entirely on the particular fruit used, its acidity and the
level at which it is used. The flavour market covers a wide range of
consumable products and extends beyond the food and beverage
industry to pharmaceuticals, animal foods and tobacco of these, by
far; the most important for flavours are soft drinks, dairy products
and savory foods (Firth, 1990).
Wightwick (1970) reported from his personal observation
that, although unflavored yoghurt is in fair demand, the most popular
forms appear to be “real fruit” yoghurt and flavoured yoghurt.
Statistics for the consumption of fruit – flavoured milk and yoghurt
showed that, in Australia the consumption of flavoured milk is
approximately 9 litres per capita per year and approximately 1-2 litres
per capita per year in New Zealand (Wright, 1992).
2.6.2 Stabilizers:
Kosikowski (1982) claimed that properly made plain yoghurt
requires no stabilizer because a stiff, smooth gel with high viscosity is
attained naturally. More of a need exist for stabilizers in flavored
yoghurt and generally the stabilizers used are gelatin, agar or complex
mixtures consisting of guar gum, arab gum and gelatin at level of 0.40.5% (Kosikowski 1982).
Different methods for adding stabilizer exist, it can be added to
the mix prior to pasteurization. In other methods a concentrated
stabilizer and fruit are added to incompletely cooled yoghurt and the
mixture is chilled in the container (Kosikowski 1982).
2.7 The nutritional value of yoghurt:
The chemical composition of food stuff provides a useful
indication of it is potential nutritional value, and the data showed in
Table 2.2 indicate the main components of some typical natural and
fruit yoghurts (Robinson, 1977). If these figures are accepted at face
value, then it is evident that yoghurt could prove an important
introduction to any diet (Robinson, 1977). At the same time, it must
be accepted that values reveal only part of the story, and even if the
almost mystical properties ascribed to yoghurt are ignored for
moment, there are some aspects of the behavior of yoghurt in the
human body that not revealed by chemical analysis (Robinson, 1977).
Several lactic acid organisms produce natural antibiotic for example L.
bulgaricus produces Bulgarican (Reddy and Shahani, 1971). It is of
some interest, therefore, to look at the constituents of yoghurt in a
little more detail, and, in particular, to asses the likely nutritional
importance of the materials concerned (Deeth and Tamime, 1981 and
Alm, 1982).
Wherever
the
final
outcome
of
various
controversies
surrounding the influence of yoghurt on the health of human beings,
there can be little doubt that it is over all nutritional value makes it a
most desirable addition to any diet. If the consumer appeal of both
natural and fruit yoghurts is also placed on record, together with its
excellent performance in respect of public health, then it is evident
why yoghurt is among the most popular fermented dairy products.
(Robinson, 1977).
2.8 Fermentation and microbiological aspects:
The classification of lactic acid bacteria by Orla Jensen (1931)
was still recognized as the standard method for classifying these
organisms. In the 7th edition of Bergey’s Manual (1957) all these
bacteria were grouped in the family Lactobacillaceae, which was
subdivided into two tribes, the Streptococceae (Coccal shaped) and
Lactobacillaceae (rod shaped). However, in the 8th edition of Bergey’s
manual (1974), lactic acid bacteria are reclassified into two families
the Streptococcaceae and Lactobacillaceae. The yoghurt starter
bacteria, S. thermophilus and L. bulgaricus are thermoduric,
homofermentative lactic acid bacteria, their general characteristics are
shown in Table 2.3. Moreover S. thermophilus is distinguished from
other members of the genus Streptococcus by its growth at 45° C and
failure to grow at 10° C. Unlike most streptococci, this species lacks a
recognizable 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 subjects of some
controversy (Davis, 1975). They also reported that the organism
appears to be basically the same as that isolated from Bulgarian
yoghurt by Metchnikoff and Coworkers in the 1900’s
2.9 Ratio of cocci: rods in starter culture:
The yoghurt starter cultures are mixed strain starters of S.
thermophilus and L. bulgaricus which are normally propagated
together at around 42° C (Bergey’s 1974). Successive subculturing
may lead to mutation or to one organism overgrowing the other.
Hence the starter strains are either monitored microscopically (Brad
smear) or examined after culturing on a solid medium, whatever the
method is used, the objective is to monitor the ratio of the cocci to
rods (Bergey’s 1974).
The ‘ratio’ as it appears in the literature, is somewhat confusing,
since it might refer to the colony: colony; clump; chain: chain or cell:
cell ratio of Streptococcus: Lactobacillus (Bergey’s 1974).
Table (2.2): Some typical values of major constituents of milk and
yoghurt:
Milk
Constituent units/100g
Yoghurt
Whole
Skim
Full fat
Low fat
Fruit
Calories
67.5
36
72
64
98
Protein (g)
3.5
3.3
3.9
4.5
5.0
Fat (g)
4.25
0.13
3.4
1.6
1.25
Carbohydrate (g)
4.75
5.1
4.9
6.5
18.6
Calcium (mg)
119
121
145
150
176
Phosphorus (mg)
94
95
114
118
153
Sodium (mg)
50
52
47
51
-
Potassium (mg)
152
145
186
192
254
Adapted from Deeth and Tamime (1981).
Table (2.3): Selected characteristics of yoghurt starter culture:
Characteristic
S. thermophilus
DNA G + C (%)
Acid from glucose
50.3
+
Gas from glucose
+
-
Growth in media containing 2% NaCl
Lactic acid in milk
L. bulgaricus
-
-
L (+)
D (-)
% Acid in milk
1.7
Ammonia from arginine
-
-
Growth at 15°c
-
-
Growth at 45°c
+
+
Serological group
×
E
Requirement for vitamins
Thiamine
-
Riboflavin
+
Folic acid
-
Vitamin B12
-
Carbohydrate utilization
Fructose
+
Galactose
±
±
Gelatin
-
Lactose
+
+
Maltose
-
-
Mannitol
-
-
Sucrose
+
-
Xylose
±
-
Sharpe et al. (1968); Rogosa (1974) and Deibel and Seeley (1974).
+ Positive reaction by 90% or more strains, - negative reaction by 90% or more
strains, ± variable slow or weak reaction, X ungrouped.
CHAPTER THREE
MATERIAL AND METHODS
3.1 Source of milk samples:
Ten stirred yoghurt samples from each of six batches processed
by Blue Nile Dairy Company (CAPO) were obtained from market.
The collection of samples was conducted during the period
from September to December 2005. Each batch was collected
separately. Samples of stirred yoghurt purchased from market are
transported to the laboratory for chemical analysis and for
microbiological examination.
3.2 Chemical analysis:
3.2.1 Fat content:
The fat content was determined by Gerber’s methods described
by AOAC (1990) as follows:
In clean dry Gerber tube, 10 ml of sulphuric acid (density 1.815
gm/ml at 20° C) were poured, and then 10.94 ml of milk samples were
added.
Amyl alcohol (1-2 ml) was added to the mixture, followed by
the addition of distilled water. The contents were thoroughly mixed
till no white particles could be seen. The Gerber tubes were
centrifuged at 1100 revolution per minutes (rpm) for 4-5 minutes, and
the tubes were then transferred to a water bath adjusted at 65° C for
three minutes. The fat percent was then read out directly.
3.2.2 Total solids:
The total solids contents were determined according to the
modified method of AOAC (1990).
Three grams of the milk samples were weighed in dry clean flat
bottomed aluminum dish and heated on a steam bath for 10-15
minutes. The dishes were placed in an oven at 100° C for three hours.
Then cooled in a desicator and weighed quickly. Weighing was
repeated until the difference between the two readings was
< 0.1 mg. The total solids (T.S) contents were calculated as fallows:
T.S% = W1/W2×100.
Where:
W1 = Weight of sample after drying.
W2 = Weight of sample before drying.
3.2.3 Ash content:
The ash content was determined according to the method
described in the AOAC (1990).
Five grams of the sample were weighed in a crucible and
evaporated to dryness on a steam bath. The crucibles were then placed
in a muffle furnace at 550-600° C until ashes were carbon free (2-3
hours), then crucibles were cooled in a desicator and weighed. The ash
content was calculated using the following equation:
Ash% =
W1 ×100
W
Where:
W1 = Weight of ash.
W = Weight of sample.
3.2.4 Protein content:
The protein content of milk samples was determined according
to Kjeldahl method as described by AOAC (1990). Ten ml of each
milk samples were weighed and poured into a clean dry Kjeldahl
flask. Kjeldahl tablets of CuSo4 and concentrated H2SO4 (25 ml) were
added to the flasks. The flasks were heated until clean solutions were
obtained (2-3 hours) and left for another 30 min. The flasks were then
removed and allowed to cool.
The digested milk samples were poured into volumetric flasks
(100 ml) and diluted with distilled water. Then 15 ml of 40% NaOH
was added to each flask and the content of the flask were distillated.
The distillate was received in conical flasks (100 ml) containing ten
ml of 2% boric acid plus three drops of indicator (bromocresol green +
phenolphthalein red).
The distillation was continued until the volume in the flasks was
75 ml then the flasks were removed from distillator. The distillate was
then titrated with 0.1 HCl until the end point (red color) was obtained.
The protein content was calculated as follows:
N% = T × 0.1 × 0.014 × 100/W
P% = N% × 6.38
Where:
T = Reading of titration.
W = Weight of original sample.
N = Total nitrogen.
P = Total protein.
3.2.5 Lactose content:
The lactose content was determined by subtracting the sum of
proteins %, fat % and ash % from total solids %.
Lactose % = T.S% – ( proteins % + fat % + ash %).
3.2.6 Solid not fat (SNF):
The solid not fat was determined by subtracting fat from total
solids.
SNF % = T.S% – fat %.
3.2.7 Titratable acidity (T.A):
The acidity of the milk samples was determined according to
the method described in the AOAC (1990).
Ten ml of each sample was placed in a white porcelain dish and
five drops of phenolphthalein indicator were added. Titration was
carried out using N/9 NaOH until a faint pink colour appeared. The
titration figure was divided by ten to get the percentage of lactic acid.
(1ml of 0.1N sodium hydroxide (NaOH) =0.009 gm of lactic acid).
3.2.8 pH – values:
The pH values were determined using pH – meter (HANNA –
instrument, model 5A520, Rench meter).
3.2.9 Determination of viscosity:
Viscosity was measured by viscometer (Visco 6–R thermo
HAAKE).
3.3 Microbiological examination:
3.3.1 Sterilization:
Petri dishes, test tubes, flasks and pipettes were sterilized by
heating in an oven (Gallen kamb) at 180° C for one hour.
3.3.2 Preparation of serial dilution:
One ml from each milk sample was taken aseptically by
sterile pipette in sterile bottle. They were mixed well with 9 ml sterile
ringers' solution. Serial dilutions were prepared (10-1 – 10-6) according
to Richardson (1985).
3.3.3 Type of culture media used for microbial examination:
3.3.3.1 Plate count agar: (KGaA642711)
This medium was prepared according to Harrigan and McCance (1976), 23.5 gms of the medium were dissolved in 1000 ml
distilled water, then it was sterilized by autoclaving at 121° C for 15
minutes. The media was then distributed using the pour plate
technique.
3.3.3.2 M – 17 medium: (DIFCO, L 110660-1)
The medium was prepared according to Harrigan and McCance,
(1976) by disolving 39.2 gms in 1000 ml distilled water. It was
sterilized by autoclaving at 121° C for 15 minutes, cooled to 50° C.
Then 50 ml of sterilized 10% lactose and agar (20 gm/liter) were
added.
3.3.3.3 MRS broth: Oxoid, (CM 495)
In 1000 ml of distilled water, 67.15 gms were disolved and
sterilized by autoclaving at 121° C for 15 minutes.
3.3.3.4 Nutrient agar: (S.d. Fine-Chem.Ltd 74065)
Twenty eight gms of the nutrient agar were disolved in 1000 ml
distilled water, sterilized by autoclaving at 121° C for 15 minutes.
3.4 Enumeration of microorganisms:
3.4.1 Streptococcus thermophilus:
This was carried out using modified (M–17) medium. 0.1 ml
from suitable dilutions were spread on the surface of sterile modified
M–17 medium. The plates were incubated at 37° C for 48 hours. The
grown colonies were considered as streptococci.
3.4.2 Lactobacillus bulgaricus:
This was carried out using modified (MRS) medium. 0.1ml
from suitable dilutions were spread on the surface of sterile modified
(MRS) medium. The plates were incubated at 37°C for 48 hours.
3.4.3 Total bacterial count:
The pour plate techniques using plate count agar medium was
used for total bacterial count. The plates were incubated at 37° C for
48 hours and the colonies were counted according to Marshal (1992).
3.5 Purification of bacterial isolates:
A part of typical well–isolated colony was picked by a sterile
wire loop and streaked on surface of Petri dish containing a fresh
solidified corresponding medium. The subculturing for each organism
was repeated until a pure colony representing the organism was
obtained. The process was repeated several times. The culture was
checked for purification by the Gram stain. The pure cultures were
streaked onto the surface of nutrient agar medium in McCartney
bottles and incubated at 37° C. The bottles were then stored at 5° C till
used (Barrow and Feltham, 1993).
3.6. Identification of organisms:
The identification of purified colonies was carried out according
to Barrow and Feltham (1993). The purified isolates were subjected to
the following tests:
3.6.1 Primary tests:
3.6.1.1 Morphological appearance:
The morphological appearance of each bacterium was recorded
according to Barrow and Feltham (1993).
Streptococci: Gram-positive cocci in pairs or chain, nonmotile.
Lactobacilli: Gram-positive rods often coryneform; chain
formation common; typically non- motile and not acid fast.
3.6.1.2 Shape of cell:
This was done by Gram stain as described by Harrigan and
McCance (1976) as follows:
Crystal violet was added to smear on slides for one minute,
followed by washing with distilled water. Lugol’s iodine was added
for one minute, and removed by washing with distilled water. The
slides were decolorized by alcohol for ten seconds and the residue was
removed by distilled water. The slides were counter – stained with
Bacto Gram Saffranin for 30-60 seconds and washed with distilled
water.
The slides were then dried with a filter paper and a drop of
immersion oil was added followed by examining under microscope.
Gram positive–organisms appeared purple, while Gram negative ones
appeared pink.
3.6.1.3 Catalase test:
The organisms to be tested were put on sterile slides. A drop of
3% hydrogen peroxide (H2O2) added to the colony and mixed.
Evolution of gas immediately or after five minutes indicated a
catalase positive result.
3.6.1.4 Oxidase test:
Two or three drops of tetra methyl – P – phenylene diamine
dihydrochloride were placed on filter paper (7 cm in diameter). Before
the drops dry, the test organism that was grown on nutrient agar
medium was removed with sterile glass rod and smeared a cross the
impregnated paper. The development of a dark purple colour within
ten seconds indicated the presence of oxidase enzyme.
3.6.1.5 Motility test:
Hanging drop technique was used. The culture was transfered
by loop to slide and examined under the microscope.
3.6.1.6 Oxidation fermentation test (OF):
Duplicate tubes of Hugh and Lifson’s medium were inoculated
by stabbing with a sterile straight wire, medium in one of the tubes
was covered with a layer of soft sterile paraffin oil to a depth of about
one cm, while the other tube was not covered. The tubes were then
incubated at 37° C and examined daily for 14 days. Colour change to
yellow in both opened and covered tubes indicated fermentative
organisms, while change in the uncovered tubes only indicated
oxidative organisms.
3.6.2 Confirmatory tests:
Fermentation of sugars:
A twenty four hours culture was inoculated into peptone broth
with sugars (manitol, lactose, xylose, maltose, glucose) and incubated
at 37° C for up to seven days. Change in colour to yellow indicated a
positive reaction. Gas was accumulated in the Durham’ tubes when
produced.
3.7 Statistical analysis:
All the data of this experiment were analyzed statistically by
using complete randomized design (CRD) and the least significant
difference test, which was used to detect difference between means
(Snedecor and Cochran, 1980). The analysis was carried out using
SPSS program (Statistical Packages for Social Science), Model
design:
Yij = M + αi + eij.
Where:
M = The over all population mean.
αi = The effect of the ith A class expressed as a deviation from
the over all mean.
eij = Random errors.
CHAPTER FOUR
RESULTS AND DISCUSSION
4.1 Chemical composition of stirred yoghurt:
Tables (1 6) show the chemical composition of stirred yoghurt
samples (fat, protein, lactose, ash, total solids, solids non fat, pH,
acidity and viscosity).
4.1.1 Fat:
Table (1) shows the fat content at days 0, 2, 4, 6, 8 and 10
which ranged from 4.00% – 4.90%, 3.10% – 4.10%, 1.60%– 2.40%,
2.50%–3.10%, 2.50%– 3.10% and 2.50% – 3.20%, respectively. The
mean values were 4.51% ± 0.27, 3.66% ± 0.38, 2.17% ± 0.26, 2.82%
± 0.23, 2.77% ± 0.22 and 2.77% ± 0.23, respectively.
Table (6) shows the statistical analysis of chemical composition
of fat by using one way ANOVA. From those results it was found the
fat content was significant at P < 0.001. The results observed
confirmed the findings of Hofi et al. (1978). There was small variation
in fat content of different samples of yoghurt because of
standardization resulting in minimal compositional variation from
sample to sample, similarly as milk composition vary from day to day
or batch to batch. However these results are not in accordance with the
findings of Athar (1986) who reported 3.6 percent fat in typical plain
yoghurt.
Table (1) Fat and protein content of stirred yoghurt samples
during storage 0, 2, 4, 6, 8 and 10 day
Fat%
Days
Mean ± Sd.
0
4.51 ±0.27
2
3.66 ±0.38
4
2.17 ±0.26
6
2.82 ±0.23
8
2.77 ±0.22
10
2.77 ±0.23
Protein%
Minimum
Maximum
Mean ± Sd.
Minimum
Maximum
d
4.00
4.90
2.6 6 ±0.70
a
1.79
3.75
c
3.10
4.10
3.16 ±0.84
a
1.61
4.11
a
1.60
2.40
3.97
±0.56
2.86
4.65
b
2.50
3.10
3.50 ±1.01
ab
1.79
4.73
b
2.50
3.10
3.97 ±1.14
b
2.14
5.36
b
2.50
3.20
2.66 ±0.54
a
1.97
3.93
ab
In this and the following tables the column being the same superscript
latter are significantly (P > 0.05) not different
4.1.2 Protein:
Table (1) shows the protein content at days 0, 2, 4, 6, 8 and 10
readings ranged from 1.79%–3.75%, 1.61%– 4.11%, 2.86% – 4.65%,
1.79%–4.73%, 2.14%– 5.36% and 1.97% – 3.93%, respectively. The
mean values were 2.66% ± 0.70, 3.16% ± 0.84, 3.97% ± 0.56, 3.50%
± 1.01, 3.97% ± 1.14, and 2.66% ± 0.54, respectively.
Table (6) shows the statistical analysis of the chemical
composition of protein. The results indicates that the protein content
was significant at P< 0.05. This result was in agreement with the
results of Tamime and Deeth (1980), who concluded that the addition
of milk powder raised the protein level of milk. These results are also
in line with findings of Duitschaver and Arnott (1972), who reported
that plain yoghurt was higher in protein content due to the absence of
dilution effect.
These results are not in line with findings of Shanley (1973)
who found that the protein and ash contents of yoghurt decreased with
the progress of storage period. This is due to the fact that the majority
of protein and mineral contents in milk decreased with storage.
4.1.3 Lactose:
Table (2) show the lactose content at days 0, 2, 4, 6, 8 and 10
were ranged from 7.03%–9.30%, 7.53%–10.29%, 6.75%–10.26%,
6.98%–10.47%, 6.79%–10.18% and 8.29%–10.92%, respectively. The
mean
values
were
8.31%±0.85,
9.02%±0.61,
8.83%±0.90,
8.59%±0.96, 8.45% ±1.18, and 9.58% ± 0.81, respectively. Table (6)
shows the statistical analysis results of lactose. These results are in
line with findings of Davis and Mclachlan (1974) who found that the
lactose calculated from protein was (5.05–8.74%).These results were
higher than those reported by El- Sadek et al (1972) for lactose in
zabady (4.5 - 4.8 %).
4.1.4 Ash:
The ash content at days 0, 2, 4, 6, 8 and 10 were shown in table
(2). The ash values ranged from 0.80%– 0.92%, 0.67%– 0.86%,
0.60%– 0.93%, 0.80%– 0.99%, 0.54%– 0.95% and 0.70%– 0.95%
respectively. The mean values were 0.84% ± 0.03, 0.73% ± 0.06,
0.78% ± 0.11, 0.92% ± 0.06, 0.86% ± 0.12, and 0.86% ± 0.07,
respectively.
Table (6) shows the chemical composition of stirred yoghurt.
The ash content was significant by different at P < 0.05, these results
are in line with finding of Shanley (1973) who found that the protein
and ash contents of yoghurt decreased with the progress of storage
period. This is due to the fact that the majority of protein and mineral
contents in milk decreased with storage. These results were supported
by the findings of Hidiroglou and Proulx (1982) who reported that
milk Ca, P and Mg contents were all highest during the first day of
storage then decreas sharply at 2nd day.
4.1.5 Total solids (TS):
Table (3) illustrates total solids content at days 0, 2, 4, 6, 8
and 10 which ranged from 15.57% – 16.90%, 16.24% – 16.92%,
14.02% – 16.69%, 15.50% – 16.42%, 15.63% – 16.28% and 14.84% –
17.74%, respectively. The mean values were 16.32% ± 0.34, 16.57%
± 0.21, 15.75% ± 0.73, 15.76% ± 0.28, 16.05% ± 0.17, and 15.88% ±
0.75, respectively.
Table (6) shows that there are no variation between total
solids (P > 0.05). These results are in line with finding of Hofi et al.
(1978). Robinson (1983) reported that the aim of total solids is
consistency improvement imparted to the yoghurt coagulum; these
results are totally different from those reported by Sarkar et al. (1996).
The results are in accordance with the findings of Athar (1986). There
was hardly any variation in total solids of different samples of stirred
yoghurt. This is most probably because of standardization of raw milk
and quality control measures taken to ensure consistency of end
product. But in case of milk used without subjecting to standardization
thus may lead to much variation as observed in total solids content.
Table (2) Lactose and Ash content of stirred yoghurt samples
during storage 0, 2, 4, 6, 8 and 10 day
Lactose content %
Ash content %
Days
Mean ± Sd.
Min.
Max.
Mean ± Sd.
Min.
Max.
Zero day
8.31 a ±0.85
7.03
9.30
0.84 ab ±0.03
0.80
0.92
2
9.02 a ±0.61
7.53
10.29
0.73 a ±0.06
0.67
0.86
4
8.83 a ±0.90
6.75
10.26
0.78 ab ±0.11
0.60
0.93
6
8.59 a ±0.96
6.98
10.47
0.92 ab ±0.06
0.80
0.99
8
8.45 a ±1.18
6.79
10.18
0.86 b ±0.12
0.54
0.95
10
9.58 a ±0.81
8.29
10.92
0.86 b ±0.07
0.70
0.95
4.1.6 Solids – non–fat (SNF):
Table (3) shows the average of solids – not – fat (SNF) content
at days 0, 2, 4, 6, 8 and 10, the SNF content ranged from 11.34% –
12.39%,
12.14%–13.52%,
11.82%–14.50%,
12.40%–
13.72%,
12.93%– 13.69% and 12.34%– 14.52%, respectively. The mean
values were 11.73% ± 0.31, 12.91% ± 0.49, 13.58% ± 0.79, 12.94% ±
0.40, 13.28% ± 0.29, and 13.11% ± 0.61, respectively.
From the results it was found that the solids– non – fat (SNF)
content were significant at P < 0.001 (Table 6). These results do not
agree with the findings of Richter (1980). There was no significant
variation in SNF content of different samples of plant made yoghurt
because raw milk is standardized to a fixed SNF content in order to
ensure consistency of end product. But in case of these results due to
raw milk is used without subjecting to standardization. Hence more
variation was observed in SNF content of yoghurt samples. These
results were in agreement with those obtained by El – Shibiny et al.
(1979a and b) who claimed that total solids content decreased
proportionally during the storage period with increase in glucose plus
galactose and these result are in line with findings of Tamime and
Deeth (1980) who found that the production of flavour components
such as acetaldehyde can arise from fat, protein or lactose and hence
their results showed that any variation of content will affected the
solids – non – fat content, so it is very important that to standardized
the milk as well as to fix the level to acceptable standard. Tamime and
Robinson (1976) found that the legal standards of yoghurt are mainly
based on the chemical composition of the product i.e. percentage of
fat, solids – non – fat (SNF) or total solids (TS). A minimum
specification for SNF or TS is included by some countries, but the
main divisions are on the basis of fat content.
4.1.7 pH:
The pH values of stirred yoghurt at days 0, 2, 4, 6, 8 and 10 as
shown in table (4) ranged from 3.73 – 4.72, 3.91 – 4.43, 4.03 – 4.40,
4.01 – 4.34, 3.82 – 4.14 and 3.52 – 4.04, respectively. The mean
values were 4.17 ± 0.29, 4.13 ± 0.17, 4.19 ± 0.13, 4.17 ± 0.11, 4.02 ±
0.10 and 3.81 ± 0.19, respectively.
Table (6) shows the result of pH were significant at P < 0.001
and the results of high acid production and decreased pH agree with
findings of Kondratenko et al. (1978) and Zobkona and Plish (1980).
These results are in line with the findings of
Eckles et al. (1952),
who reported that when acid production increased then quality of
dairy product was deteriorated. As the temperature of the refrigerator
was not suitable for the growth of acid producing bacteria so that acid
Table (3) Total solids (T.S) and Solids–non–fat (SNF) of stirred
yoghurt samples during storage 0, 2, 4, 6, 8 and 10 day
Total solids (T.S)
Days
Solids–non–fat (SNF)
Mean
Mean
Minimum Maximum
± Sd.
Minimum Maximum
± Sd.
0
16.32 ±0.34
b
15.57
16.90
11.73 ±0.31
a
11.34
12.39
2
16.57 ±0.21
b
16.24
16.92
12.91 ±0.49
b
12.14
13.52
4
15.75
ab
±0.73
14.02
16.69
13.58 ±0.79
c
11.82
14.50
6
15.76
ab
±0.28
15.50
16.42
12.94 ±0.40
b
12.40
13.72
8
16.05 ±0.17
ab
15.63
16.28
13.28
bc
±0.29
12.93
13.69
10
15.88 ±0.75
a
14.84
17.74
13.11
bc
±0.61
12.34
14.52
Production was slow and resulting in long shelf life of yoghurt
(Osborne and Pritchard 1974). The results of the study are not in line
with the findings of Salji et al. (1985) and Varnam and Sutherland
(1994), who found no significant variation in pH of different samples
of plant made yoghurt as compared to dahi because yoghurt was
incubated for specific time and temperature to attain desired pH. In
case of dahi proper fermentation condition are not fully controlled.
4.1.8 Acidity
The acidity percent of stirred yoghurt in table (4) shows at days
0, 2, 4, 6, 8 and 10 ranged from 0.89 – 0.97, 0.94– 1.50, 0.80 – 0.99,
0.90– 0.99, 0.97–1.00 and 0.99 – 1.20, respectively. The mean values
were 0.94 ± 0.02, 1.12 ± 0.21, 0.93 ± 0.06, 0.96 ± 0.03, 0.99 ± 0.01
and 1.08 ± 0.08, respectively.
From table (6) it was found that the result of acidity was
significant at P < 0.001 and these results are in line with findings of
Kamruzzaman et al. (2002). They found that acidity percentage of
plain and dahi samples increases both at room and refrigeration
temperature during storage. These results are in accordance with the
findings of Davis and Mclachlan (1974), who reported less variation
in acidity of different samples of plant made yoghurt as compared to
dahi due to controlled incubation and post production handling and
controlled storage at 4° C, so acidity remains the same through all
seasons. However in case of dahi uncontrolled incubation and post
production handling and uncontrolled storage lead to increase in
acidity during summer and subsequent decrease during winter season.
This results are in line with the findings of Kim et al. (1998) who
found that titratable acidity was 0.95 – 0.99% of Yam – yoghurt which
was prepared by addition of Yam to skim milk substrate and cultured
with a mixed cultures. Also this findings are in line with the findings
of Gregurek (1999) who found that post acidity cation showed slightly
higher acidity in yoghurt prepared with lower amounts of inoculum.
He concluded that the amount of inoculum and incubation temperature
should be properly maintained, to achieve maximum viability. From
the other side these results are not in line with the findings of Collado
et al. (1994) who found that the yoghurt drink and yoghurt like
products had 0.56% and 0.58% total titratable acidity. These results
are not in line with the findings of Dalles and Kechagias (1984) who
found that the acidity of commercial types of yoghurt in Greece
ranged from 1.02 – 2.15%.
4.1.9 Viscosity:
The viscosity at days 0, 2, 4, 6, 8 and 10 are shown in table (5)
the viscosity results ranged from 38.30% – 82.70%, 39.40%–80.10%,
49.30% – 80.20%, 49.40%–81.10%, 55.40%– 75.30% and 56.30% –
73.00%, respectively. The mean values were 64.79% ± 13.91,
Table (4) pH and acidity development of stirred yoghurt samples
during storage 0, 2, 4, 6, 8 and 10 day
pH
Days
Acidity
Mean
Mean
Minimum Maximum
± Sd.
Minimum
Maximum
a
0.89
0.97
b
0.94
1.50
a
0.80
0.99
a
0.90
0.99
a
0.97
1.00
b
0.99
1.20
± Sd.
b
3.73
4.72
0.94 ±0.02
b
3.91
4.43
1.12 ±0.21
b
4.03
4.40
0.93 ±0.06
b
4.01
4.34
0.96 ±0.03
b
3.82
4.14
0.99 ±0.01
a
3.52
4.04
1.08 ±0.08
0
4.17 ±0.29
2
4.13 ±0.17
4
4.19 ±0.13
6
4.17 ±0.11
8
4.02 ±0.10
10
3.81 ±0.19
61.98% ± 11.72, 62.57% ± 8.93, 62.96% ± 10.80, 64.34% ± 6.23 and
64.95% ± 6.07, respectively.
Table (6) shows there was no significant P > 0.05 difference in
viscosity between the samples of stirred yoghurt.
4.2 Microbiological results of stirred yoghurt:
Table (7) shows the microbiological analysis of stirred yoghurt
samples at days 0, 2, 4, 6, 8 and 10.
4.2.1 Standard plate count:
The standard plate count of bacteria at days 0, 2, 4, 6, 8 and 10
ranged from 7.00– 7.60, 7.18– 8.78, 7.18– 7.54, 7.18– 7.65, 7.30–
8.78 and 7.18– 7.70, respectively. The mean values were 7.27 ± 0.20,
7.68 ± 0.42, 7.36 ± 0.14, 7.43 ± 0.17, 7.56 ± 0.13 and 7.50 ± 0.14,
respectively.
Table (9) indicated that the results of plate count was significant
at P < 0.01. These results are in line with findings of Riadh Al-Tahiri
(2005) who found that the total bacterial count was 6 × 106 in yoghurt
produced by modern dairies in Jordan. Results of this study are also in
accordance with the findings of Kosikowski (1977) who found that
plain yoghurt may
Table (5) Viscosity of stirred yoghurt samples during storage
0, 2, 4, 6, 8 and 10 day
Days
Mean ± Sd.
Minimum
Maximum
Zero day
64.79 a ±13.91
38.30
82.70
2
61.98 a ±11.72
39.40
80.10
4
62.57 a ±8.93
49.30
80.20
6
62.96 a ±10.80
49.40
81.10
8
64.34 a ±6.23
55.40
75.30
10
64.95 a ±6.07
56.30
73.00
Table (6) Chemical composition of stirred yoghurt using one way
ANOVA analysis
Mean
square
pH
Acidity
Fat
Viscosity
T.S.
Protein
Ash
SNF
Lactose
Between groups
0.338
Within groups
0.059
Between groups
0.062
Within groups
0.009
Between groups
6.922
Within groups
0.074
Between groups
15.762
Within groups
100.494
Between groups
3.455
Within groups
1.932
Between groups
7.767
Within groups
2.871
Between groups
0.031
Within groups
0.015
Between groups
4.034
Within groups
0.261
Between groups
1.183
Within groups
0.937
NS: non significant (P > 0.05)
*: Significant at (P < 0.05)
**: Significant at (P < 0.01)
***: Significant at (P < 0.001)
F
Sig.
5.738
0.001***
6.802
0.001***
94.109
0.001***
0.157
0.977NS
1.788
0.131NS
2.705
0.030*
2.150
0.073*
15.437
0.001***
1.262
0.924NS
Contain up to one billion live L. bulgaricus and S. thermophilus cells
per ml. As yoghurt stored at 4° C becomes older, these bacteria die
and the numbers will decline to few million per ml. These results are
in line with findings of Masud et al. (1991) who found that total viable
count as well as variation was more in dahi samples.
These results are different from those reported by Davis and
Mclachlan (1974). There was no significant variation in total viable
count of different samples of plant made yoghurt as compared to dahi
because defined starter culture is used under proper condition of
fermentation for manufacture of yoghurt (Kon, 1959).
4.2.2 Identification of isolated LAB:
Table (10) shows the identification of lactic acid bacteria, for
S. thermophilus that is Gram positive, shape cocci, non motile,
catalase positive, oxidase positive, glucose positive, lactose positive,
maltose negative, sucrose positive, xylose positive and fructose
positive.
The L. bulgaricus is Gram positive, rod shape, non motile,
catalase positive and oxidase positive, glucose positive, lactose
positive, maltose negative, sucrose negative, xylose negative and
fructose positive.
4.2.3 Streptococcus thermophilus:
Table (8) show the growth of S. thermophilus in stirred yoghurt
at days 0, 2, 4, 6, 8 and 10 the results show the count of S.
thermophilus ranged from 7.00– 7.40, 7.00 – 7.60, 7.18– 7.48, 7.00 –
Table (7) Total bacteria count of stirred yoghurt samples during
storage 0, 2, 4, 6, 8 and 10 day
Days
Mean ± Sd.
Minimum
Maximum
0 day
7.27 a ±0.20
7.00
7.60
2
7.68 c ±0.42
7.18
8.78
4
7.36 ab ±0.14
7.18
7.54
6
7.43 ab ±0.17
7.18
7.65
8
7.56 bc ±0.13
7.30
8.78
10
7.50 bc ±0.14
7.18
7.70
7.54, 7.30 – 7.78 and 7.30 – 7.54, respectively. The mean values were
7.15 ± 0.15, 7.31 ± 0.16, 7.34 ± 0.11, 7.27 ± 0.18, 7.51 ± 0.15, and
7.41 ± 0.09, respectively.
Table (10) shows that the total counts of S. thermophilus were
significant at P < 0.001. These results are in accordance with the
findings of Kosikowski (1977) who reported that plain yoghurt may
contain up to one billion live L. bulgaricus and S. thermophilus cell
per ml. As yoghurt stored at 4° C becomes older, these bacteria die
and numbers will decline to few millions per ml. These results are also
in line with findings of Cousin and Marth (1976) who found that the
count of S. thermophilus ranged from 3.4 × 103 to 6.4 × 106 /ml. Also
results were in agreement with Tamime and Deeth (1980) who
reported that the quantitative standards for yoghurt bacteria differ.
It is generally accepted that the yoghurt should contain 107 cfu
of viable bacteria (Streptococcus thermophilus and Lactobacillus
bulgaricus) per ml of yoghurt (Tamime and Deeth, 1980).
Consequently there has been considerable research into ways to
maintain the viability of these organisms to ensure that the live
bacteria are still present in the yoghurt when it is consumed. The
present results differ from the findings of Jay (1986) who found that
yoghurt of the western world is cultured with Lactobacillus bulgaricus
and Streptococcus thermophilus at temperature around 43° C, as
freshly prepared yoghurt, has 100 million to 2 billion bacterial cells
per gram.
4.2.4 Lactobacillus bulgaricus:
Table (8) show the growth of L. bulgaricus in stirred yoghurt at
days 0, 2, 4, 6, 8 and 10, the result shows the count of Lactobacillus
bulgaricus ranged from 7.00 – 7.48, 7.18 – 7.78, 7.18 – 7.40, 7.00 –
7.48, 7.18 – 7.65 and 7.18 – 7.54, respectively. The mean values were
7.21 ± 0.15, 7.37 ± 0.16, 7.30 ± 0.10, 7.33 ± 0.15, 7.50 ± 0.14 and
7.41 ± 0.13, respectively.
Table (9) show the microbiological analysis and from these
results it was found that the total counts of
L. bulgaricus were significant by different (P < 0.001).
These results are in line with the findings of Ishibashi and
Shimamura (1993) who found that in Japan the fermented milk and
lactic beverages association has established a standard that requires
the presence of > 107 viable bacteria/ml in dairy products.
These results are in accordance with the 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.
These results are in agreement with the findings of Harmann
and Marth (1984) who found that after extensive study of survival of
S. thermophilus and L. bulgaricus in commercial and experimental
yoghurts recommended that yoghurt should contain at least one
million viable organisms per gram at the time of sale.
These results differ from those of Jay (1986) who found that
yoghurt of the western world is cultured with Lactobacillus bulgaricus
and Streptococcus thermophilus at temperature around 43° C and
freshly prepared yoghurt has 100 million to 2 billion bacterial cell per
gram.
Table (8) Microbiology analysis of stirred yoghurt samples during
storage 0, 2, 4, 6, 8 and 10 day
Streptococcus thermophilus
Days
Mean
Lactobacillus bulgaricus
Mean
Minimum Maximum
± Sd.
a
0
7.15 ±0.15
2
7.31
bc
4
7.34
6
7.27
Minimum Maximum
± Sd.
a
7.00
7.48
bc
7.18
7.78
ab
7.18
7.40
±0.15
7.00
7.48
7.78
7.50 ±0.14
7.18
7.65
7.54
7.41
7.18
7.54
7.00
7.40
7.21 ±0.15
±0.16
7.00
7.60
7.37 ±0.16
bc
±0.11
7.18
7.48
7.30 ±0.10
ab
±0.18
7.00
7.54
7.33
d
7.30
cd
7.30
8
7.51 ±0.15
10
7.41 ±0.09
ab
c
bc
±0.13
Table (9) Microbiology analysis of stirred yoghurt using one way
ANOVA analysis
M.S.
Between groups
Within groups
0.050
Between groups
0.154
S. thermophilus
Within groups
0.021
Between groups
0.099
L. bulgaricus
NS: non significant (P > 0.05)
*: Significant at (P < 0.05)
**: Significant at (P < 0.01)
***: Significant at (P < 0.001)
Sig.
4.263
0.002**
7.420
0.001***
5.115
0.001**
0.214
Plate count
Within groups
F.
0.019
Table (10) Identification of lactic acid bacteria
Primary test
Fermentation of sugar
Gram
Shape
Motility
Catalase
Oxidase
OF
Glucose
Lactose
Maltose
Sucrose
S. thermophilus
+
Cocci
-
+
+
F
+
+
-
+
+
+
L. bulgaricus
+
Rod
-
+
+
F
+
+
-
-
-
+
Streptococcus thermophilus
Lactobacillus bulgaricus
+: positive
-: negative
F: Fermentation
O: oxidation
Xylose Fructose
CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion:
The overall picture of stirred yoghurt quality evaluation need emphasis
on quality control during processing plants. The large number of samples
containing of either streptococci or lactobacilli indicates improper storage
conditions of yoghurt in merchandising channels.
Yoghurt is biologically active product and therefore has to be kept at
about 5° C. It is likely that inadequate storage conditions and age of sample
influenced the pH readings obtained. Generally the customers need to receive
good value products for their money. The lack of cold storage condition during
marketing responsible for variation of the product and thus influence public
acceptance.
5.2 Recommendations:
1.
More attention should be given to proper incubation temperature
and good quality control during processing.
2.
Standardization of milk for yoghurt manufacture should be observed
to meet legal standards.
3.
Adjustment of yoghurt mix should approach the standard of the
yoghurt package label.
4.
Yoghurt is biologically active product and therefore has to be kept
at about 5° C until it reaches the consumers.
5.
Cold stores with proper and efficient cooling are essential in dairy
plants and markets.
6.
Manufacturing and expiry date should be lighted on label of the
product.
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