PREPARATION AND QUALITY EVALUATION OF COCONUT

MILK INCORPORATED YOGHURT

by

Bibek Adhikari

Department of Food Technology

Central Campus of Technology

Institute of Science and Technology

Tribhuvan University, Nepal

2018
 Preparation and Quality Evaluation of Coconut Milk Incorporated

Yoghurt

A dissertation submitted to the Department of Food Technology, Central Campus of

Technology, Tribhuvan University, in partial fulfilment of the requirement for the
degree of B. Tech. in Food Technology

by

Bibek Adhikari

Department of Food Technology

Central Campus of Technology, Dharan
Institute of Science and Technology
Tribhuvan University, Nepal
March, 2018

ii
 Tribhuvan University
Institute of Science and Technology
Department of Food Technology
Central Campus of Technology, Dharan

Approval Letter

This dissertation entitled Preparation and Quality Evaluation of Coconut Milk

Incorporated Yoghurt presented by Bibek Adhikari has been accepted as the partial
fulfillment of the requirements for the B. Tech. in Food Technology

Dissertation Committee

1. Head of the Department _______________________________

(Mr. Basanta Kumar Rai, Assoc. Prof.)

2. External Examiner ____________________________________

(Mr. Birendra Yadav, Asst. Prof.)

3. Supervisor ___________________________________________

(Mr. Navin Gautam, Asst. Prof.)

4. Internal Examiner ____________________________________

(Mr. Dev Raj Acharya, Asst. Prof.)

March, 2018

iii
 Acknowledgements

I express my deep sense of gratitude to my guide Mr. Navin Gautam Asst. Prof Central
Campus of Technology, Dharan for his excellent guidance, encouragement and inspirations
throughout the work.

I am also grateful to Prof. Dr. Dhan Bahadur Karki (Campus chief, Central Campus of
Technology), Assoc. Prof Basanta Kumar Rai (HOD, Department of Food technology) and
Assoc. Prof Geeta Bhattarai (Chairperson, Central Department of Food Technology) for
their generosity and co-operation in providing an opportunity and facilities to perform this
work successfully. My sincere thanks to all my friends Pankaj Dahal, Ajit Kumar Sah,
Gajendra Bohora, Prajwal Pokharel, Nabin Parajuli, Niseel Manandhar, Nikesh Niroula
,Sanjeev neupane, Hari khatri and all who assisted me directly and indirectly, throughout
the work. Thanks to all the laboratory and library staffs of Central Campus of Technology,
Dharan for their kind co-operation. Finally, I am highly indebted to my parents and family
members for their constant encouragement, love, inspiration and moral support without
whom this work has not been completed successfully.

Date of Submission: March, 2018 Bibek Adhikari

iv
 Abstract

The current research work was conducted to measure the quality and feasibility of yogurt
prepared by using cow milk and different levels of coconut milk with 2% starter culture.
The aim of this research is to study the effect of addition of coconut milk in yoghurt.
Design expert® version 10 D-optimal design was employed for the formulating the recipe
of yoghurt. The obtained five formulations coded A (10%), B (20%), C (30%), D (40%) &
E (50%) of coconut milk incorporated yoghurt were prepared in laboratory. The samples
were subjected to sensory evaluation by quality scoring method for consumer acceptability.
Score was given by individual panelist on the basis of color, taste, aroma and texture.
Based on these quality parameter and sensory analysis score, the data were analyzed by
two ways ANOVA (no blocking) using Genstat and means were compared using LSD at
5% level of significance.

From sensory evaluation, formulation A (10% ) coconut milk incorporated yoghurt was
found to be significantly (p<0.05) best using LSD at 5% level of significance quality
whose total solid, fat, acidity, protein, carbohydrate, total ash, moisture content , lactose
content and pH were found 20%, 5.1%, 0.71%, 3.5%, 5.06%, 0.94%, 82%, 3.8% and 4.4
respectively. Shelf life of the best product was estimated in terms of acidity and total plate
count and the shelf life was found to be 2 days at room temperature and 7 days at
refrigeration.

v
 Contents

Approval Letter .................................................................................................................. iii

Acknowledgements ............................................................................................................. iv

Abstract ................................................................................................................................ v

List of tables ....................................................................................................................... xii

List of figures .................................................................................................................... xiii

List of abbreviations ......................................................................................................... xiv

1. Introduction .................................................................................................................. 1-4

1.1 General introduction ............................................................................................... 1

1.2 Statement of the problem ........................................................................................ 2

1.3 Objectives................................................................................................................ 3

1.3.1 General objectives ..................................................................................... 3

1.3.2 Specific objectives ..................................................................................... 3

1.4 Significance of the study ......................................................................................... 3

1.5 Limitation of the study ............................................................................................ 4

2. Literature review ........................................................................................................ 5-27

2.1 Historical background ............................................................................................. 5

2.2 Development of dairy industry in Nepal ................................................................. 7

2.3 Milk ......................................................................................................................... 7

2.4 Milk fermentation ................................................................................................... 8

2.5 Types of fermented milk ......................................................................................... 9

2.5.1 Traditional fermented milk ........................................................................ 9

2.5.2 Non-traditional fermented milk ................................................................. 9

2.6 Advantages of milk fermentation ............................................................................ 9

vi
2.7 The fundamental microbiology of yoghurt ........................................................... 10

2.8 Basic stages and CCPs of yoghurt manufacturing ................................................ 11

2.9 Starter culture ........................................................................................................ 12

2.10 Types of starter culture ....................................................................................... 13

2.10.1 Liquid and freeze-dried culture ........................................................... 13

2.10.2 Pure and mixed culture ........................................................................ 13

2.10.3 Mesophilic and thermophilic culture ................................................... 13

2.10.4 Physical forms of starter culture .......................................................... 14

2.10.5 Liquid cultures ..................................................................................... 14

2.10.6 Powdered cultures................................................................................ 14

2.10.7 Frozen culture ...................................................................................... 14

2.11 Selection of cultures ............................................................................................ 15

2.12 Preparation of starter culture ............................................................................... 15

2.13 Bio-technically important metabolic activities ................................................... 15

2.13.1 Acid production by microorganisms ................................................... 15

2.13.2 Flavor components in yoghurt ............................................................. 16

2.13.3 Ropiness and consistency .................................................................... 16

2.13.4 Proteolytic activity............................................................................... 16

2.13.5 Lipolytic activity.................................................................................. 17

2.13.6 Other metabolic activities .................................................................... 17

2.14 Role of thermophilic cultures in the intestine ..................................................... 17

2.14.1 Production of antibiotic substances ..................................................... 18

2.14.2 Effect on immunoglobulin level, cholesterol, lactose activity and tumor

cells ...................................................................................................... 18
vii
 2.14.3 Formation of bacteriocins and other inhibitory compounds .................. 18

2.15 Causes of slow growth ........................................................................................ 18

2.15.1 Bacteriophages..................................................................................... 19

2.15.2 Effect of chemical composition of the lactic acid flora ....................... 19

2.16 Coagulum formation in yoghurt .......................................................................... 19

2.17 Syneresis ............................................................................................................. 20

2.18 Methods for improving the body (viscosity) of yoghurt ..................................... 20

2.19 Types of yoghurt ................................................................................................. 20

2.19.1 Set type yoghurt ................................................................................... 20

2.19.2 Stirred type yoghurt ............................................................................. 21

2.19.3 Drinking type of yoghurt ..................................................................... 21

2.19.4 Frozen yoghurt ..................................................................................... 21

2.19.5 Dried yogurt ......................................................................................... 21

2.19.6 Dietetic/therapeutic yoghurt ................................................................ 22

2.20 Addition of fruit flavoring in yoghurt ................................................................. 22

2.21 Shelf-life of yoghurt ............................................................................................ 22

2.22 Techniques of shelf-life extension ...................................................................... 23

2.23 Coconut ............................................................................................................... 24

2.23.1 Description and origin ......................................................................... 24

2.23.3 Coconut fat .......................................................................................... 25

2.23.4 Coconut meat ....................................................................................... 26

2.23.5 Coconut milk ....................................................................................... 26

2.23.6 Nutritional and medicinal importance of coconut milk ....................... 27

viii
3. Material and methods .............................................................................................. 29-36

3.1 Materials................................................................................................................ 29

3.1.1 Milk ......................................................................................................... 29

3.1.2 Coconut .................................................................................................... 29

3.1.3 Milk solid not fat ..................................................................................... 29

3.1.4 Sweetener................................................................................................. 29

3.1.5 Starter Culture.......................................................................................... 29

3.1.6 Containers ................................................................................................ 29

3.1.7 Equipment and chemicals ........................................................................ 30

3.2 Method .................................................................................................................. 30

3.2.1 Preparation of coconut milk .................................................................... 30

3.2.2 Preparation of set type coconut milk yoghurt .......................................... 31

3.2.3 Chemical analysis of coconut milk .......................................................... 32

3.2.3.1 Acidity ................................................................................... 32

3.2.3.2 Fat .......................................................................................... 32

3.2.3.3 Protein .................................................................................... 32

3.2.3.4 Ash ......................................................................................... 33

3.2.3.5 pH .......................................................................................... 33

3.2.3.6 Total Solid (TS) ..................................................................... 33

3.2.3.7 Moisture ................................................................................. 33

3.2.3.8 Vitamin C .............................................................................. 33

3.2.3.9 Total sugar ............................................................................. 33

ix
 3.2.4 Chemical analysis of Milk ....................................................................... 33

3.2.4.1 Acidity ................................................................................... 33

3.2.4.2 Fat .......................................................................................... 33

3.2.4.3 Protein .................................................................................... 33

3.2.4.4 Ash ......................................................................................... 33

3.2.4.5 pH .......................................................................................... 34

3.2.4.6 Total Soluble Solid (TSS)..................................................... 34

3.2.4.7 Lactose ................................................................................... 34

3.2.5 Design expert ........................................................................................... 34

3.2.6 Analysis of yoghurt ................................................................................. 34

3.2.6.1 Sensory evaluation ................................................................. 34

3.2.6.2 Physical analysis .................................................................... 35

3.2.6.3 Chemical analysis .................................................................. 35

3.2.6.4 Microbiological examination ................................................. 36

3.2.6.5 Data analysis .......................................................................... 36

4. Results and discussions ............................................................................................ 37-50

4.1 Chemical composition of milk .............................................................................. 37

4.2 Chemical composition of coconut milk ................................................................ 38

4.3 Sensory evaluation of coconut milk yoghurt ........................................................ 38

4.3.1 Aroma ...................................................................................................... 39

4.3.2 Color ........................................................................................................ 39

4.3.3 Taste......................................................................................................... 41

4.3.4 Texture ...................................................................................................... 42

x
 4.2.5 Overall acceptability ................................................................................ 43

4.4 Chemical analysis of best product ....................................................................... 44

4.5 Physical analysis ................................................................................................... 46

4.6 Shelf-life of the product ........................................................................................ 46

4.6.1 Acidity of yoghurt at room temperature .................................................. 47

4.6.2 Total plate count of yoghurt at room temperature ................................... 47

4.6.3 Acidity of yoghurt under refrigeration .................................................... 48

4.6.4 Total plate count of yoghurt under refrigeration .................................... 49

4.7 Cost evaluation ...................................................................................................... 50

5. Conclusion and recommendations ............................................................................... 51

5.1 Conclusions ........................................................................................................... 51

5.2 Recommendations ................................................................................................. 51

6. Summary ........................................................................................................................ 52

References ................................................................................................................. 53-60

Appendix ................................................................................................................... 61-68

Color plates ................................................................................................................... .69

xi
 List of Tables

Table No. Title Page No.

2.1 Countries where coconut is abundant 24

2.2 Coconut varieties and characteristics 25

2.3 Composition of coconut milk 27

3.1 List of equipments used 30

3.2 List of chemicals used 30

3.3 Sample formulation in coded form 34

4.1 Chemical composition of milk 37

4.2 Chemical composition of coconut milk 38

4.3 Chemical analysis of best product 45

4.4 Syneresis of product 46

xii
 List of Figures

S.N. Title Page No.

3.1 Preparation of coconut milk 31

3.2 Preparation of coconut milk incorporated yoghurt 32

Mean sensory score for aroma of coconut milk incorporated

4.1 39
yoghurt

4.2 Mean sensory score for color of coconut milk incorporated yoghurt 40

4.3 Mean sensory score for taste of coconut milk incorporated yoghurt 41

Mean sensory score for texture of coconut milk incorporated

4.4 42
yoghurt

Mean sensory score for overall acceptability of coconut milk

4.5 43
incorporated yoghurt

Change in acidity under room temperature with respect to the

4.6 47
number of days

Change in TPC under room temperature with respect to the number

4.7 48
of days

Change in acidity under refrigeration with respect to the number of

4.8 49
days

Change in TPC under refregration with respect to the number of

4.9 50
days

xiii
 List of Abbreviations

Abbreviation Full form

ANOVA Analysis of variance

CCP Critical control point

LCFA Long chain fatty acid

LSD Least significant difference

MCT Medium chain triglycerides

MSNF Milk solid not fat

NFDM Non-fat dry milk

SCFA Short chain fatty acids

SMP Skim milk powder

UTH Ultra high temperature

xiv
 Part I

Introduction

1.1 General introduction

The role of fermented milk in human nutrition is well documented and the virtues of these
products were known to man even during the ancient days of civilization. These products
have long been an important component of nutritional diet. A fermented milk product has
been defined by the International Dairy Federation as the milk product prepared from
skimmed milk or not with specific cultures (Gandhi, 2000). Microorganisms employed as
starters for production of cultured dairy foods are divided into two types, based on the
optimum temperature ranges at which they operate. The lactic acid bacteria incubated at
temperatures above 35°C are referred to as thermophilic bacteria and those incubated at
20–30°C are called mesophilic starters. Yogurt is derived by culturing with thermophilic
cultures, which act in symbiosis with each other (Chandan and Kilara, 2013).

Yoghurt is a fermented product obtained through an anaerobic fermentation of lactose

in milk by relevant microorganisms most of which are classified as pro-biotic (Tull, 1996).
Yoghurt is a cultured dairy product produced by fermenting milk, with or without added
non-fat dry milk (NFDM) with Lactobacillus bulgaricus and Streptococcus thermophilus
bacteria. It usually contains 12-14% total milk solids and has soft, friable custard like
consistency, and a clear and distinct acid flavor. Yoghurt is usually produced by heating
the mix to 80-85°C for 30 min to pasteurize it and to modify the milk proteins so that they
will provide the proper viscosity and gelation with a minimum of syneresis in the product
(Morr, 1985).

Coconut (Cocos nucifera L.) is one of the oldest fruits in the world and is confined to
seacoast in the humid tropics. It has been estimated that 25% of the world’s output of
coconut is consumed as coconut milk (Gwee, 1988). Coconut milk is being used by
confectionaries, bakeries, biscuits and ice cream Industries worldwide to enhance flavor
and taste of various products (Persley, 1992). Coconut milk was found to be rich in
calcium. The milk was reported to be high in minerals and vitamin content (Nieuwentus
and Nieuwelink, 2002).
 Contrary to widely held opinion, the coconut provides nutritious sources of meat, Juice,
milk and oil. It is classified as a “functional food” because it provides many health benefits
beyond its nutritional content, due to its fiber and oil content (Sanful, 2009). The oil is
known to contribute to improved insulin secretion and the utilization of blood glucose;
reduce symptoms associated with malabsorption syndrome and cystic fibrosis; help to
relieve symptoms associated with crohn’s disease; ulcerative colitis and stomach ulcers;
improve the utilization of essential fatty acids and protect them from oxidation (Seow and
Gwee, 1997). Current trends and changing consumer needs indicate a great opportunity for
innovations and developments in fermented milks (Khurana and Kanawjia, 2007).There is
little information about fiber fortification in cultured dairy products however various fibers
like psyllium, guar gum, gum acacia, oat fiber, and soy components have potentials to be
used (Staffolo et al., 2004).

Therefore, consuming symbiotic foods that contain prebiotics (fibers) and probiotics
(lactic acid bacteria) would offer added nutritional benefits that can help boost overall
health and well-being. Therefore, the objective of this study is to produce yoghurt enriched
with coconut milk and to determine the physicochemical, microbial, sensory qualities and
overall acceptability of the product.

1.2 Statement of the problem

Yoghurt, as a fermented diary product is regarded as a probiotic carrier, is nutritionally rich

in available protein, calcium, milk fat, potassium, magnesium. It has nutritional benefits
beyond those of milk, because people who are moderately lactose intolerant can enjoy
yoghurt without ill effects, as most of the lactose in the milk precursor has been converted
to lactic acid by the bacterial culture. Yoghurt also has medical uses because of the
probiotic characteristics, in helping out on a variety of gastro intestinal conditions and in
preventing antibiotic associated diarrhea (Lourens-Hattingh and Viljoen, 2001).

The addition of fruits and flavors to yoghurt has become very popular in recent years. In
context of Nepalese market availability of fruits and flavor yoghurt is rare (Gupta, 2003).
Hence, to give a variety of product and also to improve the commercial value of yoghurt,
addition of coconut milk can be beneficial to some extent. Yoghurt obtained by using
coconut milk has been found to be delicious and a nutritional product (Imele and
Atemnkeng, 2001).

2
 Therefore, coconut milk incorporated yoghurt may be a better option to increase its
utilization along with the improvement of yoghurt quality.

1.3 Objectives

1.3.1 General objectives

The general objective is to prepare yoghurt by the addition of coconut milk in different
proportion.

1.3.2 Specific objectives

To fulfill the general objectives the following specific objectives will be done

i. To study the effect of different levels of coconut milk on the prepared yoghurt
and to evaluate its sensory properties.
ii. To analyze the coconut milk yoghurt for its proximate composition.
iii. To study the shelf life of the yoghurt.

1.4 Significance of the study

Yoghurt is the lactic-acid fermented product and has a distinct acidic, sharp flavor.
Yoghurt was first produced to preserve the milk. Yoghurt possesses the long shelf life than
milk. Different yoghurt based products are being available in the world’s market such as
drinking yoghurt, dietetic yoghurt, shrikhand etc. With an increase in population in the
world the consumption of yoghurt is being increased so the production of yoghurt should
also be increased. Considering on the standards of hygiene observed during the
manufacture of yoghurt, and the microbiological quality of the ingredients and packaging
materials, the shelf life of the yoghurt is around three weeks under refrigerated condition
(Tamime and Robinson, 1985).

For many years only natural or plain yogurt was made from whole milk available in the
market, but there has been a good demand for fruit yogurt. The scientific name of coconut
is Cocos nucifera. This coconut tree is called “The Tree of paradise” due to its various
uses. On economic point of view, coconut is very important among various fruits. It is one
of the oldest fruits cultivated by man from prehistoric times of the world. Coconut is a
highly nutrient containing fruit. It is not seasonal in nature like many other fruit crops and
in available in large quantity throughout the year. Our children, youth, pregnant women
3
and poor people are suffering from malnutrition, night blindness, anemia and various kinds
of other diseases. In such cases low cost processed supplementary food based on fruits
need to be developed (Biswas, 2013). This experiment is very important in Nepal because
coconut is available here and price is relatively cheap. Thus incorporating coconut in
yogurt could be useful to develop nutritious fermented product.

1.5 Limitation of the study

The limitations of the work are listed below:

1. Best yoghurt could not be compared with commercial yoghurt.

2. Variation of sugar and skim milk could not be carried out.
3. Shelf life of yoghurt could not be compared to control.

4
 Part II

Literature review

2.1 Historical background

Milk fermentation is one of the oldest methods practiced by the human beings to preserve
milk with an extended shelf life. The exact origination of milk fermentation is not clear;
however, it seems that it is dated back to the dawn of the civilization. It has been reported
that the early civilizations such as the Samarians, Babylonians, Pharoes and Indians were
well advanced in agricultural and animal husbandry practices (Tamime and Robinson,
1999).This can be supported by the findings of Copley et al., 2003 in which the dairy fat
residues were found in pottery fragments from Neolitic Bronze-age and Iron-age
settlements, which suggests that the practice of dairying had existed in Britain
approximately 6500 years ago (Copley et al., 2003). However, it is questionable that the
milk fermentation was practiced during this period. Therefore, the origination of the
fermented milk products including yogurt remains unsolved. It has been reported that the
Anatolian goatherds conserve their milk by thickening as they used to dry it in the sun and
transport in animal stomachs (Anon., 2013a).

The origin of yogurt is dated back to the 6000 B.C. when the Neolitic people in the
Central Asia transformed from a status of a food gatherer to a food producer where they
began the practice of milking their animals. It is generally accepted that the fermented milk
products including yogurt have been discovered accidentally when they used to store milk
in sheep-skin bags and has been evolved over centuries into commercial yogurt making
which paved the pavement for different commercially available varieties with a range of
flavors, forms and textures (Anon., 2013c). With reference to yogurt, it can be suggested
that it has been evolved in Turkey as the term “yoghurt” has been derived from a Turkish
verb, “jugurt” that means “to be curdled or coagulated” (Mahmood and Gilani, 2008). The
earliest writings about yogurt can be found from those of Pliny who lived in the first
century A. D. and wrote about ancient barbarous nations that knew how to thicken the milk
into a substance with an agreeable acidity. According to the literature, the founder of the
Mongol empire, Genghis Khan and his armies was lived on yogurt and spreading of this
news among the people had made the yogurt consumption to spread throughout the East
(Anon., 2013c). Moreover, according to the Persian tradition, Abraham owed his fecundity
and longevity to the regular ingestion of yogurt, and the emperor Francis I of France was
said to be cured of severe diarrhea by consuming yogurt made of goat milk leading to
introduce the health benefits of yogurt into the western world in 1542 (Tamime and
Robinson, 1999).

The origin of fermented dairy product dates back to the dawn of civilization. The
ancient Sanskrit scriptures of India, the Vedas, document the food value of Dahi, a
fermented milk product similar to modern yoghurt. The Bible corroborates further
evidence for the existence of soured milk as a food in early time (Chandan, 1981). The first
industrialized production of yogurt was taken place in 1919, in Barcelona, Spain at a
company named Danone (Anon., 2013c).

Yogurt was firstly introduced to the USA in the early 20th century in the form of tablets
especially designed for those with digestive intolerance. However, it became popular in the
North America when Dannon, a small-scale yogurt factory started manufacture of yogurt in
New York in 1940. Even though, yogurt has been evolved for centuries, it was subjected to
a significant and dynamic evolution process in the 20th Century to originate a vast array of
products. For instance, fruit yogurts, yogurts with fruit on bottom and blended yogurts
were introduced in 1937, 1947 and 1963 respectively (Anon., 2013a).

Yoghurt is one of the oldest known cultured milk products, with its origin in the Middle
East. It would have been made by Nomadic tribesmen, initially from the milk of goats and
sheep (Schmidt, 1992). Although yoghurt has many desirable properties, it is still prone to
deterioration, especially at ambient temperature, within a matter of days and one trend in
Middle East has been the search for simple techniques to extend keeping quality even
further. The first step was the preparation of condensed or concentrated yoghurt by
hanging the yoghurt in animal skins. The product had a total solid in the range of 25% and
acidity of greater than 2% as lactic acid. Nevertheless, even condensed yoghurt becomes
unpalatable within a week or two, and salted yoghurt was prepared which became rapidly
popular. An alternative preservation process involved the heating of yoghurt for a few
hours over low fires of special type of wood; the end product is referred to as “smoked
yoghurt” which was preserved over the winter months. However, as refrigeration become
widespread, so interest in these traditional products declined and production of new
generation of yoghurt emerged. Initially, production was confined to natural yoghurt, but

6
gradually production of fruit yoghurt gave the product an entirely fresh image (Tamime
and Robinson, 1985).

2.2 Development of dairy industry in Nepal

Traditionally dairy production was in a back-yard type enterprise in Nepal. Milk and milk
products are primarily consumed in home. The surplus milk is converted into ghee and sold
in urban areas. This is still followed in most part of the country where market for fresh
milk doesn’t exist. This situation is however different in the vicinity in urban areas where
farmers primarily sell the fresh milk instead of going for ghee production, since it reduces
the profit margin (Dahal, 2009). In Nepal, the history of dairy development is not so long.
At first, it was started in Tushal (Kabhre) at 2009 B.S. In 1956, the modern milk
processing plant was established with the financial assistance of New Zealand government
at Lainchaur, Kathmandu with a capacity of 500liters. For the implementation of effective
dairy development program, DDC (Dairy Development Corporation) was originated in
1969 under the act of Agriculture development, 2001. Similarly in 1974, modern milk
processing plant of 2000 liter capacity was established in the eastern region of Nepal,
Biratnagar. In 1974, 3000 liter capacity was established in Hetauda. Further in 1977
another plant of 5000 liter capacity was established in Balaju, Kathmandu. In addition
Pokhara milk supply scheme, Lumbini milk supply scheme and Kohalpur and Surkhet milk
supply scheme was also established (Dahal, 2009). Kathmandu dairy development scheme
is also called central dairy because milk is supplied in the dairy from all dairies of Nepal.
Beside that, the National Dairy Development Board (NDDB) has been formed as an
additional step in the development of dairy in Nepal. This body is responsible for
formulating policies, planning and development of dairy profession by being a coordinator
between the private and public sectors (Gupta, 2003).

2.3 Milk

Milk is a lacteal secretion of mammary gland of milch animals. It is composed of lipids,

carbohydrates, proteins and other many organic compounds and inorganic salts dissolved
or dispersed in water. Lipid is composed primarily of fat although there are small amount
of phospholipids, sterols, fat soluble vitamins A and D, carotene and xanthophylls. Protein
content of milk is classified as a) casein, b) lactalbumin and c) lactoglobulin. Lactose is the
carbohydrate in the milk (Meyer, 1960).

7
 Different salt and minerals are found in the milk. Plentiful vitamins are present but
vitamin C is limiting. Milk contains a number of enzymes; some of them apparently
secreted in the milk and other are formed by microorganism (Meyer, 1960).

2.4 Milk fermentation

A fermented milk product has been defined by the International Dairy Federation as the
milk product prepared from skimmed milk or not with specific cultures. The microflora is
kept alive until sale to the consumers and may not contain any pathogenic germs (Gandhi,
2000). Milk fermentation can be defined as any modification of the chemical or physical
properties of milk or dairy products resulting from the activity of microorganisms or their
enzymes. It occurs when bacteria break down milk sugars and other components of milk to
give lactic acid, alcohols, carbon dioxide etc. Lactose, fat and citric acid comprise the
important fermentable compounds of milk. Lactose a disaccharide, is the chief source of
carbon while fat and citric acid provides hydrogen and oxygen source respectively (Davies
and Barry, 1984). Fermentation in milk contributes to desirable flavor and texture in
product such as cheese and yoghurt or result in spoiled and degraded products. To ensure
development of desired fermentation, microbial cultures with known properties are added
to milk or dairy product substrate (Yulina, 2010).

Over the period, scientists have tried to isolate and study the characters of such
desirable organisms. Among the bacteria, the most important dominant group bringing
fermentation is lactic acid bacteria. The lactic acid bacteria are naturally accepted as GRAS
(Generally regarded as safe) for human consumption (Aguirre and Collins, 1993). Milk
fermentation process has been relied on the activity of lactic acid bacteria, which play a
crucial role in converting milk as raw material to fermented milk products. In milk
fermentation industry, various industrial strains of LAB are used as starter cultures. Starter
cultures of lactic acid bacteria were obtained from sequence activities and passed a process
of isolation, selection and confirmation. Several behaviors as the characteristics of each
individual selected strains of lactic acid bacteria has been established and used in the
production of fermented milk products industrially. The most important properties of LAB
are their ability to acidify milk (Mäyrä-Mäkinen and Bigret, 2004) and to generate flavour
and texture, by converting milk protein due to their proteolytic activities (Frank and Marth,
1998).

8
 Milk products also serve as the important delivery vehicles for probiotic bacteria. The
probiotic bacteria have a long history of association with dairy products. This is because
some of the same bacteria that are associated with fermented dairy products also make their
homes in different sites on the human body, including the mouth, the gastrointestinal tract
etc. Some of these microbes, therefore, can play a dual role in transforming milk into a
diverse array of fermented dairy products (yoghurt, cheese, kefir, etc.) and contributing to
the important role of colonizing bacteria. Within this sector, probiotic cultures have been
incorporated in yoghurts and fermented milk products. The probiotic bacteria used in
commercial products today are mainly members of the genera Lactobacillus and
Bifidobacterium (Eshraga et al., 2011).

2.5 Types of fermented milk

2.5.1 Traditional fermented milk

Fermented milks were prepared in India in 800 B.C. and in Vedic period. Fermented milks
have been prepared since ancient times in Mongolia, Tibet and in the Middle East. In the
sub arctic regions, the Laplanders also prepared small quantities of fermented milks. The
Yakuts (Russia) also prepared fermented milks called koumys (Tamrakar, 2017).

2.5.2 Non-traditional fermented milk

The non-traditional fermented milk appeared after 1900. They were new products under
the names: biograde, bioghurt, bifighurt, biokys, progurt, yakult, acidophilus milk, bifidum
milk cultured butter milk etc. A certain numbers of products with artificial or enzyme
acidification can be found outside the usual standards of fermented dairy products. A more
important development is the use of human intestinal germs for the preparations of
fermented milk which includes bacteria like Lactobacillus acidophilus and Bifidobacterium
bifidum (Kurmann, 1983)

2.6 Advantages of milk fermentation

The most important advantages of fermented foods are:

 Keeping milk from spoiling by undesirable bacteria which is due to the

accumulation of lactic acid and other antibacterial metabolites during fermentation.

9
  Production of variety in foods that is accomplished by change in body, texture and
flavor.
 Digestibility of fermented product, especially protein, is improved and this may be
important in people with digestive disorders.
 In some instances, the process of fermentation may reduce the bulk, the starting
material and these results in the increased storage life of products. Examples are
traditional fermented milk-cereal mixture which are dried and can be easily
transported from one area to another (Vedamuthu, 1982).
 Fermented milk products contain antibiotics produced by microorganisms used as
culture, which cause adverse effect on the harmful microorganisms present in the
intestine and controls their growth.
 Some fermented milk products are useful for the nutritional treatment of some
diseases like dysentery, gastritis, anemia, kidney stones etc.
 Fermented milk such as yoghurt has ability to increase weight than milk feeding.
(Hargrove and Alford, 1978).

2.7 The fundamental microbiology of yoghurt

The two organisms are mutually beneficial; S. thermophilus by removing oxygen and
producing weak acid conditions favoring L. bulgaricus and the lactobacillus by
hydrolyzing the lactose and the casein. S. thermophilus grow best at pH 6.5, growth
stopping at pH 4.2-4.4 and the L. bulgaricus best at pH 5.5, growth stopping at pH 3.5-3.8
(Rasic and Kurmann, 1978).

The amount of lactic acid production by L. bulgaricus is 1.7-1.8% while S.

thermophilus produces 0.6-0.8%. Lactic acid is very important in yoghurt or yoghurt like
fermented milks (Tamime and Robinson, 1985). The lactic acid contributed to the colloidal
calcium phosphate complex present in the casein micelle to the soluble calcium phosphate
fraction. This leads to gradual loss of calcium from the micelles and the consequent
coagulation of casein at pH 4.6-4.7. The lactic produced by the thermophilic cultures gives
yoghurt its sharp and acidic taste thus enhancing the flavour of the product. As reported by
many workers, S. thermophilus produces essentially L (+) lactic acid while L. bulgaricus
produces mainly D (-) lactic acid. As a consequence the yoghurt usually contains 45-60%
L (+) lactic acid and 40-55% D (-) lactic acid (Garvie, 1978).

10
 Yoghurt starter cultures are slightly proteolytic and the peptides and amino acids
produced act as precursors for the enzymic and chemical reaction, which produce flavor
compounds. Protein degradation is associated mainly with L. bulgaricus but peptidase
enzymes are produced by S. thermophilus and L. bulgaricus (Heller, 2001).

2.8 Basic stages and CCPs of yoghurt manufacturing

The basic stages of production of yoghurt are:

 Preparation of standard milk with around 12-14% milk solids not fat (MSNF).

 Heating this milk to 85-95°C, preferably in a unit that allows the temperature to be
held for some 10-30 min.

 Inoculating the milk with a culture mixture of containing Lactobacillus bulgaricus

and Streptococcus thermophilus as principle organisms.

 Incubating the inoculated milk at 42°C until a smooth coagulum has formed,
together with the desired level of acidity and flavor.

 Cooling the finished product and, unless the milk has been incubated in retail
cartons (set yoghurt), mixing with fruit or other ingredients (Tamime and Robinson,
1985).

The following critical control points (CCPs) must be followed to produce yoghurt
efficiently:

 Milk for yoghurt manufacturing must be free from penicillin, and is checked for
penicillin on receipt at the site.

 Milk must be heated to 90-95° C for 15 min before inoculation. It serves the
Following functions:

o It destroys pathogens, which is necessary to produce lactic acid at optimum

rate.

o It generates the condition for growth for starter by breakdown of milk

protein.

11
 o It creates desirable body and texture.

 If the mix is homogenized, it prevents creaming during incubation and storage of

cultured dairy products by changing colloidal characteristic of milk. The stabilizer
and other component of mix are thoroughly dispersed for optimum textural effect.

 The mix must be cooled to 43°C before starter inoculation.

 The yoghurt starter culture must have the L. bulgaricus and S. thermophilus in
correct proportion (1:1). It must be free from contaminating microorganisms and
initiate acid production within thirty minutes of inoculation.

 The inoculation must be done at correct temperature for rapid acid production. The
rate of acid production is monitored during 3.5-4 h of incubation (Chandan, 1981) .

2.9 Starter culture

A starter culture is a product with a high concentration of lactic acid bacteria, which can
activate an acidification process in milk. Starter cultures are normally manufactured in
special starter culture laboratories but may also be cultured and propagated in dairy
(Gandhi, 2006).

The starter culture for most yoghurt production is a symbiotic blend of Streptococcus
salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Although
they can grow independently, the rate of acid production is much higher when used
together than either of the two organisms grown individually. S. thermophilus grows faster
and produces both acid and carbon dioxide. The formate and carbon dioxide produced
stimulates L. bulgaricus growth. On the other hand, the proteolytic activity of L. bulgaricus
produces stimulatory peptides and amino acids for use by S. thermophilus (Desai et al.,
1994). These microorganisms are ultimately responsible for the formation of typical
yoghurt flavor and texture. The yoghurt mixture coagulates during fermentation due to the
drop in pH. The streptococci are responsible for the initial pH drop of the yoghurt mix to
approximately 5.0. The lactobacilli are responsible for a further decrease to pH 4.0. Lactic
acid, acetaldehyde, acetic acid and diacetyl are the fermentation products that contribute to
flavor. Modern industrial processes utilize “starters” in the manufacture of modern dairy
products. A starter consists of harmless microorganism, which on culturing in milk imparts

12
desirable and predictable characteristics of flavor and texture. A single strain culture
contains an individual strain of bacterial species while a mixed/multi strain culture consists
of the mixture of more than one strain or species. Starter cultures are distributed from
central laboratory to an operating plant in different forms e.g. liquid culture, frozen
concentrate, lyophilized culture etc (Chandan, 1981).

2.10 Types of starter culture

2.10.1 Liquid and freeze-dried culture

The liquid cultures are generally no longer distributed in commercial practice. To prepare a
liquid culture the organisms are propagated in a suitable medium such as milk or whey and
maintained an active condition by periodic transfers. In general, a liquid culture contains
about 109 organisms per ml of starter. Freeze-dried cultures are produced by freeze-drying
the cultures grown in milk and are preserved by lyophilization in small vials. It can be
stored at room temperature for several years but the degree of viability of organism is very
low. So reactivation of lyophilized culture is necessary for proper performance (Chandan,
1981).

2.10.2 Pure and mixed culture

A further sub-division is made into either pure cultures or mixed cultures. Pure culture
consists of only one species of lactic acid bacteria, whereas mixed cultures consist of
several species of lactic acid bacteria. Pure cultures may consist of one or more strains of
the same species. Mixed cultures are the most common type in acidification with a mixed
culture and on rare occasions on its own. DL cultures used to be cultivated as “dairy
cultures” at individual dairies, often the same culture for decades (Kurmann, 1983).

2.10.3 Mesophilic and thermophilic culture

Mesophilic cultures have optimum temperature for growth between 20 to 30°C and include
Lactococcus and Leuconostoc. These mesophillic lactic cultures are used in the production
of many cheese varieties where important characteristics are:

1. Acid producing activity

2. Gas production, and

13
3. Production of enzymatic activity for cheese ripening, e.g., proteases and peptidases
enzymes.

Thermophilic cultures have optimum temperature for growth between 37 to 45°C.

Thermophilic cultures are generally employed in the production of yoghurt, acidophilus
milk, swiss type cheese. Thermophilic cultures include species of Streptococcus and
Lactobacillus. These cultures grow in association with milk and form the typical yoghurt
starter culture. This growth is considered symbiotic because the rate of acid development is
greater when two bacteria are grown together as compared to single strains (Gandhi, 2006)

2.10.4 Physical forms of starter culture

Starter cultures can be manufactured and distributed in three different physical forms:
liquid, powder and frozen form (Neilson and Ullum, 1989).

2.10.5 Liquid cultures

The liquid cultures are generally no longer distributed in commercial practice. To prepare a
liquid culture the organisms are propagated in a suitable medium such as milk or whey and
maintained in an active condition by periodic transfers. In general, a liquid culture contains
about 109 organisms per ml of starter (Chandan, 1981). The milk used has high total solids
(TS) content. Various growth promoting substances may be added (Neilson and Ullum,
1989).

2.10.6 Powdered cultures

Powdered cultures are manufactured by freeze-drying a liquid culture cultivated to a

maximum bacterial count. Freeze drying means drying under vacuum. This is a gentle
method which minimizes the reduction in the bacterial count during manufacture. Ordinary
freeze-dried cultures must be re-inoculated into a mother culture before use (Neilson and
Ullum, 1989).

2.10.7 Frozen culture

Deep frozen cultures are prepared by deep freezing a concentrated, liquid culture at the
point of the bacteria growth at which the activity is at its highest. They are preserved by
lyophilization in small vials. Super-concentrated, deep frozen cultures are made by adding
growth-promoting substances to a milk substrate, continuously neutralizing the lactic acid
14
formed by means of ammonium hydroxide, and finally concentrating the culture in a
desludging centrifuge/ bactofuge. The concentrate is pelletized by being frozen as
individual drops in liquid nitrogen. The culture is stored at -196°C until it is dispatched to
the dairies in foamed plastic boxes containing dry ice (Neilson and Ullum, 1989).The
culture concentrate contains approximate 1011 microorganisms per ml (Chandan, 1981) .

2.11 Selection of cultures

The best method is to adopt the procedure used by cheese makers, namely to collect
cultures from various sources and access their suitability for particular purpose. Yoghurts
are judged on flavour, acidity, body, texture and homogeneity (Kurmann, 1983). It does
not follow that the quickest growing are the best for yoghurt. Too rapid growth may lead to
instability of coagulum and graininess. A slower culture is easier to control for final acidity
and may produce a better flavour (Vanderpoorten and Waes, 1972).

2.12 Preparation of starter culture

Culturing the two organisms together results in a symbiotic relationship since the growth
rate and acid production by each organism are greater than in single culture. Optimum
growth temperature for rod and coccus are 45°C and 40°C respectively. A ratio of 1:1 is
generally accepted as ideal. Using 2% inoculum and incubation at 44°C for 2.5 h produces
good yoghurt. S. thermophilus attains acidities of 0.85-0.95%, whereas L. bulgaricus
attains acidities of 1.20-1.50% (Neilson and Ullum, 1989).

2.13 Bio-technically important metabolic activities

2.13.1 Acid production by microorganisms

The main role of S. thermophilus and L. bulgaricus in yogurt manufacture is to acidify

milk by producing a large amount of lactic acid from lactose. Lactic acid reduces the pH of
the milk and leads to a progressive solubilization of micellar calcium phosphate. This
causes the demineralization of casein micelles and their destabilization, which generates
the complete precipitation of casein in a pH range of 4.6-4.7 (Fox, 1989). Amount of lactic
acid production by L. bulgaricus is 1.7-1.8% while S. thermophilus is 0.6-0.8%. Acid
contributes to the formation of gel by conversion of colloidal calcium phosphate complex
present in the casein micelle to soluble calcium phosphate fraction. This leads to loss of

15
calcium from micelles and coagulation of casein at pH 4.6-4.7. S. thermophilus produces L
(+) lactic acid while L. bulgaricus produces mainly D (-) lactic acid (Garvie, 1978).

2.13.2 Flavor components in yoghurt

The typical flavour of yogurt is due to lactic acid and various carbonyl compounds, ie
acetaldehyde, acetone and diacetyl, produced by S .thermophilus and L. bulgarius. ln
addition to carbonyl substances, many volatile compounds have also been identified in
yogurt, ie volatile fatty acids (Turcic et al., 1969) and several compounds derived from the
thermal degradation of lipids, lactose and proteins during the heat treatment of milk before
yogurt manufacture eg aldehydes, ketones, alcohols, lactones, sulfur cornpounds (Tamime
and Deeth, 1980) .

Both organisms convert nearly all the sugar to lactic acid, producing only trace amounts
of byproducts. These are very important for the characteristic yoghurt flavour, S.
thermophilus by producing diacetyl and L. bulgaricus by producing acetaldehyde (Schulz
and Hingst, 1954).

2.13.3 Ropiness and consistency

Several Gram-negative and Gram-positive bacteria, including lactic acid bacteria, produce
exocellular polysaccharides (Macura and Townsley, 1984).Mucous substances are
produced by some strains of S. thermophilus and L. bulgaricus which can have different
chemical composition. The thickening ability of the polysaccharides produced by both
organisms is different (Galesloot et al., 1968). The quantities of polymer formed by ropy
strains of both species vary considerably even under identical experimental conditions
(Cerning et al., 1990). It is difficult to establish a good correlation between the quantity of
polysaccharide produced and the corresponding viscosity. This difficulty may be due to
changes in the 3-dimensional configuration of polymers and to their interactions with some
milk compounds, mainly caseins that are precipitated at low pH (Olsen, 1989).

2.13.4 Proteolytic activity

ProteoIytic activity is greatly involved in both nutrition and interactions of yogurt bacteria,
since lactic acid bacteria cannot synthesize essential amino acids. Therefore, they require
an exogenous nitrogen source and utilize peptides and proteins in their growth medium by
more or less complete enzyme systems. S. thermophilus primarily requires glutamic acid,
16
histidine and methionine, as weil as cystine, valine, leucine, isoleucine, tryptophan,
arginine and tyrosine for growth (Shankar and Davies, 1977). The uptake of branchedchain
amine acids has been studied. lt is an active transport which requires an exogenous energy
source, depends on temperature and pH and is inhibited by L-cysteine (Akpemado and
Bracquart, 1983).

Whey protein hydrolysis is lower when the amount of cocci is higher than rods. Free
fatty acid can reduce the proteolytic activity and improve the texture of coagulum. High
proteolytic activity is observed during manufacture of lactose hydrolyzed yoghurt (Grous,
1972).

2.13.5 Lipolytic activity

Lipolysis is generally low in yogurt and is therefore not significant in terms of flavor. The
free fatty acid content of yogurt differs only slightly from that of milk (Rasic and
Kurmann, 1978).Thermophilic starter culture may contain lipolytic enzymes. During the
manufacture and storage of yoghurt, hydrolysis of fat usually occurs and the lactic acid
bacteria hydrolyze mainly long chain triglycerides (Singh et al., 1980). In general the
lipolytic activity of thermophilic starter is weak and most of the volatile acids formed in
yoghurt are derived from the hydrolysis of other compounds (Shahini and Reddy, 1979).

2.13.6 Other metabolic activities

Yoghurt starter bacteria utilize or synthesize vitamins during their growth. It has been
observed that during yoghurt production niacin, folic acid, biotin, vitamins B6 and B12 are
synthesized. It has also been observed that biotin, niacin, folic acid, vitamin B12 is
decreased during cold storage of the product or by microbial catabolism (Cerna and
Pickova, 1973). Organic acids like fumaric, succinic and benzoic show increased
concentrations in yoghurt compared to milk while hipuric acid, orotic acid are reduced by
the metabolic activity of the microorganisms (Deeth and Tamime, 1981).

2.14 Role of thermophilic cultures in the intestine

It has been suggested that S. thermophilus and L. bulgaricus can establish themselves in
the intestine and generally dominate the natural micro flora. L. bulgaricus is able to resist
the high degree of acidity so it is feasible that a percentage of bacteria ingested will reach
the intestine in viable state (Lembke, 1963).
17
 Certain strains of L. bulgaricus could survive and compete in the human intestine and
ensures the absence of putrefactive organisms and hence protect the health of the intestine
of the consumer (Acott and Labuza, 1972).

2.14.1 Production of antibiotic substances

There is generally a symbiotic relationship between yogurt bacteria, but growth inhibition
is sometimes observed (Moon and Reinbold, 1974). This should be taken into account
when selecting starters. Inhibition may be due to competition for one or more nutrients of
the growth medium (Moon and Reinbold, 1976) or to inhibitory compounds produced by
the strains, such as bacteriocins and inhibitory peptides (Pereira and Luchese, 1988).

2.14.2 Effect on immunoglobulin level, cholesterol, lactose activity and tumor cells

It has been shown that the composition of yoghurt could lead to increase level of
immunoglobulin. It has also been shown that the composition of yoghurt could lead to
decrease level of cholesterol in the blood (Mann, 1977).

In people who exhibit lactose intolerance upon ingesting milk, the milk can be replaced
by yoghurt without any allergic reactions. L. bulgaricus has been shown to have a greater
inhibitory effect on tumor cells (Mann, 1977).

2.14.3 Formation of bacteriocins and other inhibitory compounds

The study of bacteriocin production has been largely confirmed to L .acidophilus. L.

bulgaricus produces a bacteriocin called “bulgarican” and many other inhibitory
compounds (Reddy and Shahini, 1971).

2.15 Causes of slow growth

As with cheese starter, partial or complete failure may occur with yoghurt. However,
bacteriophages are generally the main causes of failure with starters, antibiotics and
preservatives or sterilants are the main causes for failure of yoghurt. S. thermophilus and L.
bulgaricus are the most sensitive of commonly used lactic cultures to penicillin (Tramer,
1973).

18
2.15.1 Bacteriophages

Knowledge of the specifie bacteriophages (phages) of thermophilic lactic acid bacteria has
been well documented, primarily on and after 1980. Specified phages may attack S
thermophilus and L bulgaricus strains during yogurt manufacture and seriously affect
product quality. Moreover, even if phage attacks do not delay acidification during yogurt
manufacture, they can lead to an important decrease in the streptococci and to a lower
flavor score of the resulting yogurt (Stadhouders et al., 1988).

In order to exert better control against attack of phages it is important to know that the
heat treatment of milk at 85ºC for 20 min. will ensure the destruction of phage particles.
Chemicals such as 0.1% quaternary ammonium, 70 to 90% ethanol, 0.5 to 1% potassium
permanganate or 50 to 100 ppm of available chlorine destroy S. thermophilus phages
(Tamime and Robinson, 1985).The phage problem will never be completely solved;
however the frequency of such infection could be reduced (Klaenhammet, 1984).

2.15.2 Effect of chemical composition of the lactic acid flora

Chemical composition can exert an important influence mainly on account of sucrose

which may vary from 0-12%. This will not affect the growth of lactic acid flora in fruit
yoghurts because the sugar is added after the souring process. Sucrose concentration
encourages those types which can ferment sucrose. L. bulgaricus is severely inhibited at
24% total solid and over (Carr et al., 1975).

2.16 Coagulum formation in yoghurt

The formation of yoghurt gel is the result of the following biological and physical action of
milk. Starter in yoghurt utilizes lactose for its energy and produces lactic acid and other
relevant compounds become inevitable. Gradual development of lactic acid destabilizes the
calcium caseinate phosphate complex. Aggregates of casein micelles and/or the individual
micelles group together and partially coalesce as the pH approaches the isoelectric point
(pH 4.6 to 4.7). It is most likely that α-lactalbumin and β-lactoglobulin interaction with the
κ-casein (linked by –SH and –SS bridges) partially protects the micelles against complete
destabilization or disruption. As a result the gel network or matrix consists of a regular
structure, which entraps within all the outer constituents of the basic mix including the
water phase (Tamime and Robinson, 1985).

19
2.17 Syneresis

Syneresis is the oozing out of water on the surface of yoghurt. Syneresis is caused by low
SNF or fat content. Insufficient heat treatment and homogenization of milk, too high
incubation temperature causes the syneresis problem. These types of problem can solved
by standardization of SNF and fat content along with reduction in incubation temperature
to 42°C and addition of stabilizers (Rayamajhi, 2011).

2.18 Methods for improving the body (viscosity) of yoghurt

Traditional yoghurt was made by heating milk in open pans, concentrating it in this way to
two-third volume. The higher solids content would also give thicker or more viscous
yoghurt. Sheep milk, if used, would also give thicker yoghurt because it is about 50%
richer in solids than ordinary cow milk (Tamime and Robinson, 1985).

If yoghurt is made from non-concentrated or unfortified cow milk an attractive gel is

obtained, but this is delicate and easily broken by vibration. SMP at 4-5% level is
incorporated to overcome this difficulty. The easiest and cheapest way is to incorporate a
carbohydrate gum such as carrageenan, alginate, agar, etc. at a level of about 0.3%. This is
harmless but does not add to the nutritive value, the milk fat is normally homogenized
(Tamime and Robinson, 1985).

Ropy strains of both S. thermophilus and L. bulgaricus can be used at same temperature
(43ºC) for ordinary yoghurt. However, the lower the temperature and the longer the time of
incubation, the higher will be the viscosity. Thus temperature of 30-32ºC with an
incubation time of 12-15 h may be used (Carr et al., 1975).

2.19 Types of yoghurt

2.19.1 Set type yoghurt

Processed milk is fed directly into the intermediate tank inoculated with starter culture
and/or flavor before it is packed in filling machine. The yoghurt cups are filled and
transferred to the incubation chamber at 42°C. After 3 hours, the cups are cooled to 15-
20°C by means of cold air in the chamber or in the cooling tunnel (Pant, 1992).

20
2.19.2 Stirred type yoghurt

It is soured in tank after which the product is stirred, cooled and packed. Stirred yoghurt
has distinct consistency, thick and smooth, and should make good eating, rather than
drinking (Gallesloot and Hassing, 1973). From 0.5-0.7% stabilizer is added in order to
impart gel structure, to ensure a smooth body and texture and to prevent wheying off or
syneresis after packaging (Chandan, 1981). The stirred type may be plain, fruit and
flavored and this form of yoghurt is more popular (Tamime and Robinson, 1985).

2.19.3 Drinking type of yoghurt

The storage of product and handling of the coagulum are similar to stirred yoghurt but fruit
syrup is used and the coagulum is homogenized after fermentation. Three different types
can be produced, firstly the coagulum is set, heated and the product has shelf-life of three
weeks at 10°C, secondly pasteurization of yoghurt at 75°C for a few seconds, followed by
cooling and packing gives the product a few weeks shelf-life at 10°C and thirdly UHT
drinking yoghurt is heated at 110°C for 4 s, cooled and filled into sterilized container under
aseptic conditions. The latter type has shelf-life of several months at ambient temperature
(Pant, 1992).

2.19.4 Frozen yoghurt

The yoghurt base is produced in the conventional way. The milk should be subjected to
UHT treatment, before fermentation with starter culture and produced natural stirred
yoghurt, then 65-80% yoghurt base, 20-35% fruit syrup base are mixed and 0.85%
stabilizer and emulsifier are added. The product is then frozen in an ordinary ice cream
freezer (outlet temperature -6°C). Finally, the yoghurt is packed at 0 to -6°C and
dispatched (Tamime and Robinson, 1985).

2.19.5 Dried yogurt

Yogurt powder is produced by fermenting non-fat milk using standard yogurt cultures until
attain the desirable pH followed by a step of drying, most probably by freeze-drying. In
addition, blended yogurt powder is manufactured by blending cultured non-fat milk,
cultured whey, cultured whey protein concentrate, cultured dairy solids, nonfat dry milk
and lactic acid which are similar to the flavor and functionality to that of the traditional
yogurt powder (Childs and Drake, 2008).
21
The main intension of manufacturing yogurt powder is to store the product in a stable and
readily utilizable state, and it can be utilized to replace fresh yogurt for beverage and dip,
and in confectionary industry as a coating material for coating of dried fruit, nuts, pretzels,
cereal and other snack items (Krasaekoopt and Bhatia, 2012).

2.19.6 Dietetic/therapeutic yoghurt

The fact that most strains of S. thermophilus and L. bulgaricus do not survive in the
intestinal tract, may be limiting factor if yoghurt is used for antibiotic therapy and/or any
other medical purpose. However, the inclusion Lactobacillus acidophilus and
Bifidobacterium bifidum as yoghurt starter culture may contravene some existing
definitions of yoghurt; the resultant milk product is reported to be of excellent therapeutic
value. E.g. lactose-hydrolyzed yoghurt is beneficial for lactose intolerant patient (Tamime
and Robinson, 1985).

Apart from these types other varieties of yoghurt are also manufactured. Some
important ones are concentrated yoghurt, carbonated yoghurt, yoghurt beverage, soy
yoghurt etc.

2.20 Addition of fruit flavoring in yoghurt

In modern technology of yoghurt manufacturing, addition of fruit and flavoring is well

popular. In the stirred type, these may be added after fermentation but in the set type, the
addition must be done before fermentation. The fruit preparations are especially designed
to meet the marketing requirements for different types of yoghurt. They are generally
present at the level of 15-20% in the final product (Chandan, 1981). Yogurts are available
in a vast array of flavors including fruit (apple, apricot, black cherry, black currant, blue
berry, lemon, mandarin, raspberry, strawberry, peach), cereal, vegetables, chocolate,
vanilla, caramel, ginger, etc (Anon., 2013b). In general, flavors are added to yogurt during
production stage and theaddition of flavors not only results a wide array of tastes, but also
increases sweetness of the product (Anon., 2013d)

2.21 Shelf-life of yoghurt

Shelf-life of a product may be defined as the number of days after production that can be
consumed while still remaining safe, retaining its quality appeal and meeting consumer

22
expectations. In other words, it should remain microbiologically safe and organoleptically
acceptable within its stated shelf-life (Ahmed, 2011).

The majority of short shelf-life yoghurts are “lives” i.e. the culture organisms are still
viable. Although their metabolic rate at 7°C is relatively low, there is nonetheless some
activity. This can be determined during shelf-life by pH measurement, by titrable acidity
determination, and by taste (Akpan et al., 2007).

Depending on the standards of hygiene observed during the manufacture of yoghurt,

and the microbiological quality of the ingredients and packaging materials, the shelf-life of
yoghurt is around 3 weeks under refrigerated condition (Goodluck et al., 2014).

At chill temperature of about 5°C yoghurt has a shelf-life of approximately 10 days,

after which the bacterial growth, although restricted, will increase the level of acidity to
such an extent as to impair the flavor, eventually rendering it unpalatable to most people.
Ultimately the bacteria are destroyed and the yoghurt becomes separated into curds and
whey. Yoghurt is particularly susceptible to attack by yeasts and molds; great care is
needed to ensure that the starter is free from these organisms and they do not gain access
during packaging (Tamime and Deeth, 1980).

2.22 Techniques of shelf-life extension

Shelf life can be prolonged by several methods:

 Stopping incubation when the pH has reached 4.6-4.8, rapid cooling and storage at
5°C.
 Aseptic operation on an enclosed production line, including aseptic addition of
sterile additives and aseptic packaging.
 Pasteurization of stirred cultured milk products with or without additives in a
continuous flow cooler, aseptic cooling, filling and sealing.
 Continuous flow heating, filling while hot, sealing the package, and cooling after a
sufficiently long pasteurization time.
 Filling while cold, sealing the package, pasteurization in the package by heating,
followed by cooling (Kessler, 2002).

23
2.23 Coconut

2.23.1 Description and origin

The coconut (Cocos nucifera L.) is a member of the family Arecaceae (palm family). It is
the only accepted species in the genus Cocos, and is a large palm, growing up to 30 m tall,
with pinnate leaves 4–6 m long and pinnae 60–90 cm long which old leaves break away
cleanly, leaving the trunk smooth. The term coconut can refer to the entire coconut palm,
the seed, or the fruit, which is not a botanical nut (Afodunrinbi and Onyeukwu, 2000).
The coconut palm is found throughout the tropics, where it is interwoven into the lives of
the local people. It is particularly important in the low islands of the Pacific where, in the
absence of land-based natural resources, it provides almost all the necessities of life-food,
drink, oil, medicine, fiber, timber, thatch, mats, fuel, and domestic utensils. For good
reason, it has been called the “tree of heaven” and “tree of life.” Today it remains an
important economic and subsistence crop in many small Pacific island states (Banzon,
1990).

The coconut's name is a bit of a misnomer, since it is botanically classified as a drupe

and not a nut. It is the largest seed known (Banzon, 1990).

Countries where coconut is abundant shown in the Table 2.1

Table 2.1 Countries where coconut is abundant

Region Countries
Burma, Indonesia, Malaysia, Philippines, Singapore,
Southeast Asia
Thailand, Vietnam.

Indian Subcontinent Bangladesh, South India, Sri Lanka, Nicobar, Seychelles.

Cameroon, Ghana, Ivory Coast, Kenya, Mozambique,

Africa
Nigeria, Tanzania
Brazil, Ecuador, Jamaica, Mexico, Trinidad and Tobago,
Central America
Venezuela.

Melanesia Fiji, Papua New Guinea, Solomon Islands, Vanuatu.

Source: Banzon (1990)

24
2.23.2 Varieties

Varieties and characteristics of coconut shown in the Table 2.2

Table 2.2 Coconut varieties and characteristics

Variety Characteristics

Thick stem with swollen base (bole). Late

flowering (5–6 yr from out planting). Little
Tall or no overlapping of male and female
phases of an inflorescence encouraging out-
crossing.
Slender stem with short internodes. Bole
slight or absent. Early flowering (3 yr from
out planting). Considerable overlapping of
Dwarf
male and female phases of an inflorescence
resulting in self-pollination

Source: Romney (1997)

2.23.3 Coconut fat

All fats and oils are composed of molecules called fatty acids. There are two methods of
classifying fatty acids. The first is based on saturation; there are saturated fats,
monounsaturated fats, and polyunsaturated fats. The other system of classification is based
on molecular size or length of the carbon chain within each fatty acid. In this system there
are short-chain fatty acids (SCFA), medium-chain fatty acids (MCFA), and long-chain
fatty acids (LCFA) (Thompson et al., 1961).

Coconut oil is composed predominately of medium-chain fatty acids (MCFA), also

known as medium-chain triglycerides (MCT). The size of the fatty acid is important
because the human body responds to and metabolizes each fatty acid differently depending
on its size. So the physiological effects of MCFA in coconut oil are distinctly different
from those of LCFA more commonly found in our foods. The saturated fatty acids in
coconut oil are predominately medium-chain fatty acids. Both the saturated and
unsaturated fat found in meat, milk, eggs, and plants (including almost all vegetable oils)

25
are composed of LCFA (Pamplona-Roger, 2007). MCFA are very different from LCFA.
They do not have a negative effect on cholesterol and help to protect against heart disease.
MCFA help to lower the risk of both atherosclerosis and heart disease. It is primarily due
to the MCFA in coconut oil that makes it so special and so beneficial. There are only a
very few good dietary sources of MCFA. The best sources of MCFA are coconut and palm
kernel oils (Pamplona-Roger, 2007) .

2.23.4 Coconut meat

Coconut meat is the edible white meat of a coconut; often shredded for use in cakes and
curries. It contains essential mineral salts particularly magnesium, calcium and phosphorus
which are of great importance to the musculoskeletal system. Though present in small
amounts (32 mg/100 g of magnesium) in coconut meat, the Magnesium content surpasses
that of all animal-based foods including meat, fish, milk and eggs (Pamplona-Roger,
2007).

2.23.5 Coconut milk

Coconut milk should not be confused with coconut water, although some studies have used
the two terms interchangeably. The aqueous part of the coconut endosperm is termed
coconut water, whereas coconut milk, also known as “santan” in Malaysia and Indonesia,
and “gata” in the Philippines, refers to the liquid products obtained by grating the solid
endosperm, with or without addition of water. Coconut milk is usually used as a food
ingredient in various traditional cooking recipes, while coconut water is served directly as
a beverage to quench thirst (Banzon, 1990).

Coconut milk is the term used to designate the liquid obtained by the manual or
mechanical extraction of grated coconut meat with or without added water. The term
coconut milk and coconut cream are used interchangeably. But coconut milk refers to the
milky fluid, freshly extracted from the coconut kernel with or without added water, and
coconut cream refer to the high fat cream like material obtained from the coconut milk by
either gravity separation or centrifugation (Banzon, 1990).

Maturity of the coconut greatly affects the yield of coconut milk. Mature brown husked
coconuts with no protruding sprouts produce higher yields of milk. Coconut milk is
generally produced from mature nuts of 12 months in age. At this stage, the meat is hard

26
and thick, with a typical composition of as follows: 50% moisture, 34% oil, 3.5% protein,
3% fiber, 2.2% ash and 7.3% carbohydrates (Banzon, 1990).

Composition of coconut milk is shown in the Table 2.3

Table 2.3 Composition of coconut milk

Components Quantity (%)

Moisture 72.88
Ash 1.7
Protein 2.02
Fat 5
Acidity (as %citric acid) 0.13
Carbohydrate 2.81

Source: USDA (2012)

2.23.6 Nutritional and medicinal importance of coconut milk

Some of the most important benefits of coconut milk are:

 A major part of the fats found in coconut milk is lauric acid, which has been found
to exhibit antibacterial, antifungal and antiviral properties. This fatty acid can boost
the immune system and its disease fighting ability.
 Lauric acid can also be helpful in maintaining the elasticity of the blood vessels and
in keeping them clean, which can lower the risk for conditions like, atherosclerosis
and heart disease.
 Coconut milk also contains several antioxidant compounds, which can provide
protection against the harmful free radicals and their damaging effects on the body
cells and tissues.
 Coconut milk can improve the health of the digestive system and promote
digestion. It can relieve the symptoms of stomach ulcers and acid reflux disease as
well.
 Coconut milk can give about 22% of the recommended daily allowance of iron.
With such a high level of iron, it can help to treat anemia caused by iron deficiency.

27
 Coconut milk health benefits are mentioned in Traditional Medicine for the human
body. It is also used for the treatment of mouth ulcers.
 Coconut is a dairy free alternative to those who are lactose intolerant and are also
allergic to animal milk. This milk is also nut free, soy free and gluten free.
 It is known to relieve the symptoms of sore throat.
 It is good for the health of your skin and hair. Many cosmetic giants use it as a base
in products for skin and hair.
 Apply coconut milk to the scalp to have dandruff free hair and condition your hair
naturally.
 Coconut milk is a reservoir of antioxidants. Antioxidants help the body fight aging,
low vision and low bone density.
 It also aids in digestion and is also used as a laxative. It can also be a remedy for
urinary and kidney problems.
 Coconut milk is an excellent source of Vitamin E. It helps in the nourishment of the
skin.
 The saturated fat content in coconut is made up of short and medium chain fatty
acids. These fatty acids are quickly converted in to energy instead of storing as fat
in the body.
 The medium chain fatty acids present in coconut milk are full of lauric acid. Lauric
acid is anti fungal, anti viral and anti microbial. Lauric acid present incoconut milk
helps to keep the arteries of the heart clean and healthy (Banzon, 1990).

28
 Part III

Material and methods

3.1 Materials

The materials collected for the preparation of coconut milk incorporated yoghurt were as
follows:

3.1.1 Milk

The standardized (3% fat and 8% SNF) and pasteurized milk was collected from Bharaha
department store of Dharan produced by Dairy Development Corporation (DDC).

3.1.2 Coconut

Coconut was collected from local market of Amarhatt Dharan.

3.1.3 Milk solid not fat

Skim milk powder was used as the source of MSNF and it was bought from the Bharaha
department store of Dharan. Manufactured by Singhania Industries, Shreepur, Birgunj-16.

3.1.4 Sweetener

Sugar was used as a sweetener. It was bought from the Bharaha department store of
Dharan.

3.1.5 Starter Culture

Starter culture a liquid culture containing L. bulgaricus and S. thermophilus in correct

proportion (1:1) was collected from the Kamdhanu Dairy Tharahara.

3.1.6 Containers

Plastic cup as ice cream packaging materials were bought from Bharaha department store
of Dharan. The size of cup was 100 ml and plain in design.
3.1.7 Equipment and chemicals

Table 3.1 List of equipment used

Physical apparatus Physical apparatus

Heating arrangement Refrigerator
Electric balance (Phoenix) Daily routine glassware
Gerber centrifuge Muffle furnace (Accuma, India)
Thermometer Hot air oven (Vitco, India)
Refractrometer (Hand refractometer model
Stainless steel vessels
WYT- 32, Zhongyou Optical Instruments).
Titration apparatus Incubator (Vitco, India)
Desiccators
Gerber butyrometer
Kjeldahal digestion and distillation set

Table 3.2 List of chemicals used

Chemicals
40 % Formaldehyde
Culture medium (plate count agar)
Starter culture
Saturated potassium oxalate
0.0005% Fuchsin solution

3.2 Method

3.2.1 Preparation of coconut milk

The coconut was de-husked. The de-husked nut was cracked open into halves. The split
nuts were de-shelled to separate the coconut meat (kernel). Coconut meat was washed and
comminuted using an electric blender with water. This was then pressed through a muslin
cloth and strained to obtain coconut milk.

30
Flow chart for the preparation of coconut milk is shown in Fig. 3.1

De-husked whole coconut Drilling Coconut water

De-watered, de-husked whole coconut

De-shelling, grinding Splitting

Comminuted coconut meat

Milk extract

Coconut milk Coconut residue

Fig. 3.1 Preparation of coconut milk

Source: (Tamrakar, 2017)

3.2.2 Preparation of set type coconut milk yoghurt

The standardized and pasteurized milk from DDC (Dairy Development Corporation) was
taken for the preparation of yoghurt. The milk was mixed with 4% SMP (Skim milk
powder) and 3% sugar at 45°C. Heating of milk was further continued till the temperature
reached to around 65-70°C for certain period. After that the heated milk was cooled to
around 43-44°C. After cooling, five formulations of the samples were made by adding 10,
20, 30, 40, and 50% of coconut milk per 100 ml yoghurt mix. Then the starter culture is
added at the rate of 2% to each formulations. Then the mix is placed in plastic cups. The
yoghurt mix was then kept in an incubator which was maintained at a temperature of about
43°C and was kept for 3.5-4 h until the coagulum is formed. Now the prepared yoghurt
was immediately cooled to 5-7°C and stored at that temperature in a refrigerator.

31
Flow chart for the preparation of coconut milk incorporated is shown in Fig. 3.2

Standardized milk (3%fat, 8% SNF)

Pre-heating to 45°C

Addition of 4%SMP and 3% sugar

Heating to 65-70°C

Cooling until (43-44°C)

Addition of 10, 20, 30, 40, and 50% coconut milk by volume

Inoculation of 2% culture

Placing mix in several plastic cups

Incubation at 43°C for 4 h

Set type coconut milk yoghurt

Cooling and storage (5-7°C)

Fig. 3.2 Preparation of coconut milk incorporated yoghurt

Source: Biswas (2013)

3.2.3 Chemical analysis of coconut milk

3.2.3.1 Acidity

Acidity was determined by titrimetric method as Pearson (1981) .

3.2.3.2 Fat

Fat content in milk was determined by Gerber method as described by Kharel (1999).

3.2.3.3 Protein

Protein was determined kjeldahl method as described in AOAC (2005).

32
3.2.3.4 Ash

Ash content was determined as described in Ranganna (2000).

3.2.3.5 pH

The pH value was determined by the direct reading with the digital pH meter as given in
KC and Rai (2007).

3.2.3.6 Total Solid (TS)

Total solid was determined by subtracting the moisture from the 100 according to
Ranganna (2000)

3.2.3.7 Moisture

Moisture content was determined as per the methods described by Ranganna (2000).

3.2.3.8 Vitamin C

Vitamin C was determined following the method of KC and Rai (2007).

3.2.3.9 Total sugar

The total sugar was determined following the method of Ranganna (2000).

3.2.4 Chemical analysis of Milk

3.2.4.1 Acidity

Acidity and was determined by titrimetric method as per Pearson (1981).

3.2.4.2 Fat

Fat content in milk was determined by Gerber method as described by Kharel (1999).

3.2.4.3 Protein

Protein content was determined by formal titration method as described by Kharel (1999).

3.2.4.4 Ash

The ash content was determined as described by Ranganna (2000).

33
3.2.4.5 pH

The pH value was determined by the direct reading with the digital pH meter as given in
KC and Rai (2007).

3.2.4.6 Total Soluble Solid (TSS)

The total soluble solid of milk was determined by using Hand refractometer (Model WYT-
32, Zhongyou Optical Instruments).

3.2.4.7 Lactose

Lactose content was determined by Lane and Eynon method as per Pearson (1981).

3.2.5 Design expert

Using Design expert version 10, five formulations are designed under mixed condition
which are then coded alphabetically as given in Table 3.3

Table 3.3 Sample formulation in coded form

Sample Milk Coconut milk

A 90% 10%
B 80% 20%
C 70% 30%
D 60% 40%
E 50% 50%

3.2.6 Analysis of yoghurt

3.2.6.1 Sensory evaluation

Sensory evaluation was carried out using 9-point hedonic scale described by Ranganna
(2000). Sensory panelists were semi trained panelists from Central Campus Of
Technology, Dharan. Sensory evaluation was carried out on the quality attributes viz.,
colour and appearance, taste, body and texture, flavour and overall acceptability. The
specimen of the evaluation of card is shown in Appendix A.

34
3.2.6.2 Physical analysis

3.2.6.2.1 Syneresis

Degree of syneresis, expressed as proportion of free whey was measured by a method used
by Lee and Lucey (2004). A 100 g sample of yoghurt was placed on a filter paper resting
on the top of a funnel. After 10 min of drainage in vacuum condition, the quantity of the
remained yoghurt was weighed and syneresis was calculated as follows:

Wt. of initial sample –wt. of sample after filtration ×100

% Free whey (g/100g) =
Wt. of initial sample

3.2.6.3 Chemical analysis

3.2.6.3.1 Fat

Fat content was determined by the Gerber method as described in AOAC (2005).

3.2.6.3.2 Lactose

Lactose content was determined by the Lane and Eynon method as described in Ranganna
(2000).

3.2.6.3.3 pH

The pH value was determined by the direct reading with the digital pH meter as given in
KC and Rai (2007).

3.2.6.3.4 Titrable acidity

Titrable acidity was determined by titrimetric method given in AOAC (2005).

3.2.6.3.5 Protein

Protein was determined kjeldahl method as described in AOAC (2005).

3.2.6.3.6 Ash

Ash content was determined as described in Ranganna (2000).

35
3.2.6.3.7 Total carbohydrate

Total carbohydrate contents of samples were calculated by difference, that is the

percentage of moisture, ash, protein and fat was subtracted from 100 % Pearson (1981).

3.2.6.4 Microbiological examination

Total plate count (TPC) was carried out by using plate count agar as described in IDF
(1991).

3.2.6.5 Data analysis

Analysis of variance (ANOVA) was carried out for data from sensory evaluation. The
significant differences between them were studied by using L.S.D. at 5% level of
significance using Genstat release 12.1 software program developed by VSN International
Ltd.(VSNi) in the collaboration with practicing statisticians at Rothamsted Research and
the organization in Australia, New Zealand, Switzerland and the UK. t-Test: Two-Sample
Assuming Equal Variances was carried out using Microsoft Excel 10 to evaluate the
significant difference between the synerisis of the two samples.

36
 Part IV

Results and discussions

Coconut milk yoghurt was prepared at CCT, Dharan, in a laboratory for the present study.
The coconut milk incorporated yoghurt samples were prepared by incorporating 10, 20, 30,
40,and 50% coconut milk. The milk was mixed with 4% SMP (Skim milk powder) and
3% sugar at 45°C. Heating of milk was further continued till the temperature reached to
around 65-70°C for certain period. After that the heated milk was cooled to around 43-
44°C. After cooling, five formulations of the samples were made by adding 10, 20, 30,
40,and 50% of coconut milk per 100 ml yoghurt mix.Then the starter culture is added at
the rate of 2% to each formulations.Then the mix is placed in plastic cups. The yoghurt
mix was then kept in an incubator which was maintained at a temperature of about 43°C
and was kept for 3.5-4 h until the coagulum is formed. Coconut milk yoghurt with the
different levels of cococnut milk was subjected to sensory evaluation and compared with
each other to assess its acceptable level.

4.1 Chemical composition of milk

The proximate composition of the milk collected from DDC (Dairy Development
Corporation) is presented in the Table 4.1. The collected milk was standardized and
pasteurized.

Table 4.1 Chemical composition of milk

Parameters *Values (% dry basis)

Total soluble solid 8.4 (0.057)
Acidity as lactic acid (%) 0.14 (0.005)
pH 6.5 (0.057)
Lactose (%) 4.5 (0.057)
Protein (%) 3.4 (0.057)
Fat (%) 3.0 (0.057)
Ash (%) 0.72 (0.005)

*Values in the table are arithmetic mean of triplicate samples. Figure in the parentheses
indicates standard deviation.
 The composition of DDC milk in Table 4.1 has little variation over the composition of
milk analyzed by Dahal, (2009). The milk analyzed by Dahal is produced from Kamdhenu
Dairy Co-operative (KDDC). This variation in composition of milk may be due to the
species, nutritional aspect of animal, stage of lactation and feeding of animals. The
variation may also be due to different processing standard and specification of dairies.

4.2 Chemical composition of coconut milk

Coconut milk was analyzed. The results of analysis of coconut milk in dry basis are
tabulated in the Table 4.2.

Table 4.2 Chemical composition of coconut milk

Parameters Values
Acidity (% as citric acid) 0.13 (0.005)
Ash (% dry basis) 0.7 (0.057)
Fat (% dry basis) 4.2 (0.057)
Moisture (%) 91.2 (0.75)
pH 6.4 (0.057)
Protein (% dry basis) 2.8 (0.057)
Total solid (% dry basis) 8.2 (0.11)
Vitamin C (mg\100g) 3 (0.057)
Total sugar (% Dextrose) 3.1 (0.057)

*Values in the table are arithmetic mean of triplicate samples. Figure in the parentheses
indicates standard deviation.

There was little variation in the value obtained during this work from the value given by
Tamrakar (2017) and Biswas (2013). This may be due to difference in variety, climatic
conditions of cultivation, fruit maturity.

4.3 Sensory evaluation of coconut milk yoghurt

Sensory evaluation of all five formulation of the product which were carried out by a group
of seven semi-trained panelists evaluating aroma, color, texture and overall acceptance of
prepared coconut milk yoghurt. The Analysis of Variance (ANOVA) was carried out using
least significant difference (LSD) at 5% level of significance.

38
4.3.1 Aroma

Regarding aroma of coconut milk incorporated yoghurt, the analysis shows that the mean
sensory score for sample A, B, C, D, and E were found to be 7.57 , 7.57 , 7.14 , 6.71 and
6.71 respectively. Statistical analysis shows that effect of different coconut milk portion
on aroma of the product was not significantly different at 5% level of significance.

10

9 a a
a a a
8

7
Mean sensory score

0
A B C D E
Sample
Fig. 4.1 Effect of coconut milk on aroma of yoghurt

Fig. 4.1 represents the mean sensory scores for aroma of coconut milk yoghurt. Values on
the top of the bars bearing similar superscript are not significantly different at 5% level of
significance. Vertical error bar represent ±standard deviation of scores given by panelist.

4.3.2 Color

Regarding color of cococnut milk incorporated yoghurt, the analysis shows that the mean
sensory score for sample A, B, C, D, and E were found to be 8.143 , 7.571 , 6.571 , 6.429
and 6.286 respectively. Statistical analysis shows that effect of different coconut milk
portion on color of the product was significant (p<0.05). LSD shows that sample A and B,
A and C, B and C, A and D, B and D, A and E, B and E were significantly different but

39
there was no significant difference between samples C, D and E. Among five samples,
sample A got the high mean score, due to optimum acceptance of panelist.

As the proportion of cow milk was decreased the color preference became decrease. It
may be due to addition of coconut milk. Similar results were reported by Biswas (2013).

10

9 c b
8 a
a a
7
Mean sensory score

0
A B C D E
Sample

Fig. 4.2 Effect of coconut milk on color of yoghurt.

Fig. 4.2 represents the mean sensory scores for color of coconut milk yoghurt. Values on
the top of the bars bearing similar superscript are not significantly different at 5% level of
significance. Vertical error bar represent ±standard deviation of scores given by panelist.

40
4.3.3 Taste

Regarding taste of coconut milk incorporated yoghurt, the analysis shows that the mean
sensory score for sample A, B, C, D, and E were found to be 8 , 7.571 , 6.857 , 6.143 and
5.857 respectively. Statistical analysis shows that effect of different coconut milk portion
on taste of the product was significant (p<0.05). LSD shows that sample A and C, A and
D, A and E, B and C, B and D, B and E were significantly different but there was no
significant difference between samples A and B and C and D.

Among five samples, sample A and B got the high mean score, due to optimum
acceptance of panelist.

As the proportion of cow milk was decreased the taste preference became decrease. It
may be due to addition of coconut milk. Similar results were reported by Biswas (2013).

10

9 c c
b
8
b a
7
Mean sensory score

0
A B C D E
Sample

Fig. 4.3 Effect of coconut milk on taste of yoghurt

Fig. 4.3 represents the mean sensory scores for taste of coconut milk yoghurt. Values on
the top of the bars bearing similar superscript are not significantly different at 5% level of
significance. Vertical error bar represent ±standard deviation of scores given by panelist.

41
4.3.4 Texture
Regarding texture of coconut milk incorporated yoghurt, the analysis shows that the mean
sensory score for sample A, B, C, D, and E were found to be 8.29 , 7.57, 6.42 , 5.71 and
5.14 respectively. Statistical analysis shows that effect of different coconut milk portion
on texture of the product was significant (p<0.05). LSD shows that sample A and B, A and
C, A and D, A and E, B and C, B and D, B and E were significantly different but there was
no significant difference between samples D and E.

Among five samples, sample A got the high mean score, due to optimum acceptance of
panelist.

As the proportion of cow milk was decreased the texture preference became decrease. It
may be due to addition of coconut milk. Similar results were reported by Biswas (2013).

10
d
9 c

8 b
7 a
Mean sensory score

a
6

0
A B C D E
Sample

Fig. 4.4 Effect of coconut milk on texture of yoghurt

Fig. 4.4 represents the mean sensory scores for texture of coconut milk yoghurt. Values on
the top of the bars bearing similar superscript are not significantly different at 5% level of
significance. Vertical error bar represent ±standard deviation of scores given by panelist.

42
4.2.5 Overall acceptability

Regarding overall acceptability of coconut milk incorporated yoghurt, the analysis shows
that the mean sensory score for sample A, B, C, D, and E were found to be 8 , 7.71, 6.85 ,
6.14 and 5.71 respectively. Statistical analysis shows that effect of different coconut milk
portion on overall acceptability of the product was significant (p<0.05). LSD shows that
sample A and C, A and D, A and E, B and C, B and D, B and E, C and D, C and E were
significantly different but there was no significant difference between samples A and B and
D and E.

Among five samples, sample A and B got the high mean score, due to optimum
acceptance of panelist.

As the proportion of cow milk was decreased the overall acceptability preference
became decrease. It may be due to addition of coconut milk. Similar results were reported
by Biswas (2013)

10
c
9 c
8 b
a a
7
Mean sensory score

0
A B C D E
Sample

Fig. 4.5 Effect of coconut milk on overall acceptability of yoghurt

43
Fig. 4.5 represents the mean sensory scores for overall acceptability of coconut milk
yoghurt. Values on the top of the bars bearing similar superscript are not significantly
different at 5% level of significance. Vertical error bar represent ±standard deviation of
scores given by panelist.

There was significant difference between the samples A with respect to other samples
but there was no significant difference between other samples.The overall acceptability of
sample A was higher due to improvement in color, taste and texture with respect to other
samples. From the sensory evaluation of the product conducted on the attributes like
aroma, color, taste, texture and overall acceptability, the product containing 10% coconut
milk and 90% cow milk by volume was rated as best in all attributes.

4.4 Chemical analysis of best product

The standardized and pasteurized milk from DDC (Dairy Development Corporation) was
taken for the preparation of yoghurt. The milk was mixed with 4% SMP (Skim milk
powder) and 3% sugar at 45°C. Heating of milk was further continued till the temperature
reached to around 65-70°C for certain period. After that the heated milk was cooled to
around 43-44°C. After cooling, five formulations of the samples were made by adding 10,
20, 30, 40,and 50% of coconut milk per 100 ml yoghurt mix.Then the starter culture is
added at the rate of 2% to each formulations.Then the mix is placed in plastic cups.The
yoghurt mix was then kept in an incubator which was maintained at a temperature of about
43°C and was kept for 3.5-4 h until the coagulum is formed.

44
Chemical analysis of best product was done. The values of the chemical analysis are shown
in Table 4.4

Table 4.4 Chemical analysis of coconut milk incorporated yoghurt.

Parameters Yoghurt

Acidity (% lactic acid) 0.71 (0.01)

Ash (% dry basis) 0.94 (0.04)

Total Carbohydrate (% dry basis) 5.06 (0.08)

Fat (% dry basis) 5.1 (0.1)

Moisture 82 (1.5)

pH 4.4 (0.1)

Protein (% dry basis) 3.5 (0.1)

Total solid (% dry basis) 20 (1)

Lactose (% dry basis) 3.8 (0.2)

*Values in the table are arithmetic mean of triplicate samples. Figure in the parentheses
indicates standard deviation.

The chemical analysis of the best product was done. Percent acidity was to be 0.71%.It is
within the range (0.6-0.9%) reported by Yulina (2010). Desai et al. (1994) reported that
addition of fruit juice/pulp increases percent acidity. Ash content of the product was found
to be 0.94%.The result is within the range of value (0.8-1.5%) showed by Ahmed (2011).
Mahmood and Gilani (2008) reported that addition of fruit juice/pulp increases ash content
which may be due to insoluble solids and fiber content which may contribute in increasing
the ash content. Carbohydrate content was found to be 5.06% which was similar to the
result obtained by Afodunrinbi and Onyeukwu (2000). Fat content was found to be 5.1%

45
which is nearly same to the result obtained by Biswas (2013).Percentage fat is higher than
normal dairy yoghurt which is nearly(3-4%),this may be due to higher percent of fat
content in coconut milk than cow milk (Biswas, 2013).
Moisture content in the yoghurt was found to be 82% that is approximately similar to
the yogurts prepared from camel milk by Eshraga et al. (2011).PH was found to be 4.4
which agrees quite well with the results of Akpan et al. (2007).Protein content was found
to be 3.5% which was approximately similar to the result obtained by Afodunrinbi and
Onyeukwu (2000).Total solid was found to be 20%, which is nearly similar to the result
obtained by Biswas (2013).Lactose content was found to be 3.8% which was similar to the
result obtained by Mahmood and Gilani (2008) during the preparation of yoghurt blended
with apple and banana.

4.5 Physical analysis

The physical analysis of the commercial yoghurt and the best product was performed. The
values obtained for the syneresis are shown in Table 4.5

Table 4.5 Syneresis of yoghurt

Samples Syneresis (%)

Commercial yoghurt 19.26a

Best product 20.28a

t-Test was carry out to evaluate the significant difference between the two samples
.There were no significant difference between commercial and best product (P<0.05) as
shown in Appendix C. The results are similar to the work done by Rayamajhi (2011).

4.6 Shelf-life of the product

Best product which was found best with respect to aroma, color, taste, texture and overall
acceptability hence was used for further study. Hence it was subjected for chemical
analysis with respect to acidity and microbial count in laboratory.

46
4.6.1 Acidity of yoghurt at room temperature

The acidity of yoghurt increased from 0.71% to 1.19% within 7 days of storage under room
temperature. The increase in acidity of the yoghurt could be due to the acid production by
culture bacteria during storage period. Moreover increase in value of acidity is due to
adition of coconut milk which increases availability of higher nutrients for the production
of lactic acid. Yoghurt sample remain suitable for consumption up to 2 days. The results
are in agreement with Ahmed (2011).

1.4

1.2
Acidity (as % lactic acid)

0.8

0.6

0.4

0.2

0
0 2 4 6 8
Days

Fig. 4.6 Change in acidity under room temperature with respect to the number of days
In fig. 4.6 vertical error bars represents standard deviation

4.6.2 Total plate count of yoghurt at room temperature

TPC of yoghurt increases from 4.2×104 to 18.2×10 4CFU/ml within 7 days of storage under
room temperature. The increase in TPC of yoghurt is due to the production of lactic acid
bacteria which increases with the addition of coconut milk. The results are in agreement
with Ahmed (2011). Similar results was obtained by Goodluck et al. (2014) for the
consumable range of total baterial count as in the range of ( 3.0 x 103-10.5 x 104 cfu\ml).
Yogurt samples were suitable for consumption up to 2 days.

47
 25

20
Microbial Count (×104)

15

10

0
0 2 4 6 8
Days

Fig. 4.7 Change in TPC under room temprature with respect to the number of days
In fig. 4.7 vertical error bars represents the standard deviation

4.6.3 Acidity of yoghurt under refrigeration

The acidity of chilled yoghurt increased very slowly from 0.71 to 0.91 % within 7 days of
storage under refrigeration. The increase in acidity of yoghurt could be due to the acid
production by culture bacteria during storage period owing to their activity even at low
temperature. Acidity in yogurt samples also increased in refrigeration temperature but not
as rapid as at room temperature due to temperature effect . Yoghurt samples were suitable
for consumption up to 7days. The results are in agreement with Ahmed (2011)

48
 1.2

1
Acidity (as % lactic acid)

0.8

0.6

0.4

0.2

0
0 2 4 6 8
Days

Fig. 4.8 Change in acidity under refregration with respect to the number of days.

In fig. 4.8 vertical error bars represents the standard deviation

4.6.4 Total plate count of yoghurt under refrigeration

TPC of yoghurt increased slowly from 4.4×104 to 7.6×104 CFU/ml within 7 days of storage
under refrigeration. The increase in TPC of yoghurt is due to the production of lactic acid
even at low temperature.Though the rate of increase was not as that of room temperature
due to the fact that the rate of increase in lactic acid bacteria decreases in low temperature.
The results are in agreement with Ahmed (2011). Similar results was obtained by
Goodluck et al. (2014) for the consumable range of total baterial count as in the range of
(3.0 x 103-10.5 x 104 cfu\ml). Yoghurt samples were suitable for consumption up to 7 days.

49
 9

7
Microbial count (×104)

0
0 2 4 6 8
Days

Fig. 4.9 Change in TPC under refregration with respect to the number of days

In fig. 4.9 vertical error bars represents the standard deviation

4.7 Cost evaluation

The total cost of the best coconut milk incorporated yoghurt was calculated. It is shown in
the Appendix B. The price for 100 ml coconut milk incorporated yoghurt was found to be
Rs.10.47 which was cheaper than commercial yoghurt.

50
 Part V
Conclusion and recommendations

5.1 Conclusions

On the basis of the work conducted, the following conclusion can be concluded:

 From the sensory evaluation of the product conducted on the attributes like aroma,
color, taste, texture and overall acceptability, the product containing 10% coconut
milk and 90% cow milk by volume was rated as best in all attributes.

 From the storage analysis the product at refrigerated condition had increased self
life.
 The coconut milk incorporated yoghurt thus prepared was found to be cheaper and
more nutritional than the commercial yoghurt.
 The synerisis was not affected by the incorporation of coconut milk in yoghurt.

5.2 Recommendations

 Yoghurt can be prepared by blending different proportion of sugar and MSNF.

 Thermal treatment of coconut milk yoghurt can be studied to extend the shelf life of
the product.

 Coconut milk based ice cream could be prepared and their quality studied.

51
 Part VI
Summary
Coconut milk incorporated yoghurt is a cultured dairy product produced by fermenting
milk and coconut milk, with or without added non-fat dry milk (NFDM) with
Lactobacillus bulgaricus and Streptococcus thermophilus bacteria.

The standardized and pasteurized milk from DDC (Dairy Development Corporation)
was taken for the preparation of yoghurt. The milk was mixed with 4% SMP (Skim milk
powder) and 3% sugar at 45°C. Heating of milk was further continued till the temperature
reached to around 65-70°C for certain period. After that the heated milk was cooled to
around 43-44°C. After cooling, five formulations of the samples were made by adding 10,
20, 30, 40,and 50% of coconut milk per 100 ml yoghurt mix.Then the starter culture is
added at the rate of 2% to each formulations.Then the mix is placed in plastic cups.The
yoghurt mix was then kept in an incubator which was maintained at a temperature of about
43°C and was kept for 3.5-4 h until the coagulum is formed.

Sensory evaluation of five products was carried out. The sensory evaluation revealed
that the product containing 10% coconut milk and 90% milk by volume was found to be
best whose total solid, fat, acidity, protein, carbohydrate, total ash, moisture content,
lactose and pH were found to 20%, 5.1%, 0.71%, 3.5%, 5.06%, 0.98%, 82%, 3.8% and 4.4
respectively.Shelf life of the best product was estimated in terms of acidity and total plate
count and the shelf life was found to be 2 days at room temperature and 7 days at
refrigeration.

From the overall analysis of the result it is clear that good quality yogurt could be
prepared by adding coconut milk with cow milk. This new product will help to utilize
coconut to some extent. Coconut is available through the year for yogurt preparation. It is
expected that consumer will be interested to consume this type of yogurt day by day.
Considering commercial point of view, production of maximum volume yogurt from
minimum volume of cow milk by incorporating coconut milk make this business
profitable.
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 Appendix

Appendix-A

Sensory evaluation Card (specimen)

(Hedonic Rating test)

Name: ........................................ Date: ………………………….

Product: Coconut milk incorporated yoghurt

Observe the product by tasting. Use appropriate scale to show your attitude by checking at
the point that best describes you feeling of the product. An honest expression of your
personnel feeling will help to choose right product.

Quality Description

9 = Like extremely 6 = Like moderately 3 = Dislike moderately

8 = Like very much 5 = Neither like nor dislike 2 = Dislike very much

7 =Like moderately 4 = Dislike slightly 1 = Dislike extremely

Table A.1 Sensory panelist is requested to give ranks on their individual choice.

Parameters Sample A Sample B Sample C Sample D Sample E

Aroma

Color

Taste

Texture

OA

Comments if any: ……………………………………………………………………………

Signature
 Appendix B

Table B.1 Cost evaluation of 100ml of 10% coconut milk incorporated yoghurt

Particulars Quantity (g) Rate (Rs.) Amount (Rs.)

Milk 90 (ml) 80/liter 7.2

Whole Coconut 15 50/kg 0.75

SMP 40 300/kg 1.2

Sugar 30 80/kg 0.24

Overhead cost (20%) 1.08

Total 10.47

62
 Appendix C

Table C.1 t-Test: Two-Sample Assuming Equal Variances

Commercial Best
Mean 19.48666667 20.42
Variance 0.043433333 0.0436
Observations 3 3
Pooled Variance 0.043516667
Hypothesized Mean Difference 0
Df 4
t Stat -5.479672404
P(T<=t) one-tail 0.002699888
t Critical one-tail 2.131846786
P(T<=t) two-tail 0.005399775
t Critical two-tail 2.776445105

63
 Appendix D

Table D.1 Two way ANOVA no blocking for Aroma

Source of d.f s.s m.s V.r F pr.

variation

Formulation 4 5.143 1.286 1.28 0.304

Panelist 6 29.086 4.848 4.84 0.002

Residual 24 24.057 1.002

Total 34 58.286

Table D.2 LSD for Aroma

Table Formulation Panelist

rep.
7 5

d.f
24 24

l.s.d
1.105 1.307

64
Table D.3 Two way ANOVA no blocking for Color

Source of d.f s.s m.s V.r F pr.

variation

Formulation 4 18.5714 4.6429 22.16 <.001

Panelist 6 22.4000 3.7333 17.82 <.001

Residual 24 5.0286 0.2095

Total 34 46.0000

Table D.4 LSD for Color

Table Formulation Panelist

rep. 7 5

d.f 24 24

l.s.d 0.5050 0.5975

65
Table D.5 Two way ANOVA no blocking for Taste

Source of d.f s.s m.s V.r F pr.

variation

Formulation 4 23.2571 5.8143 23.48 <.001

Panelist 6 24.3429 4.0571 16.38 <.001

Residual 24 5.9429 0.2476

Total 34 53.5429

Table D.6 LSD for Taste

Table Formulation Panelist

rep. 7 5

d.f 24 24

l.s.d 0.5490 0.6495

66
Table D.7 Two way ANOVA no blocking for Texture

Source of d.f s.s m.s V.r F pr.

variation

Formulation 4 47.0286 11.7571 32.92 <.001

Panelist 6 16.5714 2.7619 7.73 <.001

Residual 24 8.5714 0.3571

Total 34 72.1714

Table D.8 LSD for Texture

Table Formulation Panelist

rep. 7 4

d.f 24 21

l.s.d 0.659 0.780

67
Table D.9 Two way ANOVA no blocking for OA

Source of d.f s.s m.s V.r F pr.

variation

Formulation 4 26.9714 6.7429 18.75 <.001

Panelist 6 21.9429 3.6571 10.17 <.001

Residual 24 8.6286 0.3595

Total 34 57.5429

Table D.10 LSD for OA

Table Formulation Panelist

rep. 7 5

d.f 24 24

l.s.d 0.661 0.783

68
 Color plates

Plate no 1. Sensory evaluation of yoghurt Plate no 2. Lab analysis

Plate no 3. Lab analysis

69