BL Aackbookword

EFFECT OF INCORPORATION OF HOLY BASIL ON PHYSICO
CHEMICAL PROPERTIES OF MILK
by
Bishakha Adhikari
Department of Science and Technology
Lalitpur Valley College
Institute of Science and Technology
Tribhuvan University, Nepal
2023
EFFECT OF INCORPORATION OF HOLY BASIL ON PHYSICO
CHEMICAL PROPERTIES OF MILK
A dissertation submitted to the Department of Food Technology, Lalitpur Valley

College, Tribhuvan university, in a partial fulfillment of the requirements for the degree
of B. Tech in Food Technology
by
Bishakha Adhikari
Department of Food Technology
Lalitpur Valley College
Affiliated to Tribhuvan University
Jawalakhel, Lalitpur
2023
Tribhuvan University
ii
Institute of Science and Technology
Department of Food Technology
Lalitpur Valley College, Lalitpur
Approval letter
This dissertation entitled Effect of incorporation of holy basil on physico chemical

properties of milk presented by Bishakha Adhikari has been accepted as the partial
fulfillment of the required for the degree of B.Tech in Food Technology
Dissertation Committee
1. Head of Department
Dr. Sitaram Joshi
2. External Examiner
3. Supervisor
Mr. Sandeep Raj Pradhan
4. Co- Supervisor
Mr. Abhitosh Dhungel
February, 2023
iii
Acknowledgement
This thesis completion is the end of my research for obtaining my Bachelor’s degree in
Food Technology and finally, this achievement has been possible with the kind support and
inspiration from numerous people.
First and foremost, I would like to express my gratitude to my respected guide, Mr.
Sandeep Raj Pradhan for his invaluable guidance and dynamic supervision throughout this
dissertation work. I would also like to extend my cordial gratitude towards Dr. Sitaram
Joshi, college chief and Lalitpur Valley College for the support in this research. I am
greatly indebted to Ms. Puja Shrestha for providing necessary laboratory equipment,
environment and other faculty members for their encouragement and support to complete
my thesis in innovative way on time. I would also like to thank my classmates especially
for their continuous support & cooperation at the time of need. The college experience
would not have been the same without my classmates. Finally, I must pay my sincere
admiration and gratitude to my loving and supportive parents for their blessings and
continuous encouragement.
Date of submission:
Bishakha Adhikari
iv
Abstract
Ocimum Sanctum Linn., also known as holy basil, is a Lamiaceae family herbal plant. It
has an antimicrobial effect against microorganisms, which aids in the preservation of food
products when added to them. Herbal milk is a type of flavored milk. It includes the
addition of herbal plants such as Tulsi, Khajoor, Cumin, Ginger and others. Flavored milk
has a high nutritional profile and serves as a link between children and nutrition. The
objective of this study was to evaluate the physico-chemical, sensory and microbiological
characteristics of herbal milk. Tulsi powder was added in the ratio of 0%, 0.5%, 0.6% and
0.7% in Sample A, B, C and D respectively.
The proximate composition of herbal milk indicated a significantly higher content of

moisture in Sample D (87.45±0.03) than Sample A (86.21±0.04). The proximate
composition i.e., protein, fat, ash, specific gravity and lactose of Sample B was observed as
3.37±0.01, 2.84±0.05, 0.66±0.04, 1.06±0.01, 5.15±0.01 and Sample D was observed as
3.25±0.06, 2.62±0.04, 0.57±1.03, 1.02±0.01 and 5.04±0.03 respectively. The
microbiological analysis showed no presence of coliform, yeast and mold. However, the
total plate count in Sample A, B, C and D were found to be 4.19, 3.76, 3.88 and 3.98
respectively. The best results for sensory analysis were observed in Sample B according to
the panelists whereas Sample D showed close resemblance to Sample A thereby it can be
selected as the replacement of Sample A. The addition of Tulsi showed improved results in
acidity and pH of herbal milk resulting in increased shelf life.
v
List of Tables
Table 1: Typical Chemical Composition of Milk of Different Species ............................... 9

Table 2: Protein Composition of Bovine Milk ....................................................................
10
Table 3:Vitamins content in fresh milk ...............................................................................
13
Table 4: Types of pasteurization ......................................................................................... 20
Table 5:Proximate composition of Tulsi juice .................................................................... 21
Table 6:Chemical composition of cow milk........................................................................ 22
Table 7:Formulation of Tulsi incorporated milk .................................................................
29
Table 8:Proximate composition of Tulsi .............................................................................
33
Table 9: Effect of Tulsi on acidity of milk ..........................................................................
41
Table 10: Effect of Tulsi on pH of milk ..............................................................................
42
Table 11: Microbial analysis of herbal milk ........................................................................
49
Table 12: Cost evaluation of 1 liter of herbal milk ..............................................................
50
Table 13:Mean sensory score for different concentration of Tulsi incorporated milk ........
81
Table 14: Proximate analysis of Tulsi incorporated milk ....................................................
81
vi
List of Figures
Figure 1: Chart of preparation of flavored milk ..................................................................

15
Figure 2: Flow chart for preparation of herbal milk using Tulsi .........................................
28
Figure 3: Fat content of herbal milk ....................................................................................
35
Figure 4: Protein content of herbal milk ..............................................................................
36
Figure 5: Ash content of herbal milk ...................................................................................
37
Figure 6: Specific gravity of herbal milk............................................................................. 38
Figure 7: Carbohydrate of herbal milk ................................................................................
39
Figure 8: Moisture content of herbal milk ...........................................................................
40
Figure 9: Mean sensory score for color ...............................................................................
43
Figure 10: Mean sensory score for mouth feel ....................................................................
45
Figure 11: Mean sensory score for odor ..............................................................................
46
Figure 12: Mean sensory score for taste ..............................................................................
47
Figure 13:Mean sensory score for overall acceptability ......................................................
48
vii
List of photographs
Photograph No. Title Page No.
Photograph 1 Standardized milk 79
Photograph 2 Test for caustic soda 79
Photograph 3 Herbal milk samples 79
Photograph 4 Samples for sensory evaluation 79

Photograph 6 Microbial analysis 80
viii
List of abbreviation
Abbreviation Full form
ANNOVA Analysis of variance
et.al And others
AOAC Association of Official Analytical Chemist
DDC Dairy Development Corporation
DFTQC Department of Food Technology and quality control
FAO Food and Agriculture Organization
FDA Food and Drug Administration
IDF International Dairy Federation
TP Tulsi Powder
% Percentage
SNF Solid Non-Fat
SD Standard Deviation
SPSS Statistical Package for Social Science
TS Total Solid
WHO World Health Organization
FDA Food and Drugs Administration

CI Calving Interval
ix
Table of Contents
Approval letter....................................................................................................................ii
Acknowledgement..............................................................................................................iii
Abstract...............................................................................................................................iv
List of tables.........................................................................................................................v
List of figures......................................................................................................................vi
List of photographs...........................................................................................................vii
List of abbreviations………………………………………………………...……….… viii
1.Introduction ...................................................................................................................... 1
1. 1.General
Introduction .................................................................................................... 1
1.2 Statement of problem ...................................................................................................

2
1.3 Objectives of study ......................................................................................................

2
1.3.1Specific objectives ..................................................................................................

3
1.4 Significance of work ....................................................................................................

3
1.5 Limitations: ..................................................................................................................

3
2. Literature review .............................................................................................................

4
2.1. Background .................................................................................................................

4
2.1.1 Milk ...........................................................................................................................

4
2.1.2 Basil .......................................................................................................................

4
x
2.2 Relation between milk and Ayurvedic herbs ...............................................................
5
2.3 Varieties in Flavored milk ...........................................................................................

6
2.3.1 Chocolate flavored milk ........................................................................................

6
2.3.2. Sterilized flavored milk ........................................................................................

7
2.3.3. Fruit flavored milk ...............................................................................................

7
2.3.4. Herbal flavored milk ............................................................................................

8
2.3.5. Pasteurized flavored milk .....................................................................................
8
2.4 Chemical composition of milk .....................................................................................

8
2.5. Constituents of milk ....................................................................................................

9
2.5.1 Water .....................................................................................................................

9
2.5.2 Milk Fat .................................................................................................................

9
2.5.3. Milk Protein ........................................................................................................

10
2.5.4. Lactose ...............................................................................................................

11
2.5.5. Enzymes in milk .................................................................................................

11
2.5.6. Gases in milk ......................................................................................................

12
2.6. Flavor Component ....................................................................................................

12
2.7. Minor component ......................................................................................................

13
xi
2.7.1 Vitamin ................................................................................................................
13
2.8. Effect of Heat on Milk Proteins ................................................................................

14
2.9. Flavored milk among children ..................................................................................

14
2.11. Nutrient contribution of flavored milk ....................................................................

15
2.11.1. Total milk intake and nutrient contribution from flavored milk ......................
17
2.12. Tulsi ........................................................................................................................

18
2.12.1. Health benefits of Tulsi ....................................................................................

19
2.12.2. Stress resilience ....................................................................................................

19
2.12.3. Common cold and fever ...................................................................................

19
2.12.4. Antibiotic property ...........................................................................................

19
2.12.5. Hepatoprotective, Reno protective and Neuroprotective activities ..................

19
2.13. Types of pasteurization ...........................................................................................

20
2.13.1. Micro-organisms killed by pasteurization ........................................................

20
2.14 Ingredients................................................................................................................
21
2.14.1. Tulsi ..................................................................................................................

21
2.14.3. Milk ..................................................................................................................

21
2.15. Packaging ................................................................................................................
22
xii
3. Materials and Methods ................................................................................................. 23
3.1. Materials ...................................................................................................................

23
3.1.1. Raw material collection ......................................................................................

23
3.1.2. Laboratory instruments and chemicals ..................................................................

23
3.1.2.1 List of equipment used .....................................................................................

23
3.1.2.2. List of chemicals used .....................................................................................

23
3.1.3. Glassware ...............................................................................................................

23
3.1.4. Laboratory facilities ...............................................................................................

23
3.2. Analysis of Tulsi .......................................................................................................

23
3.2.1. Determination of Moisture Content ...................................................................

23
3.2.2. Determination of protein ....................................................................................

24
3.2.3. Determination of fat ...........................................................................................

24
3.2.4. Determination of Crude fiber .............................................................................

24
3.2.5. Determination of total ash ..................................................................................

24
3.2.6. Determination of carbohydrate ...........................................................................

24
3.2.7. Determination of acidity of Herbal milk ............................................................

25
3.2.8. Determination of pH ...........................................................................................

25
xiii
3.3. Analysis of Milk .......................................................................................................
25
3.3.1. Acidity ................................................................................................................

25
3.3.2. Fat .......................................................................................................................

25
3.3.3. Solid not Fat Content (SNF) ...............................................................................

26
3.3.4. pH .......................................................................................................................
26
3.3.5. Protein ................................................................................................................

26
3.3.6. Moisture .............................................................................................................

27
3.4. Preparation of Herbal milk........................................................................................

28
3.5. Product Formulation .................................................................................................

29
3.6. Proximate Analysis of Tulsi incorporated milk ........................................................
29
3.6.1. Moisture content .................................................................................................

29
3.6.2. Fat content ..........................................................................................................

29
3.6.3. Protein content ....................................................................................................

30
3.6.4. Ash content .........................................................................................................

30
3.6.5. Carbohydrate ......................................................................................................

30
3.7. Microbial analysis .....................................................................................................

31
xiv
3.7.1. Preparation ofSample Dilution ...........................................................................
31
3.7.2. Enumeration of total bacteria .............................................................................

31
3.7.3 Coliform analysis of Herbal milk ........................................................................

31
3.7.4. Enumeration of yeast and molds in Herbal milk ................................................

31
3.8. Sensory Analysis of Herbal milk ..............................................................................

32
3.9. Cost evaluation..........................................................................................................

32
3.10. Statistical Analysis ..................................................................................................

32
4. Result and Discussion ....................................................................................................

33
4.1.1. Moisture Content ................................................................................................

33
4.1.2. Crude Protein ......................................................................................................

33
4.1.3. Crude Fat ............................................................................................................

34
4.1.4. Crude Ash ...........................................................................................................

34
4.1.5. Crude Fiber .........................................................................................................

34
4.1.6. Carbohydrate ......................................................................................................

34
4.3. Proximate analysis of tulsi incorporated milk ..........................................................

35
4.3.1. Fat content ..........................................................................................................

35
xv
4.3.2. Protein content ....................................................................................................
36
4.3.3. Ash content .........................................................................................................

37
4.3.4. Specific gravity ..................................................................................................

38
4.3.5. Carbohydrate ......................................................................................................

39
4.3.6. Moisture content .................................................................................................
40
4.3.8. Effect of Tulsi on pH of milk .................................................................................

42
4.4 Sensory Analysis ........................................................................................................

43
4.4.1. Color ...................................................................................................................

43
4.4.3. Odor ....................................................................................................................

46
4.4.4. Taste ...................................................................................................................

47
4.4.5. Overall acceptability ..........................................................................................

48
4.5. Microbial Analysis ....................................................................................................

49
4.5.1. Total Plate Count ................................................................................................

49
4.5.2. Coliform count ...................................................................................................

49
4.5.3. Yeast and mold count .........................................................................................

50
4.6. Cost evaluation..........................................................................................................

50
xvi
4.7. Calorific Value ..........................................................................................................
50
5. Conclusions and Recommendation ..............................................................................

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

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

51
6. Summary ........................................................................................................................
52
Appendixes .........................................................................................................................
61
Appendix A ......................................................................................................................
61
Appendix B ......................................................................................................................
63
xvii
Part I
Introduction
1.1.General Introduction
Milk is a balanced food that contains a lot of the vital elements. Milk helps us stay healthy
and wards off a number of ailments. In essence, milk is an emulsion of fat, protein,
carbohydrate, minerals, and vitamins in water. About 80.7% of whole cow's milk is made
up of water. Protein, fat, carbs, vitamins, and minerals are present in the remaining 13%
(anyon, 2011). It is a valuable source of carbohydrates, proteins containing essential amino
acids, immunoglobulins, essential fatty acids and other micronutrients (Hossain et al.,
2011).
One or more healthy cows must be fully milked five days after and 15 days before
parturition to produce milk, which is described as the lacteal secretion that is almost free of
colostrum. Buffalo milk contains 70.6% fat, 3.8% protein, 4.9% lactose, 0.78% ash and
10.7% total solids. Cow milk contains 40.5% fat, 3.8% protein, 4.9% lactose, 0.72% ash
13.9% total solids. That is why milk is an important part of the diet of all age group
including expectant mothers (Bettoni, 2013). By centrifugation or naturally occurring
creaming, the milk is divided into the two fractions (cream and skim milk). While skim
milk is almost fat-free, the cream contains a lot of fat.
The same vital components found in plain milk, such as protein, calcium, potassium,
phosphorus, vitamin A, vitamin B12, iodine, and riboflavin, are also present in flavoring
milk. It has a longer shelf life because it has been sterilized. Herbal milk has several health
advantages, including better bone health, lowered risk of heart disease, cancer, ulcers, type
2 diabetes, allergy and asthma, enhanced immunity, among many more. The demand for
milk and milk products is rising quickly as a result of population expansion, changes in the
socioeconomic standing, income, distribution, and tastes and preferences of the populace.
In light of the significance of natural components, attempts have been made to make herbal
milk (Gaur et al., 2019).
Tulsi, also known as holy basil or Tulsi in Hindi and Sanskrit, is a highly regarded culinary
and medicinal aromatic herb from the Lamiaceae family that is native to the Indian
subcontinent. It has been used in Ayurvedic medicine for more than three thousand years.
Tulsi is frequently referred to as an "Elixir of Life" in the Ayurvedic tradition because of
1
its curative properties and track record of treating a wide range of common health issues.
Tulsi leaf extracts are recommended in the Indian Materia Medica for the treatment of
pyrexia, rheumatism, and bronchitis. Treatment of epilepsy, asthma or dyspnea, hiccups,
cough, skin and hematological illnesses, parasite infections, neuralgia, headache, wounds,
inflammation, and oral problems are some more reported medicinal uses. For earaches, the
leaf juice has been applied as a drop, and the tea infusion has been used to treat liver and
gastric disorders. Three types of tulsi are commonly described. Ocimum tenuiflorum (or
Ocimum sanctum L.) includes 2 botanically and phytochemically distinct cultivars that
include Rama or Sri tulsi (green leaves) and Krishna or Shyama tulsi (purplish leaves),
while Ocimum gratissimum is a third type of tulsi known as Vana or wild/forest tulsi (dark
green leaves) (Jamshidi, 2017).
1.2 Statement of problem

Herbal milk has been very popular among health-conscious people but in the context of
Nepal, the availability of Herbal milk is rare. Most of the people are unaware about the
Tulsi incorporated milk although it is present in almost every household. Tulsi can not only
be used for religious purpose but also commercially as it has many health benefits (Dangol,
2018). Herbs are well known for their preservative properties by hindering microbial
growth in food (Negar et al., 2017). Due to this preservative nature of Tulsi, its addition
would really help in enhancing the shelf life as well as creating flavorful milk. Even though
Tulsi has many positive aspects, it is not used much for product development. Due to easy
availability, Tulsi can be used in milk to improve its nutritional and sensory values.
Palthur et al (2014b) and Gaur et al (2019) have done research on Tulsi and other different
herbs being focused on limited parameter and not much focused on sensory and storage
evaluation which is must for commercial production.
Nutritional benefit as well as addition of sensory value is seen during the making of Tusli
flavored milk which is good so this dissertation is important to know the added benefit of
Tulsi in human health.
1.3 Objectives of study

The general objective of the study was to improve the flavor and texture and to lengthen
the shelf life of milk by adding herbal flavor.
2
1.3.1 Specific objectives
1. To determine the physicochemical properties of Tulsi powder and herbal milk like
moisture, pH, acidity, carbohydrate, total ash, protein content, ash content.
2. To perform sensory evaluation of the product.
3. To perform microbial evaluation of product.
4. To evaluate the cost of the product.
1.4 Significance of work

This study can be beneficial for dairy industries. This study can be useful for producing
flavored milk with good mouth-feel and texture at minimal cost. This can decrease the
import of flavored milk and increase the market of flavored milk produced inside the
country. The significance of study are as follows:
1. Quality evaluation of the Herbal milk shall be done on different basis like
physicochemical, sensory and microbial analysis.
2. Once the study is completed, it will largely be beneficial to dairy industries that are
working on the development of Herbal milk.
3. It creates the scope for further study and research.
1.5 Limitations:
1. Microbial analysis couldn’t be done every day.
2. Micro nutrients like vitamins, calcium, magnesium etc. couldn’t be determined.
Part II
Literature review
2.1. Background
2.1.1 Milk
Humans probably first domesticated animals and began using milk and basic milk products
between 6000 and 8000 B.C. Even around 4000 B.C. The Egyptians kept dairy cattle, and
milk production in ancient India at around 2000 B.C. In addition to being consumed as
3
food, milk and butter were also employed in antiquity for cultic and cosmetic purposes. It
was not until 1541 that K. Gessner, a scientist from Zeurich, wrote a book on dairy
farming. A century later, milk was first studied scientifically; lactose's characteristics were
first reported in 1633 by F. Bartoletti, while fat globules and lactalbumin were found in
milk in 1673 and 1841, respectively by A. Van Leeuwenhoek and Th. A. Quevenne. Milk
was examined scientifically for the first time a century later; the properties of lactose were
described in 1633 by F. Bartoletti and. The presence of fat globules in milk were
discovered in 1673 and 1841 by A. Van Leeuwenhoek and lactalbumin Th. A. Quevenne
respectively.
The first methods for the conservation of milk were developed in the 19th century. F.
Appert sterilized milk for the first time in 1804; Newton introduced the process of
evaporating milk with added sugar in 1835 and G. Borden developed the process of
evaporating sweetened condensed milk under vacuum in 1853. The milk separator was
invented in 1859, and the first condensed milk plant in Europe was built in 1866 (Jost.,
2012).
2.1.2 Basil
Ocimum sanctum Linn. (Also known as Ocimum tenuiflorum) has been used for thousands
of years in Ayurveda for its diverse healing properties. Tulsi, the Queen of herbs, the
legendary ‘Incomparable one’ of India, is one of the holiest and most cherished of the
many healing and healthy giving herbs of the orient. Marked by its strong aroma and
astringent taste, it is regarded in Ayurveda as a kind of ‘elixir of life’ and believed to
promote longevity. Traditionally, Ocimum sanctum L. is taken in many forms, as herbal
tea, dried power or fresh leaf. For centuries, the dried leaves of Tulsi have been mixed with
stored grains to repel insects. Ocimum sanctum L. (Tulsi) is an erect, much branched
subshrub 30-60 cm tall, with simple opposite green or purple leaves. The chemical
composition of Tulsi is highly complex, containing many nutrients and other biologically
active compounds, the proportions of which may vary considerably between strains and
even among plants within the same field. The leaf volatile oil contains eugenol (1-
hydroxy2-methoxy-4-allylbenzene, euginal (also called eugenic acid), urosolic acid
(2,3,4,5,6,6a,7,8,8a,,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid]),
carvacrol (5-isopropyl-2-methylphenol), linalool (3,7-dimethylocta-1,6-dien-3-ol),
limatrol, caryophyllene (4,11,11-trimethyl-8-methylene-bicyclo[7.2.0]undec-4-ene),
methyl carvicol (also called Estragol: 1-allyl-4-methoxybenzene) while the seed volatile oil
4
have fatty acids and sitosterol; in addition, the seed mucilage contains some levels of
sugars and the anthocyans in green leaves. The sugars are composed of xylose and
polysaccharides. The stem and leaves of holy basil contain a variety of constituents that
may have biological activity, including saponins, flavonoids, triterpenoids, and tannins
(Pattanayak, 2010). It has over 150 different species and varieties and is found in the
tropical regions of Asia, Africa, Central and South Africa. The Ocimum (Basil) family
contains some of the world's most popular herbs. The plant itself purifies the air around
any dwelling in which it is planted, and modern researchers have discovered that it
produces ozone (Tewari1 et al., 2012).
2.2 Relation between milk and Ayurvedic herbs

Milk's health benefits have been enhanced since Vedic times by the use of herbal infusions.
This correlation is used for disease prevention and to overcome nutritional deficiencies.
Because nutritional deficiency is nearly impossible to avoid in modern times, natural
supplements aid in overall growth, development, and immunity. Herbs can also aid in the
removal of toxins from the body. According to the World Health Organization, more than
80% of the world's population, or 4.3 billion people rely on traditional plant-based medical
systems for primary health care (Mohd et al., 2011).
Ayurvedic system has the oldest and one of the most developed herbal systems in the world
– it has been in continuous use for at least 5,000 years. The ancient Vedic seers in India
compiled and classified medicinal and healing properties of hundreds of herbs into an
herbal healing system that can be accessed and utilized by anyone. One of the latest things
is the production of new flavored milk products using herbs. Given below is the list of
ayurvedic herbs that can be added to milk to make flavored Milk. Brahmi, Tulsi, Ginger,
Shatvari, Turmeric, Alfafa, Stevia, Khajoor, Aloe Vera, Cumin, Coriander, Cinnamon,
Black pepper, Mace, Bay leaf, Poppy seeds, Liquorice or Mulethi, Jatamansi or Spikenard
and Pueraria tubersosa). Recently a new product by the name of memory milk has been
developed by a major brand in the market under the category of sterilized homogenized
flavored toned milk that contains combination of various ayurvedic herbs like Brahmi,
Shankpushpi, Tulsi, Ashwagandha and other herbs (Kishore et al., 2020).
2.3 Varieties in Flavored milk

Flavored milk is milk that has had some flavor added to it. Flavored milks typically begin
with a pleasant flavor. When the term "milk" is used in the preparation of flavored milk, it
5
means that the product should contain a percentage of milk fat from the milk from which it
was prepared. When the fat content is lower (1-2%), the term drink is used. These milk
beverages are made by standardizing dairy and nondairy ingredients in appropriate
concentrations. In the production of flavored milks, artificial flavors and colors are added.
Fat and SNF levels are adjusted to the desired level by adding cream to standardize fat and
reconstituted skim milk powder to standardize SNF content. After standardization, milk is
homogenized in two stages at 65°C. Because cocoa particles settle during storage,
homogenization is required in the preparation of chocolate-flavored milk. Filtration is an
unavoidable step that must be completed after homogenization. Then, pasteurization at
71˚C for 30 minutes or 80˚C for 15 seconds and cooling to around 4°C (Tiwari et al.,
2017).
2.3.1 Chocolate flavored milk

Chocolate milk is sweetened milk with chocolate or cocoa flavoring that has a dark color
and a chocolaty flavor. The raw materials used in the preparation of chocolate-flavored
milk range from low-fat milk to full-cream milk, sugar, cocoa powder, and stabilizer are
standardized and blended together before being homogenized and pasteurized/sterilized. To
flavor the milk, sugar and cocoa powder are used, and carrageenan is used as a stabilizer in
chocolate-flavored milk to keep these flavorings in suspension. Depending on the
composition and raw materials used, various types of chocolate milk can be prepared. The
following ingredients are commonly used in its preparation: cocoa powder - 1 to 1.0.5%,
and stabilizer - 0.2% and minimum legal standard for milk fat content. In order to make
chocolate-flavored milk, the milk is first standardized to the desired fat and solidsnot fat
levels for drink. It is preheated to 35-40˚C for effective filtration/clarification and
standardization. Following preheating, cocoa powder (1-1.0.5% w/v), sugar (5-0.7% w/v),
and sodium alginate (stabilizer) (0.2% w/v) are gradually added with continuous stirring to
ensure proper mixing. After mixing, the milk is heated to 60˚C, homogenized at 2500
pounds per square inch, and clarified. The mixture is then pasteurized at 71°C/30 minutes,
rapidly cooled to 5°C, bottled, and refrigerated (5°C) until used (Tiwari et al., 2017).
2.3.2. Sterilized flavored milk

Milk sterilization, also known as flavoring milk sterilization, is a high heat treatment
process used to kill all pathogenic microorganisms found in milk. Sterilized products have
excellent keeping qualities and can be stored at normal room temperatures for longer
periods of time. The most common sterilized dairy product is flavored milk. This milk
6
combines the benefits of both sterilized and flavored milk. Sterilized flavored milk has a
long shelf life. According to FSSAI, “Sterilization when used in association with milk,
means heating milk in sealed container continuously to a temperature of either 115˚C for
15 minutes or at least 130˚C for a period of one second or more in a continuous flow and
then packed under aseptic condition in hermetically sealed containers to ensure
preservation at room temperature (Tiwari et al., 2017).
2.3.3. Fruit flavored milk

Permitted fruit flavors or essences, as well as permitted colors and sugar, are used in the
preparation of fruit-based flavored milk. The preparation method is similar to that of
chocolate milks/drinks. Strawberry, orange, lemon, pineapple, banana, vanilla, carrot juice,
apricot, and peaches are some popular flavors. Strawberry-flavored milk is also well-liked
by consumers Pure fruit juices or syrups can be successfully combined with milk to create
milkshakes. Mango and banana are commonly used in the preparation of milkshakes in
India, especially during the summer months. Fruit-flavored milks are made by combining
fruit pulp or fruit juices with sugar in the milk. In general, these beverages are made by
combining milk solids (either liquid whole/skim milk, whey, buttermilk, or their
concentrated or anhydrous counterpart) with fruit juice, sugar solution, stabilizer (generally
pectin, carrageenan, CMC, gelatin, etc.) and an edible acid (citric, lactic, malic or tartaric
acid). Natural fruit juices such as apple, grape, and pineapple have also become more
popular in the preparation of this type of flavored milk (Tiwari et al., 2017).
2.3.4. Herbal flavored milk

As people become more aware of the benefits of herbal components, an increasing number
of people are turning to herbal food products for their health benefits. Certain food
products may be the best medium for communicating the medicinal benefits of herbs.
There is also a need to develop new technologies for adding value to milk. Several
medicinal herbal plants or their components, such as Aloe vera, cinnamon, and tulsi, are
added to certain dairy and food products. Along with its herbal value, herbal-flavored milk
contains several other essential nutrients, making it a potential food supplement for adults
and children. These are flavored with various herbal plants or components to improve the
nutritional and medicinal properties (Tiwari et al., 2017).
7
2.3.5. Pasteurized flavored milk
Pasteurized milk products are liquid milk and cream products intended for direct
consumption by consumers. Whole milk, skim milk, standardized milk, flavored milks, and
various types of cream are all included in this category. According to FSSAI, ͞ ”
Pasteurization, pasteurized and similar terms shall be taken to refer to the process of
heating every particle of milk of different classes to at least 63˚C and holding at such
temperature continuously for at least 30 minutes or heating it to at least 71.5˚C and holding
at such temperature continuously for at least 15 seconds or an approved temperature time
combination that will serve to give a negative Phosphatase test. All pasteurized milk of
different classes shall be cooled immediately to a temperature of 10˚C, or less (Tewari et
al., 2012).
2.4 Chemical composition of milk

Milk is the whole, fresh, clean lacteal secretion obtained from milking one or more healthy
cows completely. Milk must contain at least 3% milk fat and at least 8.20.5% milk SNF.
Milk can be standardized by adding or removing cream or by adding skim milk. When so
standardized, milk must contain at least 3.20.5% milk fat and at least 8.20.5% milk SNF.
Milk cannot be processed 15 days before and 5 days after calving.
2.5. Constituents of milk
Table 1: Typical Chemical Composition of Milk of Different Species (% Composition)

Species Water Fat Protein Lactose Ash
Ass 90.0 1.3 1.7 6.5 0.5
Buffalo 84.2 6.6 3.2 5.2 0.8
Camel 86.5 3.1 4.0 5.6 0.6
Cow 86.6 4.6 3.4 4.9 0.5
Ewe 79.4 8.6 6.7 4.3 1.0
Goat 86.5 4.5 3.5 4.7 0.8
Human 87.7 3.6 1.8 6.8 0.1
Source: Kailasapathy (2015)
2.5.1 Water
All of the additional milk ingredients (total solids) are suspended or dissolved in water.
Milk contains trace amounts of water that are chemically or physically hydrated into
lactose, salts, and proteins. Milk's shelf life is extended by reducing water activity when
8
water is removed, as is the case with concentrated and dried milk products (Kailasapathy,
2016).
2.5.2 Milk Fat

One of the most crucial components of milk is fat, which is also thought of as the
economics of milk and milk products. The majority of milk price plans are mostly
determined by milk fat content. It provides a plentiful energy source (around 9 kcal per
gram) and a transporter for fat-soluble vitamins (A, D, E, and K). Significant amounts of
necessary fatty acids are present. As well as imparting soft, smooth, and rich tastes and
overcoming watery, flat, harsh, and gritty features that are typically found in their absence,
milk fats play a significant role in the creation of acceptable or disagreeable flavors in dairy
products (Singh, 2014).
The range of fat levels is substantially wider than that of any milk component, ranging
from less than 3.0% to more than 6.0%. The majority of milk fat (96–98%) is made up of
triglycerides. A chiral carbon is located at the sn-2 position of the glycerol skeleton in
triacylglycerols, which are composed of three distinct fatty acids. Bovine lipids include
more than 400 distinct fatty acids (Damodaran et al., 2007).
2.5.3. Milk Protein

Milk contains a mixture of colloidal suspension and solution-based proteins. Whey protein
(also known as serum proteins) and caseins are the two main forms of milk proteins. Over
80% of the protein in milk is casein, though the ratio of whey protein to casein fluctuates
depending on the stage of lactation. The five primary classes of caseins are s1, s2, gamma,
and s-caseins. Caseins are globular proteins having phosphoserine residue. Phosphoserine
residues are grouped together and are what give rise to the hydrophobic regions with highly
negative charges. The most hydrophobic component is seen in -casein, which also forms
aggregates with its hydrophilic N-terminus exposed to solvent and hydrophobic interior.
Due to the presence of phosphate groups, -s-caseins are sensitive to calcium and precipitate
in the presence of calcium ions at a pH of 7.0. A charged oligosaccharide moiety and
possessing just one phosphoserine group set -casein apart from - and -caseins.
Whey protein comprises β-lactoglobulin and α-lactalbumins, proteose-peptones (partially
derived from hydrolysis of β-caseins) and small quantities of the blood-derived proteins,
serum albumin, and immunoglobulins. Whey proteins (WPN) are typical compact globular
9
proteins, with a relatively uniform sequence distribution of nonpolar, polar, and charged
residues (Kailasapathy, 2016).
Table 2: Protein Composition of Bovine Milk

Protein Components Weight (g/Kg)
Total protein 35.1
Total casein 28.6
Whey protein 6.1
α S1-Casein 11.5
α-S2-Casein 3.0
κ-Casein 3.4
γ-Casein 1.2
α-Lactoglobulin 1.2
β- Lactoglobulin 3.1
Serum albumin 0.4
Immunoglobulin 0.8
Proteose-peptones 1.0
Source: Kailasapathy (2016)

2.5.4. Lactose
O-D-galactopyranosyl-(1–4)-Dglucopyranose, or lactose, is the primary sugar in milk and
makes for 4.2–0.5% of the milk's weight. The two sugars that make up lactose, a
disaccharide, are -D-glucose (or -D-glucose) and -D-galactose. The C1 atom of glucose can
readily transition from the α- to the β-form via aldehydes form; this process is known as
mutarotation and will finally result in equilibrium. At 20 C, -lactose and -lactose have
different specific rotations to polarized light, which are +89.4 or +35.0, respectively. An
aqueous lactose solution with an optical rotation of +55.7 at 20 C includes 37.3% α-lactose
and 62.0.7% β-lactose (Huppertz, 2009).
The equilibrium ratio for lactose at 20 C is 1/1.68, and it occurs in both α- and β-forms.
Either of the two types of lactose is typically present in dairy products.
1. Crystalline forms: (a) α-hydrate or α-lactose monohydrate and (b) anhydrous β-lactose
2. Amorphous “glass” mixture of alpha and beta lactose
10
Aqueous lactose solution is concentrated to supersaturation to produce alpha hydrate, also
known as -lactose monohydrate, which is then crystallized at a moderate pace below 93.5
degrees Celsius. It is lactose in its stable solid state, and its crystals are stiff, less soluble,
and gritty in the mouth, like sand particles. Both the amount and size of crystals affect this
sandiness flaw. Crystals of anhydrous β-lactose are soft, more soluble, and sweet. The
rapid drying of a lactose solution produces the anhydrous lactose glass (also known as
amorphous non-crystalline glass), whose viscosity rises so quickly that crystallization is
prevented. This is highly hygroscopic, quickly absorbs moisture from the air, and gets
sticky, causing a problem (cakiness) in milk powder that has been stored (Mehta, 2015).
2.5.5. Enzymes in milk

These chemical catalysts promote a variety of intricate anabolic and catabolic processes. It
doesn't participate in chemical alterations. They are proteineous and colloidal, and they are
categorized based on the chemical changes they cause, such as hydrolyses (hydrolyzing
enzymes), oxidizing enzymes (enzymes), and reductases (reducing enzymes). They can be
categorized as proteases (which split proteins), lipases (which split fats), amylases (which
split starches), etc. depending on the substrate that they work with. Heat, light, and pH
fluctuations are all potentially harmful to enzymes. Coenzymes and anti-enzymes are
substances that increase or decrease the activity of an enzyme, respectively. A variety of
enzymes are produced in milk as it is drawn from beneath itself, while others enter as a
result of bacterial contamination. Fresh milk contains peroxidase, catalase, reductase,
phosphatase, and lipase, while other enzymes enter due to bacterial contamination (Walstra
et al., 1999).
2.5.6. Gases in milk

When milk is drawn from the cow's udder, it contains dissolved gases up to 8% by volume,
the majority of which is carbon dioxide. However, when milk is exposed to air, its volume
drops by about 0.6%. This volume change is not only quantitative, but also qualitative.
Carbon dioxide is expelled, whereas oxygen and nitrogen are gained. The oxidation of fat
and ascorbic acid is caused by dissolved oxygen. A combination of heat and vacuum
treatment can help to reduce it (Walstra et al., 1999).
2.6. Flavor Component

The main flavor compounds in milk are lactose and dissolved salts, which produce a sweet
and salty taste, respectively. If the CI/lactose ratio is high, as in mastitis milk, the sweet
11
flavor prevails, while the salty flavor prevalent. Other compounds responsible for the
distinctive flavor of fresh milk include dimethyl sulphide, diacetyl, 2-methyl butanol-1, and
some aldehydes. Fresh milk may contain off-flavors from the feed. The compounds
responsible for the off-flavors enter the milk via the cow, the air, or both. Examples are
clover and garlic flavors. When a cow is in ketosis, such as from a protein-deficient diet,
milk contains higher concentrations of ketones (including acetone). As a result, the milk
has a distinctive cow flavor. If such flavor compounds are hydrophilic, vacuum heating
may remove some of them.
Milk spoilage, particularly microbial spoilage, can result in flavor defects such as acid,
unclean, fruity, ester, malty/burnt, phenolic, bitter, rancid flavors, and so on. Lipolysis is an
enzymatic spoilage. A cardboard flavor occurs in milk as a result of auto oxidation of
phospholipids; it can be detected in skim milk as well. Plasma phospholipids appear to
oxidize more easily. This can easily result in a metallic flavor in sour-cream buttermilk if
the defect is weak, and a sharp (pungent) flavor if the defect is strong. Light can also
include flavor defects in milk. The tallowy flavor developed gradually as a result of light
autoxidation. However, when milk is exposed to light, additional flavor compounds can
form. Riboflavin is required for the development of this "sunlight flavor" (vitamin B2).
More intense heat treatments, such as 80-100˚C for 20s, result in "cooked flavor," which is
primarily caused by 𝐻2S. Maltol, furanone compounds (formed from lactose), and aliphatic
methyl ketones and lactones are the main "sterilization flavors" of high-temperature treated
milk (Walstra et al., 1999).
2.7. Minor component

Among the trace elements found in milk, zinc has the highest concentration (3 mg/kg of
milk), while others have lower concentrations. Al, As, Ba, B, Br, Cd, Cs, Cr, Co, F, I, Fe,
Pb, Li, Mn, Hg, Mo, Ni, Rb, Se, Si, Ag, Sr, Sn, Ti, V are the others. These are naturally
occurring components of milk. Some of these elements can be concentrated in milk by
increasing their level in the cow's feeding ration. As a result, their concentration in milk
can vary greatly. Se, for example, can range from 4 to 200 g/kg of milk (Walstra et al.,
1999).
2.7.1 Vitamin
Table 3: Vitamins content in fresh milk
Vitamins mg per 100 ml Range
12
Vitamin A 1.59 136-176
Vitamin D 2.21 0-10.9
Carotenoids 0.03 0.025-0.60
Vitamin E 0.1 0.02-0.18
Vitamin K 0.00467 0.0-0.0160
Vitamin C 2.09 1.57-2.75
Biotin 0.003 0.0012-0.0060
Chlorine 13.7 4.3-28.5
Folacin 0.0059 0.0038-0.0090
Myo-inositol(total) 11 6.0-18.0
Niacin 0.09 0.03-0.20
Pantothenic acid 0.34 0.26-0.49
Riboflavin 0.17 0.08-0.26
Thiamine 0.04 0.02-0.08
Vitamin B-6 0.06 0.02-0.08
Vitamin B-12 0.00042 0.00024-0.00074
Source: Walstra et al (1999)
2.8. Effect of Heat on Milk Proteins
Casein micelles are extremely stable at temperatures up to 140 °C. In contrast, WPN are
relatively heat-labile, with extensive denaturation occurring at 80 °C. Because of its one
free sulfhydryl group, β-lactoglobulin is more heat labile than α-lactalbumin, allowing the
initiation of autocatalytic disulfide exchange reactions (Kailasapathy, 2016).
2.9. Flavored milk among children

Children are drinking less milk, and one of the main reasons for this is an increase in soft
drink consumption. Flavored milk is a nutritious alternative to plain milk that can help
children consume more dairy. Furthermore, children who consume flavored milk have
higher intakes of vitamin A, calcium, phosphorus, magnesium, potassium, and saturated fat
(Pelsmaeker et al., 2014). Nonetheless, because of its unique nutritional composition, milk
is an important part of a healthy diet for children. Milk and dairy products are high in
calcium and thus beneficial for bone health and hypertension prevention, among other
things (MacDonald, 2008). Children and adolescents who consume flavored milk have
higher calcium intakes than non-flavored milk consumers (Johnson et al., 2002).
13
When deciding what to eat, children place a high value on the taste and sensory properties
of food products. Personal taste and liking preferences are important determinants of food
choice in children. Dislike of the taste is mentioned as one of the major reasons why
children do not consume fruit and vegetables. Based on a sample of Swedish children, Berg
et al (1999) conducted research into food choices using the examples of plain milk and
bread. Children's attitudes toward consumption were influenced by their beliefs about the
health and taste aspects of these products. Little is known about children's perceptions of
the health and taste benefits of flavored milk versus plain milk. As a result, this study
investigates whether children value taste over health when drinking plain or flavored milk.
Children are exposed to food promotion on a daily basis, and food advertisements influence
which foods children consume. Food promotion can be found everywhere, from the corner
store to television and the Internet. Food marketers target children because of their
influence on their parents' purchases and because of their future role as food. Taking into
account that food advertisements benefit the entire product category, it is hypothesized that
children will consume more flavored milk if their brand awareness is higher (Pelsmaeker et
al., 2014).
2.10. General flow chart for the preparation of flavored milk (Source: DDC)
Receiving milk
Standardization (3% fat & 8% SNF)
Pre-heating (35-40˚C)
Addition of color, flavor and Stabilizer
Homogenization (175 kg/𝑐𝑚2)
14
Filling and capping
Sterilization (120˚C for 30 minutes)
Cooling (Room temperature)
Storage (Room temperature)
Figure 1: Chart of preparation of flavored milk
2.11. Nutrient contribution of flavored milk

Flavored milks, like unflavored milks, are nutrient dense foods with a high nutrient-
toenergy ratio. Flavored milks have an excellent nutritional profile, including significant
amounts of high-quality protein, calcium, riboflavin, magnesium, phosphorus, niacin
equivalents, vitamin B12, vitamin A, and, when added, vitamin D. A comparison of the
nutrient content of chocolate milk and unflavored milk at various fat levels reveals that
most nutrients are present in comparable amounts.
Many population groups, according to government data, do not meet the National Academy
of Sciences' daily calcium recommendations. For example, 70% of preteen girls and 60%
of preteen boys aged 6 to 11, as well as nearly 90% of teenage girls and nearly 70% of
teenage boys aged 2 to 9, do not meet their calcium requirements. Getting enough calcium
throughout life helps to reduce the risk of bone fractures in childhood and adolescence, as
well as osteoporosis later in life.
Recognizing children's low calcium intake, the American Academy of Pediatrics issued a
policy statement urging pediatricians to recommend milk, cheese, yogurt, and other
calcium-rich foods for their children's daily diets in order to help build bone mass and
prevent rickets. Because eating habits formed during childhood tend to be followed
throughout life, this policy statement recommends that children meet their calcium needs
through food first. Other experts agree that eating calcium-rich foods is the best way to
meet calcium requirements. Chocolate and other flavored milks contain naturally high
levels of calcium. Dairy foods, such as chocolate and other flavored milks, provide
15
essential nutrients that are important for health, and their consumption improves the diet's
overall nutritional adequacy.
Vitamin D-fortified flavored milk, as well as unflavored milk, is an excellent source of the
vitamin. This vitamin improves calcium absorption and helps to prevent rickets, a
boneweakening disease caused by a lack of vitamin D. Although rickets was nearly
eradicated in the United States by the 1960s as a result of vitamin D fortification of cow's
milk, this disease is making a comeback in young children It has been demonstrated that
providing flavored milk as part of school meal programs such as the National School
Lunch Program (NSLP) and the School Breakfast Program (SBP) increases milk and
nutrient intake. Providing low and nonfat milks, including flavored milks, in school
vending machines is a relatively new approach to increasing students' milk and nutrient
intake. Although chocolate milk has been discovered to be the most popular flavor, tastes
vary geographically (NDL, 2016).
2.11.1. Total milk intake, sugar intake, and nutrient contribution from flavored milk
Several studies have measured and reported a link between flavored milk consumption and
total milk consumption among various groups of milk consumers. According to these
studies, flavored milk consumers had higher total milk intakes than plain milk consumers,
higher total milk intakes than non-consumers of flavored milk, and higher total milk
intakes than plain milk drinkers; additionally, flavored milk consumers of all ages and both
genders consumed more total milk than exclusively plain milk consumers.
Furthermore, data from urban families who purchased flavored milk revealed that per
capita milk consumption in families who purchased chocolate milk was 15.4%-21.0.7%
higher than in families who did not, regardless of family income. According to the
research, people who drink flavored milk consume more milk overall. Higher milk
consumption can be attributed to increased consumption of flavored milk as well as the
sum of flavored milk consumption plus plain milk consumption, because flavored milk
consumers do not always consume flavored milk exclusively In terms of micronutrient
intake, flavored milk consumption was associated with higher energy-adjusted calcium
intake,10 accounting for 22% of total calcium intake,95 and certain age groups of flavored
milk consumers reported higher calcium intake than plain milk consumers. 12,65 In a
representative sample of Australian children, 60% of children aged 9 to 16 were not
meeting their daily calcium targets. Fayet et al. (2013) reported that flavored milk drinkers
16
were 1.7 times more likely to meet their calcium targets than plain milk drinkers. Effect on
nutrient intake.
The authors investigated the nutrient density of plain milk and flavored milk and found that
both types of milk provided a higher percentage of six nutrients (calcium, vitamin A,
vitamin D, magnesium, phosphorus, and potassium) than calories (energy). As a result, the
percentages of six essential nutrients provided by plain and flavored milk consumption
were higher than the percentage contribution of added sugar.
Overall, nutrient contribution research indicates that flavored milk consumption is

associated with higher calcium intakes, a higher likelihood of meeting calcium targets, and
similar or higher intakes of other micronutrients. In terms of energy intake, some
subgroups report a higher contribution of energy from flavored milk than plain milk, while
others report no difference or a lower contribution of energy from flavored milk than plain
milk (Moore, 2015).
2.12. Tulsi
Tulsi contains several Phyto-constituents such as cardinene, cubenol, borneol, linoleic
acid, orientin, linolenic acid, steric acid, oleic acid, palmitric acid, eugenol, vallinin,
vicenin, vitexin, vllinin acid, circineol, gallic Acid, vitamin A, vitamin C, phosphorus and
iron due to which it possess multifarious medicinal properties such as antiviral, antifungal,
antibacterial, antimalarial, anthelmintic, anti-oxidant, anti-cataract, anti-inflammatory,
chemo preventive, radio protective, hepato-protective, neuro-protective, cardio-protective,
anti-diabetic, anti-hypercholesterolemia, anti-hypertensive, anti-carcinogenic, analgesic,
anti-pyretic, anti-allergic, immunomodulatory, anti-asthmatic, diaphoretic, anti-thyroid,
anti-fertility, anti-ulcer, anti-emetic, Anti-spasmodic, anti-arthritic, adaptogenic properties
(Parle et al., 2012).
Tulsi's chemical composition is extremely complex, containing numerous nutrients and

other biologically active compounds. These constituents change dramatically with time,
cultivation process, and storage. Because the nutritional and pharmacological properties of
the whole herb in its natural form, as it has traditionally been used, result from the
synergistic interaction of many different active phytochemicals, the overall effects of Tulsi
cannot be fully duplicated with isolated compounds or extracts. Tulsi active component
standardization has been difficult due to its inherent botanical and biochemical complexity.
However, eugenol (an essential oil) and ursolic acid are two of the most well-known active
17
components that have been identified and extracted. Many scientific studies have been
conducted to determine the pharmacological effects of steam distilled, petroleum ether, and
benzene extracts of various parts of the Tulsi plant and their active ingredient eugenol on
various systems such as the immune system, reproductive system, central nervous system,
cardiovascular system, gastric system, urinary system, and blood. Tulsi is commonly used
as a vitalizer and to increase physical endurance.
Tulsi has traditionally been consumed in a variety of forms, including cold, hot, or dried
leaf tea (herbal teas), powdered leaf, alcohol tinctures, and oil (ghee) preparations, as well
as seed, root, and stem formulations, which are used both systemically and topically. In
human clinical studies and animal experiments, isolated compounds are administered by
injection in addition to various extracts (Mohan et al., 2010).
2.12.1. Health benefits of Tulsi 2.12.2. Stress resilience

When tested against mice and rats, the plant Ocimum sanctum was found to have
adaptogenic properties. Basil leaves improve coping ability, adaptability to stressful
environments, and provide significant stress protection (adaptogen). Even healthy people
can benefit from chewing 12 basil leaves twice a day to reduce stress (Mohan et al., 2010).
2.12.3. Common cold and fever

Tender Tulsi leaves boiled with tea act as a preventive medication against malaria and
dengue fever, which are common during the rainy season. An Ayurvedic preparation
containing Ocimum sanctum, Piper nigram, and Curcuma longa has been shown to have
antimalarial activity against Plasmodium vivax and to be highly effective against
Plasmodium falciparum. This preparation has been found to alleviate the clinical symptoms
of malaria caused by these species. In malarial fever, a decoction prepared from the roots
of the Tulsi plant is used as a diaphoretic. Tulsi is an important ingredient in many
Ayurvedic cough syrups and expectorants. It aids in the mobilization of mucus in cases of
bronchitis and asthma (Mohan et al., 2010).
2.12.4. Antibiotic property

Essential oils extracted from the leaves of Ocimum sanctum L. were found to inhibit the
growth of E. coli, Bacillus anthracis, and Pseudomonas aeruginosa in vitro, demonstrating
antibacterial activity. Ocimum sanctum also has antifungal activity against Aspergillus
Niger, and an aqueous extract of it has been shown to be effective in patients with viral
encephalitis. Tulsi leaf paste has been found to be very effective in the treatment of ring
18
worm infections. Tulsi has powerful natural antibacterial, antiviral, and antifungal
properties, making it useful in the treatment of many serious systemic diseases as well as
localized infections (Mohan et al., 2010).
2.12.5. Hepatoprotective, Reno protective and Neuroprotective activities

Ocimum sanctum leaf extract was found to be hepatoprotective against the hepatotoxic
paracetamol by significantly lowering serum enzymes aspartate aminotransferase (AST),
alanine aminotransferase (ALT), and alkaline phosphatase (ALP) in rats, as well as a
significant reduction in fatty liver degeneration on histopathological examination. When
compared to the gentamicin-only treated group, administration of a combination of. In
albino rats, Ocimum sanctum leaf extract prevents stress-induced dendritic deficiency in
hippocampal neurons. Tulsi appears to have a very high safety margin and a very low toxic
profile, providing safe beneficial effects at low doses with no undesirable side effects,
according to research. In Ayurvedic medicine, Tulsi has therapeutic potential either alone
or in combination with other plants in various clinical conditions like eye disorders
(glaucoma, cataract, & chronic conjunctivitis), catalepsy, snake and scorpion bites etc.
(Mohan et al., 2010).
2.13. Types of pasteurization

Table 4: Types of pasteurization
Temperature Time Pasteurization type
63°C 30 minutes Vat Pasteurization
72°C 15 seconds High temperature short time
Pasteurization (HTST)
89°C 1.0 seconds Higher- Heat Shorter Time
(HHST)
(HHST)
(HHST)
(HHST)
(HHST)
19
138°C 2.0 seconds Ultra-Pasteurization (UP)
Source: Watts (2016)
2.13.1. Micro-organisms killed by pasteurization

Streptococci, Lactobacilli, Micro bacteria, Coliforms, Micrococci Coliforms, gas producers
Clostridium butyricum, Torula cremoris Ropy or stringy fermentation Alcaligenes
viscolactis, Enterobacter aerogenes Bacillus spp., Pseudomonas spp., Proteus spp.
Streptococcus liquefaciens Lipolytic organisms Pseudomonas fluorescens, Achromobactor
lipolyticum, Candida lipolytica, Penicillium spp (Watts, 2016).
2.14 Ingredients
2.14.1. Tulsi
Tulsi was chosen because it reduces the risk of heart disease, as well as relieves fever,
headache, and sore throat. Milk is one of the most important carriers that has been used
successfully to deliver phytochemicals found in traditional herbs. (Primarily polyphenols)
for specific health benefits in traditional Indian medical science (primarily polyphenols) for
specific health benefits in traditional Indian medical science (Sawale et al., 2015).
Table 5:Proximate composition of Tulsi juice

Constituents Tulsi juice Percent
Moisture 96.13
Dry Matter (TS) 3.87
Fat 0.62
Titrable Acidity 0.17
Specific gravity 1.0096
Source: Hingne (2021)
2.14.3. Milk
Milk has been mentioned for centuries as a wonder drink with magical health benefits.
Despite the fact that milk is a complete food in and of itself, a new category of drinks
known as Nutraceuticals has emerged (ÖZER et al., 2010).
Cow's milk is an extremely nutritious food for a growing child. It has a well-balanced fat,
carbohydrate, and first-rate protein composition. It is high in mineral salts, particularly
calcium and phosphorus, and contains essential vitamins (Wilson, 1943). Approximately
Casein accounts for 80% of milk proteins (as1, as2-, b- and k casein). Casein molecules are
20
precursors to several bioactive peptides that have antimicrobial activity as well as vector
properties in the body for calcium, zinc, copper, iron, and phosphate ions (Ebringer et al.,
2008). Milk is a source of macro- and micronutrients, as well as a number of active
compounds that play an important role in nutrition and health protection.
Table 6:Chemical composition of cow milk

Total solids 11.36
Protein 2.82
Fat 3.42
Ash 0.65
Lactose 4.47
Source: Sanz et al (2008)
2.15. Packaging
One of the benefits of glass is that it is chemically inert. Because glass is impermeable to
gases and water, it is ideal for long-term storage of foods or beverages that are susceptible
to spoilage due to oxygen and moisture exposure. Its transparency is useful in situations
where product visibility is critical. Glass can be recycled by simply heating it until it is
molten and then reforming it into a container with no loss of strength or quality or the
production of harmful by-products. Glass's smooth, hard, inert surface makes it relatively
easy to clean, and its heat resistance makes it simple to sterilize. There is also the cost of
transporting empty bottles back to the packer filler, as well as the capital investment
required for cleaning and inspection equipment. Thus, glass packaging can be used for
flavored milk packaging (Grayhurst, 2012).
Part III Materials and Methods

3.1. Materials
3.1.1. Raw material collection
1.Milk
Standardized milk (3%fat and 8% SNF) was collected from market of Jawalakhel produced
by Dairy Development Committee (DDC).
21
2.Tulsi
Tulsi was collected from Lubhu and Naxal area and oven dried at 50˚C for 4 hours.
3.1.2. Laboratory instruments and chemicals

3.1.2.1 List of equipment used
The equipment used were available in Lalitpur Valley College. The list of equipment used
are shown in Appendix A
3.1.2.2. List of chemicals used

The chemicals used were available in Lalitpur Valley College. The list of equipment is
shown in Appendix A
3.1.3. Glassware
Pippete, Burette, Measuring cylinder, beaker, volumetric flask, Test tubes, Conical flask,
Funnel, Wash bottle, Glass rod, Petri plate, Silica crucible, Whatman filter paper.
3.1.4. Laboratory facilities

The glassware and equipment were used as per available in Lalitpur Valley College,
Jawlakhel.
3.2. Analysis of Tulsi powder

3.2.1. Determination of Moisture Content
The moisture content of sample was determined by weight loss during heating in the
thermostatically controlled hot air oven at 105±5°C as described in AOAC (2005). This
method consists of measuring the weight loss by foods due to evaporation of water. 3g
Tulsi was spread in a flat-bottom glass dish and dried in thermostatically controlled hot air
oven at 105±5°C and cooled in desiccators. The moisture content was calculated as:
Moisture content % = 𝐼𝑛𝑡𝑖𝑎𝑙 𝑤𝑡−𝐹𝑖𝑛𝑎𝑙 𝑤𝑡 × 100%

𝐼𝑛𝑡𝑖𝑎𝑙 𝑤𝑡.
3.2.2. Determination of protein

The crude protein of sample was determined by estimating nitrogen content in the Sample
By micro Kjeldahl method as described by Ranganna (2007). In Kjeldhal method nitrogen
content was established by the determination of the amount of reduced nitrogen present in
the sample, The nitrogenous compounds were converted into ammonium sulphate by
boiling with concentrated sulphuric acid. The ammonium sulphate formed was
22
decomposed with an alkali (NaOH), and the ammonium liberated was absorbed in excess
of neutral boric acid and then titrated with standard acid.
3.2.3. Determination of fat

The fat content was determined by using Soxhlet extraction apparatus using petroleum
ether (boiling point: 40-60°C) as a solvent as described in Ranganna (2007). In brief,
solvent soluble materials in Tulsi (3g) were extracted from an oven-dried sample in a
Soxhlet extraction process. The recycling was done for a number of times until extraction
was completed. The solvent was evaporated and the residue was weighted.
3.2.4. Determination of Crude fiber

Crude fiber was determined by using chemical process, the sample was treated with boiling
dilute sulphuric acid, boiling sodium hydroxide and then with alcohol as standard method
of Ranganna (2007)
3.2.5. Determination of total ash

Ashing of the samples were carried out in a muffle furnace at a a temperature not
exceeding 525°C for 5-6 hours, as described in Ranganna (2007). The total ash was
calculated by incinerating all the organic matter of the food sample. Tulsi (3g) was used.
Ashing was done in muffle furnace not more than 525°C for 5-6 hours. The crucible was
cooled and weighed in desiccators. The difference in weights gives the total ash content
and was expressed as percentage:
𝐴𝑠ℎ
Ash% (wet basis) = ×100
𝑆𝑎𝑚𝑝𝑙𝑒(𝑔)
3.2.6. Determination of carbohydrate

Carbohydrate content was determined by difference method as described in Ranganna
2007)
Total carbohydrate (%) = 100 – (moisture + curde protein + crude fat + crude fiber + total
ash) %
3.2.7. Determination of acidity of Herbal milk

The determination of acidity in sample involves preparation of aqueous suspension solution
of the food and titration with standard NaOH using phenolphthalein indicator. The result is
expressed as dominant acid. 10 ml sample was pipetted out in a conical flask. The sample
was titrated with 0.1 NaOH using 2 drops of phenolphthalein indicator to a persistent pink
23
end point. The acidity was calculated in terms of lactic acid anhydrous as follows: (AOAC,
2005)
Total acid% (TA) = 𝑣𝑜𝑙 𝑜𝑓 𝑡𝑖𝑡𝑟𝑎𝑡𝑒×𝑊𝑡 𝑜𝑓 𝑐𝑖𝑡𝑟𝑖𝑐 𝑎𝑐𝑖𝑑 × 100

𝑣𝑜𝑙 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒×1000
3.2.8. Determination of pH
The measurement of pH is carried out by using pH indicators (available as solution or
paper strip) or by electronic pH meters. In very sensitive and accurately calibrated pH
meters, a precision of up to ±0.01 units can be obtained. When pH meter is inserted in the
milk or juice an electrical circuit is established due to protons (H+). The instrument is
calibrated to directly reflect the negative logarithm of hydrogen ion concentration in the
mol/liter. When pH indicators are used, the constituent chemical respond to hydrogen ion
concentration by changing their color that corresponds to the pH unit. The pH meter was
calibrated with buffers of pH 4.0 and 7.0. It was then washed with distilled water several
times to remove buffer. The probe of pH meter was dipped in Sample And reading was
taken (Horwitz et al., 2010)
3.3. Analysis of Milk

3.3.1. Acidity
Acidity present in milk was determined by the acid titration method, titrating against 0.1N
NaOH with reference to the Laboratory Handbook for Dairy Industries NDDB (2001). It is
expressed in terms of lactic acid.
3.3.2. Fat
Fat content was determined by Gerber’s method with reference to the Laboratory
Handbook for Dairy Industries NDDB (2001). 10ml of sulphuric acid was transferred into
a butyrometer. 10.75 ml of the milk sample was transferred carefully in the same
butyrometer and 1 ml of amyl alcohol was transferred to it. The butyrometer was closed
with stopper and shaken carefully until the contents are thoroughly mixed and transferred
into a water bath of temperature 65°C and left for not less than 5 min. Then, it was
centrifuged for 5 min and again transferred into a water bath of temperature 65°C. Then,
fat content was read by inverting stem upward.
3.3.3. Solid not Fat Content (SNF)

SNF content of milk was determined by using lactometer reading method with reference to
the Laboratory Handbook for Dairy Industries NDDB (2001). Milk was poured into a
24
lactometer jar without the formation of air bubbles. Lactometer was gently inserted in a
milk jar and allowed to remain steady in the milk. Reading was taken within 30 seconds
and the temperature of the milk was noted. The corrected lactometer reading (CLR) was
obtained by applying the correction factor from the temperature correction chart and SNF
was calculated by using formula;
SNF% = CLR/4 + 0.25F + 0.5
Where, CLR= Corrected Lactometer Reading
3.3.4. pH
The pH of the milk was determined by using the digital pH meter with reference to the
NDDB (2001). The milk sample was poured into a clean and dry beaker. The pH meter
was dipped into the Sample And reading was taken.
3.3.5. Protein
Protein Content was determined by the formal titration method (Crowhurst, 1956). 20
grams of sample were weighed into a 100ml beaker and 0.8 ml of saturated potassium
oxalate was added. The sample was mixed and few drops of phenolphthalein was added. It
was titrated with standard 0.1 N NaOH until pink end point. 4 ml of 40% formaldehyde
were added and sample was titrated back to same endpoint. The net formal titration was the
milliliters of NaOH required to titrate the Sample After formaldehyde was added. The
protein content was calculated by using the formula;
P= (ml ×N×9.04)-0.14.
P is percent total protein; ml is net milliliters of NaOH for the formal titration; N is
normality; 9.04 is the product of the equivalent weight of NaOH×0.226 and 0.14 is the
intercept.
3.3.6. Moisture
The moisture content of milk was determined by using a hot air oven as per described by
Ranganna (2007).
25
3.4. Preparation of Herbal milk
Receiving milk
Platform test
Pre-heating(35˚C)
Mixing of Tulsi powder at 0.5%, 0.6% and 0.7% with milk
Cooling (Room temperature)
Agitation
Filling and capping
Sterilizing(121˚C/30min)
Cooling (15-20˚C)
Storage in refrigerated condition (4˚C)
Figure 2: Flow chart for preparation of herbal milk using Tulsi
The standardized milk from DDC was taken for the preparation of Herbal milk. Platform
test of milk was conducted. The milk was pre heated at 35°C and tulsi powder was added
to the milk at different concentrations. The prepared samples were cooled followed by
26
agitation. Now, the bottles were filled and crown capping was done. In bottle sterilization
was carried out at 121˚C for 30 min. The bottles were cooled and stored at refrigerated
condition. The milk was prepared and for each of the four treatments, 1 liter of
standardized milk was taken. The Tulsi powder were added at 0.5%, 0.6% and 0.7%.
3.5. Product Formulation

The experiment was conducted with four treatments. The blend of different proportions of
Tulsi powder and milk were used as treatment. Previously, milk sample was prepared with
0.4%TP. This didn’t provide satisfactory results in sensory. Hence, samples were prepared
with 0.5%TP. 0.6%TP and 0.7%TP. The flavored milk with Tulsi was previously prepared
by Gaur et al (2019) with the use of Tulsi, turmeric and ginger. Here, Tulsi was used in the
form of liquid at 3%. Therefore, a modification was made with Tulsi powder with different
percentage for the preparation of our flavored milk. The treatments used in experiment
were:
Table 7: Formulation of Tulsi incorporated milk

Sample Part of milk (ml) Part of Tulsi powder (mg)
A 1000 0
B 1000 5000
C 1000 6000
D 1000 7000
3.6. Proximate Analysis of Tulsi powder incorporated milk

3.6.1. Moisture content
Moisture content of the sample was determined by using hot air oven method as standard
method of AOAC ( 2005).
Moisture content = 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑡−𝑓𝑖𝑛𝑎𝑙 𝑤𝑡 ×100

𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑡
3.6.2. Fat content

Fat content of the Herbal milk was determined by Gerber’s method according to NDDB
(2001). 10ml of a well-mixed sample was transferred in the butyrometer. 10ml of a
wellmixed sample was transferred carefully in the same butyrometer and 2ml of amyl
alcohol was transferred to it. The butyrometer was closed with stopper and shook carefully
27
until contents are thoroughly mixed and transferred into a water bath of temperature 65°C
and left for not less than 5 min. Then, it was centrifuged for 5 min and again transferred
into a water bath of temperature 65°C. Then, the fat column on butyrometer was adjusted
and reading was taken.
3.6.3. Protein content

Protein content of the Herbal milk was determined by formal titration method NDDB
(2001). 20 grams of sample was weighed into a 100ml beaker and 0.8 ml of saturated
potassium oxalate was added. The sample was mixed ad few drops of phenolphthalein was
added. It was then titrated with standard 0.1N NaOH until pink end point. 4ml of
40%formaldehyde were added and sample was titrated back to same end point. The net
formal titration was the milli liters of NaOH required to titrate the Sample After
formaldehyde was added.
3.6.4. Ash content

Ash content was determined by muffle furnaces according to Ranganna (2005). 5g of
weighted sample in silica crucible was charred in a hot plate till no smoke raise from it and
finally, ashing was done in muffle furnace at 550°C to the constant weight. The difference
in weight was total ash content remaining in crucible, under standardized conditions
(Ranganna, 2005)
𝐴𝑠ℎ
Ash content = × 100
𝑆𝑎𝑚𝑝𝑙𝑒(𝑔)
3.6.5. Carbohydrate
Total carbohydrate content in Herbal milk was determined by difference method as per
Ranganna (2005).
3.6.6. Specific gravity
Specific gravity was determined according to AOAC (2005). The milk sample was heated
to 40˚C and held for 5 minutes. The milk sample was mixed thoroughly, not vigorously to
avoid any air bubbles. The milk was placed in a cylinder sufficiently until overflow.
Lactometer was gently inserted in the cylinder and let to equilibrium. The lactometer and
temperature reading were taken. Specific gravity= (L/1000) +1
28
3.7. Microbial analysis
3.7.1. Preparation of Sample Dilution
A representative sample of Herbal milk (1 gm) was diluted 1:10 with sterile distilled water,
diluted serially (10) and one milliliter from each. The selected dilution after thoroughly
mixing were carefully transferred into petri dishes using sterile pipettes (NDDB, 2001)
3.7.2. Enumeration of total bacteria
For each dilution, 1ml sample was aseptically transferred into sterile petri plates in
triplicate, followed by adding 10-12 ml of standard plate count agar at 45-46°C. The petri
dishes were covered and mixed by gentle rotation then allowed to solidify. The plates were
inverted and incubated at 37°C for 24 hours.
The developed colonies were counted, plates with 25-250 or less than 25 colonies were
selected. The average number of colonies in each dilution was multiplied by the reciprocal
of the dilution factor and recorded as colony-forming units/gm.
3.7.3 Coliform analysis of Herbal milk
Coliform analysis of the Herbal milk is determined by pour plate technique on violet red
vile agar (VRBA) technique as per the procedure of NDDB ( 2001)
From each dilution, 1 ml sample was aseptically transferred into sterile petri dishes
followed by addition of 10-15 ml Violet Red Bile Agar medium at 44-46°C. The contents
were allowed to solidify (5-10 minutes) on a leveled surface. The plates were then inverted
and incubated at 37°C for 24 hours.
3.7.4. Enumeration of yeast and molds in Herbal milk
Yeast and molds were enumerated by a pour plate method using PDA media by incubating
at 25°C for 5 days as per the procedure of Laboratory Handbook for Dairy Industries
NDDB (2001).
For each dilution, 1 ml sample was aseptically transferred into sterile petri plated in
triplicate; molten Potato Dextrose Agar was poured at 45°C to each inoculated dish and
mixed well. Tartaric acid was added just before pouring into inoculated dishes. 1 ml of
sterile quarter Ringer’s solution was inoculated as “control”. The petri plates and “control”
were inverted and incubated at 25°C for 48 hours. The colonies were counted selecting the
dishes with 10-150 colonies.
29
3.8. Sensory Analysis of Herbal milk
The 9-point hedonic rating forms were developed to evaluate the milk of different
treatments by using the organoleptic test tools. The freshly prepared sample of the milk
were subjected to sensory evaluation. All four treatments were coded first and each
treatment was offered 12 panelists.
The samples were evaluated by using the hedonic test for their mouthfeel, color, odor, taste
and overall acceptability. Randomization for the sample was carried out by providing a
different 3-digit number for every sample for every panelist. A nine-point hedonic scale
adopted by Peryam (1957) and Ranganna (1992) were used for evaluation. The samples of
sensory evaluation card are presents in Appendix B. Intensity scores of each attribute for
different treatments were analyzed statistically with analysis of variance (ANOVA) method
by using SPSS.
3.9. Cost evaluation
A partial budgeting approach was implied to estimate the simple cost ratio for the milk
manufactured from different treatments. Some cost of milk, water, electricity cost etc. were
taken into consideration while calculating the cost of milk. Milk was bought in Nrs. 90 per
liter. The calculations were done for a liter of milk and the cost of production of 1 liter of
Herbal milk was calculated.
3.10. Statistical Analysis
IBM SPSS Statistics 25.0 and Microsoft Office Excel 2013 were used for the data analysis
and data interpretation. The data analyzed using Analysis of variance (ANNOVA).
Average was compared using post hoc multiple comparison LSD (List Significance
Difference) test. Significance differences were determined at p <0.05 level. The results
were expressed as mean expressed as mean value ± standard deviation of triplicate
samples.
Part IV
Result and Discussion

The research was carried out for the formulation and quality evaluation of herbal milk.
Four different treatments viz, Sample A, Sample B, Sample C and Sample D were prepared
using the varying composition of Tulsi powder at 0%, 0.5%, 0.6% and 0.7% respectively
and packed in glass bottles. All samples were analyzed for physicochemical and
30
microbiological tests. The test samples were also analyzed for sensory test and further
stored under refrigerated temperature (4±1˚C) for 14 days and quality was evaluated.
4.1. Proximate composition of Tulsi powder
Table 8:Proximate composition of Tulsi, (Mean± SD)

SN Parameters Observation
1 Moisture 10.46±1.16
2 Crude Protein 15.92±0.32
3 Crude Fat 1.73±0.04
5 Total Ash 10.37±1.08
6 AIA 0.76 ±0.07
7 Crude Fiber 6.01±0.20
8 Calorific Value 298.25±2.50
9 Carbohydrate 54.75±0.39
*Values are mean ±SD of triplicate sample
4.1.1. Moisture Content

In the present study, moisture content was found to be 10.46±1.16% which is slightly less
than that to that of Akah et al (2017) where it was reported that the moisture content of
oven dried Tulsi at 50°c as 11.21 ±0.01. Okunlola et al (2018) reported moisture content of
6.5% in O. gratissimum. This may be due to the difference in variety of Tulsi. The
variation could be due to different climatic conditions of growth and the type of Tulsi used
to carry out the experiment.
4.1.2. Crude Protein

The crude protein content of Tulsi was found to be 15.92±0.32 which is slightly less than
the value of 18.20±0.00 reported by Akah et al (2017). The protein content varied due to
the difference in region of growth and type of Tulsi used to carry out the experiment.
Okunlol et al (2018) observed protein content of 14.73% in variety O. gratissimum which

is slightly less than our observation.
4.1.3. Crude Fat

The crude Fat content in Tulsi was found to be 1.73±0.04%. This amount was slightly
lower than the value 1.97±0.03% reported by Akah et al (2017). Similarly, Okunlola et al
(2018) observed fat content of 2.25% in variety O. gratissimun which is higher than our
31
observation. This might be due to climatic conditions, difference in variety, season,
temperature and other similar factors.
4.1.4. Crude Ash

Ash content was found to be 10.37±1.08% which is lower than Crude Ash 13.64±0.05% as
found in research by Akah et al (2017). The Ash content is a reflection of the amount of
minerals in a food. Munde et al (2018) showed ash content was 13.8 which is slightly
higher than our findings. Okunlola et al (2018) observed ash content of 2.25% which is
very lower than our observation. This might be due to climatic conditions, difference in
variety, season, temperature and other similar factors.
4.1.5. Crude Fiber

The Crude Fiber was found to be 6.01±0.20%. However, Akah et al (2017) reported a
higher value for Crude Fiber at 9.36±0.01%. Okunlola et al (2018) observed crude fiber
content of 5.95% in variety O. gratissimum which is lower than our observation. This
variation might be due to environmental factors such as; temperature, light, climate and the
variety of Tulsi used to carry out the experiment.
4.1.6. Carbohydrate
The carbohydrate was found to be 54.75±0.39 which was similar to the findings of Munde
et al (2018) where they found the carbohydrate content was 58.4. Akah et al (2017)
reported carbohydrate content of 45.61 which is very low than our finding. Okunlola et al
(2018) observed carbohydrate of 66.34% in variety O. gratissimum which is very high than
our observation. This might be due to climate, temperature, light and the variety if Tulsi
used to carry out the experiment.
32
4.3. Proximate analysis of Herbal milk 4.3.1.
Fat content
Figure 3: Fat content of herbal milk
Fat content
3.1
c
3
bc
2.9
b
2.8
2.7 a
2.6
2.5
2.4
2.3
A B C D
From the above result, the fat content of Sample D was significantly different from Sample
A, Sample B and Sample C (p<0.05). The minimum value for fat content (2.62±0.04) was
found to be in Sample D. Similarly, maximum value (2.93±0.06) was found in Sample A.
Sample A showed significant difference from Sample C and D. Here, Sample A and B
showed no significant difference. In the same way, Sample B and C were not significantly
different (p>0.05).
A similar observation was reported by Sayyad (2022) for fat percentage in flavored milk.
In the same way, Palthur et al (2014b) observed fat content of 2.16% which was lower than
our findings when milk was prepared by partial substitution of Ocimum sanctum powder.
Similarly, Trivedi et al (2014) observed that there was a marginal decline in fat content in
the experimental samples on addition of basil powder compared to control. Kumar et al
(2017) observed fat content of 1.42% in milk prepared by addition of wheat grass juice
which was lower than our findings.
33
4.3.2. Protein content
Figure 4: Protein content of herbal milk
Protein content
4.5
4 b
a a a
3.5
3
2.5
2
1.5
1
0.5
0
A B C D
The addition of herbal preparations had significant effect on the protein content of herbal
milk. Sample A was significantly different to Sample B, Sample C and Sample D (p<0.05).
Similarly, Sample B, Sample C and Sample D showed no significant difference (p>0.05).
The minimum value for protein content (3.25±0.06) was found in Sample D. Similarly,
maximum value (3.93±0.07) was found in Sample A.
Palthur et al (2014b) observed protein content of 3.45% which was similar to our findings
when milk was prepared by partial substitution of Ocimum sanctum powder. Trivedi et al
(2014) observed that there was a decline in protein content in the experimental samples on
addition of basil powder compared to control which is in accordance with our findings.
Palthur et al (2014a) observed protein content of 3.48% which was higher than our
findings when milk was prepared by using ginger juice. Chavan (2021) discovered the
protein content of 3.40% which was similar to our observations.
34
4.3.3. Ash content
Figure 5: Ash content of herbal milk
Ash content
0.8 c
0.7 b b
0.6 a
0.5
0.4
0.3
0.2
0.1
0
A B C D
The crude fiber content in herbal milk showed Sample A was significantly difference to
Sample B, Sample C and Sample D (p<0.05). The ash content ranged from (0.72±0.01) in
Sample A to (0.57±0.03) in Sample D. The minimum value for ash content in Herbal milk
was found in Sample D (0.57±0.03). Similarly, maximum (0.72±0.01) was found in
Sample A. Similarly, Sample B was significantly different to Sample A and D. However,
Sample B and C showed no significant difference (p>0.05).
These results were found similar to the result of Gaur et al (2019). In the same way, Palthur
et al (2014a) observed ash content of 0.67% which was lower than our findings when milk
was prepared by using ginger juice. Johri (2014) observed ash content of 0.78% which was
slightly higher than our findings when misthi doi prepared with Tulsi extract. Palthur et al
(2014b) observed ash content of 0.69% which was in accordance with our findings when
milk was prepared by partial substitution of Ocimum sanctum powder. Hingne (2021)
observed gradual decrease in ash content of flavored milk in comparison to ash content of
normal milk.
35
4.3.4. Specific gravity
Figure 6: Specific gravity of herbal milk
Specific gravity
1.1
1.08 c
bc
1.06 ab
1.04 a
1.02
1
0.98
0.96
A B C D
The addition of herbal preparations had significant effect (P<0.05) on the specific gravity
of herbal milk. The minimum value for specific gravity in herbal milk was found in Sample
D (1.02±0.01). Similarly, highest value for specific gravity was found in sample D
(1.07±0.01). Sample A showed no significant difference with Sample B. Similarly, Sample
C showed no significant difference with Sample D (p>0.05).
Palthur (2014b) observed specific gravity of 1.085 which was slightly higher than our
findings when milk was prepared by partial substitution of Ocimum sanctum powder.
Accordingly, Raiesi et al (2014) reported that the specific gravity of milk increases with
increase in the concentration of date palm juice which is not in accordance with our
findings. Priyanka & Chavan (2021) discovered the specific gravity to be 1.034 to 1.039
which was similar to our observation.
36
4.3.5. Lactose
Figure 7: Lactose of herbal milk
Lactose
5.3
5.25 a
5.2 b
5.15 ab
5.1 a
5.05
5
4.95
4.9
4.85
A B C D
The addition of herbal preparation on milk showed Sample B was significantly different to
Sample A and Sample D (p<0.05). However, Sample A, Sample C and Sample D showed
no significant difference (p>0.05). The minimum lactose content was found to be in
Sample A (5.04±0.03). Similarly, a maximum value (5.20±0.02) was found in Sample D.
Our findings were similar to that of Kailasapathy (2015).
37
4.3.6. Moisture content
Figure 8: Moisture content of herbal milk
Moisture
88
d
87.5 c
b
87
86.5 a
86
85.5
85
A B C D
LSD test for mean comparison of moisture showed that the moisture content of all samples
was significantly different from each other (p<0.05). The moisture content of herbal milk
ranged from 82.02% to 82.53%. This was dependent on the proportion of Tulsi powder
used. Sample A had the lowest moisture value (86.21±0.04) and Sample D had the highest
moisture value (87.45±0.03). Sample A was significantly different to Sample B, C and D.
Similarly, Sample B was significantly different to Sample A, C and D. In the same way,
Sample C showed significant difference to Sample A, B and D (p<0.05).
Sayyad (2022) observed similar results of increase in moisture with addition of Tulsi with
the moisture content of 84.67% This might be due to the increase in the proportion of Basil
leaves extract which has more moisture content as compare to cow milk. Priyanka &
Chavan (2021) discovered that the moisture content ranged from 87.11 to 87.42% during
the incorporation of aloe vera and tulsi juice. This finding was similar to our observation.
38
4.3.7. Effect of Tulsi on acidity of milk
Table 9: Effect of Tulsi on acidity of milk

Days 0 3 6 9 12
Sample A 0.153±0.01𝑎 0.160±0.01𝑎𝑏 0.182±0.01𝑎 0.20±0.01𝑏 0.228±0.01𝑏
Sample B 0.198±0.02𝑏 0.189±0.01𝑎 0.197±0.01𝑏 0.198±0.01𝑏 0.208±0.01𝑎
Sample C 0.194±0.05𝑏 0.180±0.01𝑎 0.189±0.01𝑎 0.19±0.04𝑎𝑏 0.198±0.01𝑎
Sample D 0.192±0.01𝑏 0.18±0.01𝑎 0.186±0.01𝑎 0.189±0.01𝑎 0.19±0.01𝑎𝑏
*Similar alphabets in the same row shows not significantly different from each other
Here, Sample A, Sample B, Sample C and Sample D represents the presence of 0%, 0.5%,
0.6% and 0.7% Tulsi powder respectively in milk. From the results, significant difference
(p<0.05) was observed in the acidity level of most of the milk samples. On day 0, the
titrable acidity ranged from 0.153 on Sample A to 0.192 in Sample D. Acidity of milk
increased right after addition of basil powder. T On day 3 the acidity of Sample A showed
gradual increase with 0.160 acidity whereas the milk samples with basil powder showed
decrease in acidity. The acidity appeared to be 0.189, 0.180 and 0.18 of milk samples A, B
and C respectively. On 9th day the acidity of Sample A was 0.20 whereas Sample B, C and
D showed the acidity 0.198, 0.19 and 0.189 respectively. The milk samples showed
gradual increment in acidity. These results were similar to the findings of Sayyad (2022)
who showed increment of acidity with addition of basil in lassi. The normal milk could no
longer be consumed after 9th day whereas the herbal milk showed the result of 0.198, 0.19
and 0.189 respectively. On the 12th day, curdling of milk was seen in Sample A. Thus, it
could no longer be consumed. In the same way, 0.208, 0.198 and 0.19 acidity were
observed in Sample A, B and C respectively. The best results were observed in Sample D
where the milk had very good quality compared to the other samples.
The acidity of the strawberry milk was ranged between 0.15 to 0.19 during the period of
storage according to the findings of Hossin (2021) which was similar to our findings.
Dhande (2020) observed the acidity of wheatgrass flavored milk ranged from 0.16 to
0.18%. Dalim (2012) observed the acidity of chikoo as 0.18 and banana flavored milk
beverage as 0.20. Our obtained values of acidity of herbal milk have lower value than the
work of Hassan et al (2015) who reported the acidity of fruit flavored milk acidity ranged
from 0.20 to 0.28 in the 7days of storage. The acidity increases results of growth of
microorganism during storage or conversion of milk lactose into lactic acid by bacteria.
39
(Hassan et al., 2015). The following phenolic actives have been identified in Tulsi, which
exhibits antioxidant and anti-inflammatory activities, Rosmarinic acid ((2R)-2-[[(2E)-
3(3,4-Dihydroxyphenyl)-1-oxo-2-propenyl]] oxy]-3-(3,4-dihydroxyphenyl) propanoic acid,
apigenin (5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, cirsimaritin
(5,4’dihydroxy-6,7-dimethoxyflavone), isothymusin (6,7-dimethoxy-5,8,4’-
trihydroxyflavone) and isothymonin. The oil shows good antibacterial activity against
Staphylococcus aureus, Bacillus pumius and Pseudomonas aeruginosa, where S. aureus
was the most sensitive organism. (Pattanayak, 2010).
4.3.8. Effect of Tulsi on pH of milk

Table 10: Effect of Tulsi on pH of milk
Days 0 6 12
Sample A 6.38±0.01𝑐 5.86±0.01𝑎 5.93±0.03𝑎
Sample B 6.37±0.01𝑐 6.04±0.01𝑏 6.03±0.02𝑏
Sample C 6.33±0.01𝑏 6.13±0.01𝑐 6.03±0.01𝑐
Sample D 6.27±0.01𝑎 6.19±0.02𝑑 6.15±0.01𝑑
*Similar alphabets in the same row shows not significantly different from eachother
From the results, significant difference (p<0.05) was observed in the acidic level of most of
the milk samples. On day 0, pH ranged from 6.38 on Sample A to 6.27 in Sample D. The
pH of Sample A ranged from 6.38 to 5.93 in 12 days of interval. Similarly, Sample B
ranged the pH from 6.37 to 6.03, Sample C ranged from 6.33 to 6.03 and Sample D ranged
from 6.27 to 6.15. Here, the best results were seen in Sample D with best quality in 12 days
interval period. The pH decreased along with the gradual increase in acidity. This could be
attributed to the continuous conversion of lactose to lactic acid during storage.
Similar tendencies for pH values have been seen in the observation of Hossin et al
(2021).During the storage of milk sugar (lactose), the lactic acid bacteria produce lactic
acid thus lowering the pH. This is because the bacteria convert milk sugar or lactose into
lactic acid which ultimately increases the qacidity of the final product (Puteri et al., 2014).
Similar observation was showed by Sayyad (2022) where the pH of basil incorporated lassi
ranged from 4.36 to 3.92. Thus, pH directly effects the shelf life of food products.
40
4.4 Sensory Analysis
The observed mean value of different sensory properties of Herbal milk and their statistically
significant difference are tabulated on Appendix C.
4.4.1. Color
The mean sensory score for color was found to be 4.83, 6.41, 6.75 and 7.75 on a 9-point
hedonic rating scale for the Herbal milk formulation Sample A, Sample B, Sample C and
Sample D respectively.
Color
10
9 c
8 b bc
7
6 a
5
4
3
2
1
0
A B C D
Figure 9: Mean sensory score for color
The highest value was found in Sample D (7.75±1.13), the lowest value of color was found
in Sample A (4.83±4.8). The results showed that Sample A was significantly different to
Sample B, C and D (p<0.05). The highest score was obtained in Sample D, while least
score was obtained by Sample A. Similarly, Sample B was significantly different to
Sample A and C (p<0.05). Here, Sample B and Sample C showed no significant difference
(p>0.05). Sample D was the most preferred with respect to color. The panelist showed a
preference for brighter color of the Sample D. Panelists showed similar interest in color of
Sample B and C. The preference for brighter color that tulsi impacts on food products are
consistent with other studies like milkshakes.
From the results, it is clear that the sensory scores for color were affected by the incorporation of
Tulsi with milk. The increasing Tulsi juice level imparted green color.
41
Kumar (2013) observed the highest score of color and appearance at 3.0% level of Tulsi
extract; the level of Tulsi extract increases, decrease the color and appearance score of the
ice cream sample. The results showed the preference for brighter color compared to others.
42
4.4.2. Mouth feel
Mouth feel
10
9 c
8 b
ab
7
a
6
5
4
3
2
1
0
A B C D
Figure 10: Mean sensory score for mouth feel
The mean sensory score for mouth feel was found to be 5.25, 6, 6.91 and 8 on a 9-point
hedonic rating scale for Sample A, Sample B, Sample C and Sample D respectively. The
results showed that there was no significant difference (p>0.05) in mouth feel between
Sample A and Sample D. Here, Sample B, Sample C and Sample D showed significant
difference with each other (p<0.05). The highest value was found in Sample B (8.0±1.2)
and the lowest value for mouthfeel was found in Sample D (5.25±1.28). Since, Sample D
showed close resemblance to Sample A, it was selected as best sample according to graph.
According to the panelists, Sample B was selected as the best sample.
Gaurb et al (2019) reported that the scores decreased with the addition of Tulsi juice. In the
same way, Trivedi et al (2014) reported decrease in preference of basil incorporated ice
cream. Thus, we can conclude that addition of excess amount of basil in food products
leads to disagreeable mouthfeel of the panelists.
43
4.4.3. Odor
Odour
9
b
8
ab
7 a a
6
5
4
3
2
1
0
A B C D
Figure 11: Mean sensory score for odor
The mean sensory scores for the odor of Herbal milk were found to be 6.0, 6.67, 7.75 and
6.16 on a 9-point hedonic rating scale for Sample A, Sample B, Sample C and Sample D
respectively. The highest value was found in Sample C (7.75±1.13), the lowest value of
color was found in Sample A (6.0±2.0). The results showed that there was no significant
difference (p>0.05) in aroma between Sample A, Sample B and Sample D. However, there
was a significant difference (p<0.05) in aroma between Sample A and Sample C. Since,
Sample D showed close resemblance to Sample A, it was selected as best sample
according to graph. Among four samples, Sample C had a better preference based on the
panelist’s acceptance.
These results were similar to the results of Gaur et al (2019). They showed that the
preference increased with the addition of Tulsi juice. Our results showed that the panelists
preferred Sample C other samples. Here, the Tulsi percentage was greater than Sample A
and B but less than Sample D. Thus, we can agree with the results obtained be Gaur et al
(2019). In the same way, Trivedi et al (2014) reported the odor was affected with the
addition of Tulsi in ice cream. These might be responsible for the pleasing and delicate
flavor of ice cream containing basil.
44
4.4.4. Taste
Taste
9 c
8 bc
7 ab
a
6
5
4
3
2
1
0
A B C D
Figure 12: Mean sensory score for taste
The mean sensory scores for the taste of Herbal milk were found to be 6.33, 8.0, 7.08 and
5.58 on a 9-point hedonic rating scale for Sample A, Sample B, Sample C and Sample D
respectively. The results showed that there was no significant difference in taste between
Sample A and Sample D. Here, Sample B showed significant difference with Sample A
and D (p<0.05). The highest value was found in Sample A (8.0±1.27) and the lowest value
for taste was found in Sample D (5.5±1.97). Since, Sample D showed close resemblance to
Sample A, it was selected as best sample according to graph. Among four samples, Sample
B had a better preference based on the panelist’s acceptance.
Gaur et al (2019) reported that the score decreased with the stronger flavor by the addition
of basil juice. The panelists seemed to love the taste of Sample B. The panelists preferred
Sample B over Sample D and Sample A. Thus, it can be concluded that the score decreased
with the addition of basil. Hence, our results were similar to that of the findings of Gaur et
al (2019). Tulsi was found to improve the flavor score significantly over the control
according the findings of Trivedi et al (2014) which was similar to our observation.
45
4.4.5. Overall acceptability
OA
9 c
8 b
7 a
a
6
5
4
3
2
1
0
A B C D
Figure 13:Mean sensory score for overall acceptability
The mean sensory scores for the overall acceptability of Herbal milk were found to be
6.08, 7.91, 7.0 and 5.67 on a 9-point hedonic rating scale for the Herbal milk formulation
of Sample A, Sample B, Sample C and Sample D respectively. The highest value was
found in Sample B (7.91±0.79), the lowest value of overall acceptability was found in
Sample D (5.67±1.15). Here, Sample A, Sample B, Sample C and Sample D were
significantly different with each other (p<0.05). However, Sample A and Sample D
showed no significant difference (p>0.05). The high score for Sample B may indicate the
fact that the panelists loved the flavor and texture of Tulsi incorporated milk. Since,
Sample D showed close resemblance to Sample A, it was selected as best sample according
to graph. Among four samples, Sample B had a better preference based on the panelist’s
acceptance.
The results were similar to that of Gaur et al (2019) who showed the acceptability for
samples with low Tulsi juice. The color changes were not found to have any hitch on the
acceptability. On the other hand, the body and texture and melting quality score tended to
decrease with increase in level of incorporation of basil juice in ice cream according to
Trivedi et al (2014). This was similar to our observation of overall acceptability of herbal
milk.
46
4.5. Microbial Analysis
Table 11: Microbial analysis of herbal milk
Samples Total plate count Coliform count Yeast and mold
10^3 (CFU per g) count
Sample A 4.19𝑎 Nil Nil

Sample B 3.98𝑏 Nil Nil
Sample C 3.88𝑐 Nil Nil
Sample D 3.76𝑑 Nil Nil
4.5.1. Total Plate Count

The total plate count in all the herbal milk samples was significantly different form each
other. The total plate count in Sample A was found to be 4.19* 10^3cfu/gm. The result is
slightly higher than that reported by Acharya et al (2018). Similarly, TPC in Sample B, C
and D were found to be 3.98* 10^3cfu/g, 3.88* 10^3cfu/g and 3.76* 10^3cfu/g
respectively. This result also indicates that highest level of total plate was found in Sample
A (4.19* 10^3). Similarly, lowest total plate count was found in Sample D (3.76* 10^3).
The total plate count is therefore the most important group of micro-organisms responsible
for the spoilage of dairy products. Standard plate count indicates that the product might
have been produced and handled under unhygienic conditions. It could be observed that
herbal milk with Sample D recorded the lowest total bacterial count. Thus, the increase in
tulsi powder decreased the TPC in Herbal milk.
From the table it was observed that total plate count decreased with increase in Tulsi. This
result is in close agreement with findings of Hossin et al (2021). Our results showed higher
total plate count than that of Palthur et al (2014b).
4.5.2. Coliform count

The coliform count from Herbal milk with varying proportions of Tulsi powder along with
controlled Herbal milk was summarized in the table. The coliform count in different four
treatments Sample A, Sample B, Sample C and Sample D were found to be nil which may
be due to strict hygienic measures being practiced during the process of manufacturing.
The absence of coliform bacteria shows that the milk samples are free from fecal
contamination due to hygienic conditions employed during production. It may also be due
antimicrobial effect of Tulsi (Gaur et al., 2019).
47
The result of the coliform test revealed that the microbial quality of milk for all the
proportions of basil powder was excellent. Absence of coliform in Herbal milk signifies a
safe and high-quality product. This could be due to the sterilization process during
processing. Thus, the results agree with the results of Gaur et al (2019).
4.5.3. Yeast and mold count

Yeast and mold count for milk sample with varying proportions of Tulsi powder along
with controlled milk were summarized in the above table. The presence of yeast and old
mold in Sample A, Sample B, Sample C and Sample D were found to be nil which may be
due to strict hygienic measures being practiced during the process of manufacture.
Yeast and mold were observed negative for all the treatments. Absence of yeast and mold
in the milk signifies a safe and high-quality product. This could be due to sterilization
process during processing. Thus, the results agree with the results of Gaur et al (2019) who
reported no signs of yeast and mold during the preparation of flavored milk.
4.6. Cost evaluation

Table 12: Cost evaluation of 1 liter of herbal milk
Treatment Cost of milk Cost of Tulsi Overhead cost Total cost
powder (20%)
Sample A Rs.90 Rs.0 Rs.18 Rs.108
Sample B Rs.90 Rs.10 Rs.20 Rs.120
Sample C Rs.90 Rs.12 Rs.20.4 Rs.122.4
Sample D Rs.90 Rs. 14 Rs.20.8 Rs.124.8
4.7. Calorific Value

The calorific value of Sample A, control sample was found to be 62.89Kcal/100gm. The
calorific value of Sample B, Sample C and Sample D were found to be 59.64Kcal/100gm,
Kcal/100gm, 58.81Kcal/100gm and 56.74Kcal/100gm.
Part V
Conclusions and Recommendation
48
5.1. Conclusions
On the basis of the work conducted, the following conclusion can be drawn:
1. All the Tulsi incorporated milk showed low fat content compared to Sample A, thus Tulsi
has the potential to be used in milk for health-conscious consumers.
2. The best results for sensory analysis were observed in Sample B according to the
panelists. Sample D showed close resemblance to Sample A thereby it can be selected as
the replacement of milk since Sample D showed similar organoleptic properties close to
control i.e., milk.
3. Sample D showed the best results for microbiological analysis and acidity having the best
shelf life among all the samples.
4. Incorporation of herbal plants can be a positive step towards product diversification.
Hence, the producer of the related field can explore and expose the opportunity lagging
behind.
5.2. Recommendations
1. To study the effect of incorporating other herbal plants in milk.
2. To add other various herbal plants in milk and milk products 3. Microbial tests
can be done frequently to observe the changes.
4. Standardized milk can be replaced with soy milk.
Part VI Summary
Ocimum sanctum Linn, commonly known as holy basil is an aromatic perennial plant in
family Lamiaceae. It has used in Ayurvedic medicine for more than 3000 years. It
possesses multifarious medicinal properties such as antiviral, antifungal, antibacterial,
antimalarial, anthelmintic, anti-oxidant, anti-cataract, anti-inflammatory, chemo
preventive, radio protective, hepato-protective, neuro-protective, cardio-protective,
49
antidiabetic etc. Tulsi has antimicrobial effect against micro-organisms like Candida
albicans, E. coli and Staphylococcus aureus.
Herbal milk is a type of flavored milk which generally starts with a pleasant flavor. Flavor
milk has excellent nutritional profile including high amount of calcium, riboflavin, vitamin
A, Vitamin B12. Herbal milk can be the best medium between children and nutrition.
The aim of the present work was to study the moisture, protein, fat, ash, specific gravity
and lactose content of herbal milk. The sensory and microbiological analysis oh herbal
milk was conducted. Tulsi powder was added in the ratio of 0%, 0.5%, 0.6% and 0.7% in 1
liter of milk and labelled as sample A, B, C and D respectively. The proximate
composition of Tulsi powder was analyzed. The effect of Tulsi powder on the physical and
chemical parameters of the milk samples were investigated.
The moisture content of Sample A, B, C and D showed significant difference and were
observed as 86.21±0.04, 86.84±0.04, 87.06±0.02 and 87.45±0.03 respectively. Here, ash
content was observed as 0.72±0.01, 0.66±0.04, 0.64±0.01 and 0.57±0.03 respectively.
Sample B, C and D showed no significant difference in protein content. The protein
content was observed as 3.93±0.07, 3.37±0.01, 3.36±0.02 and 3.25±0.06 in sample A, B, C
and D respectively. Similarly, the fat content was observed as 2.93±0.06, 2.84±0.05,
2.77±0.04 and 2.62±0.04 respectively. The specific gravity was not varied in huge amount
and reported as 1.07±0.01, 1.06±0.01, 1.04±0.01 and 1.02±0.01 in Sample A, B, C and D.
In the same way, the lactose content was observed as 5.02±0.02, 5.15±0.01, 5.11±0.04 and
5.04±0.03. The calorific value of Sample A, B, C and D was found to be
62.89Kcal/100gm. 59.64Kcal/100gm, 58.81Kcal/100gm, 56.74Kcal/100gm respectively.
The proximate composition of Tulsi powder was analyzed. The moisture content, Crude
protein, Crude fat, ash content, acid insoluble ash, Crude fiber, Calorific value and
carbohydrate in Tulsi powder were observed. They were as: 10.46±1.16, 15.92±0.32,
1.73±0.04, 10.37±1.08, 0.76±0.07, 6.01±0.20, 298.25±2.50 and 54.75±0.39 respectively.
The best results for sensory analysis were observed in Sample B according to the panelists.
Sample D showed close resemblance to Sample A thereby it can be selected as the
replacement of milk since Sample D showed similar organoleptic properties close to
control i.e., milk. The panelists preferred mouthfeel, taste and overall acceptability of
Sample B over other samples. However, Sample D showed close resemblance to Sample A
in terms of mouthfeel, odor, taste and overall acceptability. The microbiological analysis
50
showed no presence of coliform bacteria, yeast and mold. However, the total plate count in
Sample A, B, C and D was observed as 4.19, 3.76, 3.88 and 3.98 respectively. The results
showed decrease in TPC which is due to the anti-microbial effect of Tulsi.
Similarly, acidity and pH were observed for 12 days. The samples with Tulsi powder (B,
C, D) showed control in acidity in compared to normal milk. Sample D had the best results
with the acidity of 0.19 in 12 th day of observation. According to the sensory,
microbiological and physico chemical analysis result, Sample D was found to have the best
results. Thus, the addition of Tulsi powder significantly affected various parameters of
milk.
References
Akah, N. E. (2017). Proximate composition, total phenol content and sensory properties of
sweet basil. (Ocimum Basilicum L.) leaves dried using different methods. Journal of
Tropical Agriculture, Food, Environment and Extension, 23-28.
AOAC. (2005). Official Method of Analysis, 18th ed. Washington D.C: Association of Official
Analytical Chemist.
Arati H. Munde, H. R. (2018). Effect Of Drying On Basil Leave By Using Microwave.

International Journal of Innovative Research and Creative Technology, 38-40.
Belitz, H., Grosch, W., & Schieberle, P. (2009). Milk and dairy products. Food chemistry.
Springer, 489-545.
Berg, C., I. J., & M.Conner. (1999). Understanding choice of milk and bread for breakfast
among Swedish children aged 11-15years: an application of the Theory of Planned
Behaviour. An application of theory of planned behaviour, 5-19.
Brijesh Kumar, V. P. (2018). Shelf life assessment of natural antioxidant-treated milk smoothies
stored under refrigeration . Nutrition and food science, 1000-1013.
51
Dalim Dalim, M., Khaskheli, M., Baloch, M. H., Soomro, A. H., Khaskheli, G. B., Mangsi,
A. S., & Barham, G. S. (2012). Production and comparison of banana and chikoo
falvored milk-based beverages. Pakistan Journal of Nutrition, 600-604.
Dalim, M. K. (2012). Production and comparison of Banana and Chikoo Flavoured milkbased
Beverages . Pakistan Journal of Nutritions, 600-604.
Dangol, P. (2018). Ppepatation and quality evaluation of flavored milk (strawberry) milk by
optimising its parameters. Lalitpur: Tribhuvan University, 1-53.
Devesh Tewari, H. P. (2012). Pharmacognostical, Biochemical and Elemental investigation

of Ocimum basilicum plants available in western Himalayas. International Journal
of Research in Pharmaceutical and Biomedical Sciences , 840-845.
Dhande, S. C. (2020). Evaluation of Physico-Chemical properties of lemongrass flavoured milk.

International Journal of Chemical Studies, 2127-2130.
Ebringer, L., Ferencik, M., & Krajcovic, J. (2008). Beneficial health effects of milk and fermented
dairy products. Folia Microbiologica, 378-394.
El-Kamary, S. S., D.Shardell, M., Abdel-Hamid, M., Ismail, S., El-Ateek, M., Metwally,
M., Strickland, G. (2009). A randomized controlled trial to assess the safety and
efficacy of silymarin on symptoms, signs and biomarkers of acute hepatitis.
ScienceDirect, 391-400.
Fayet-Moore, F. (2015). Effect of flavored milk vs plain milk on total milk intake and nutrient
provision in children. Nutrition Reviews, 1-17.
FlaviaFayet, A.Ridges, L., K.Wright, J., & PeterPetocz. (2013). Australian children who
drink milk (plain or flavored) have higher milk and micronutrient intakes but
similar body mass index to those who do not drink milk. Nutrition Research,
95102.
Gaur, G. K., Rani, R., & Khushal, C. N. (2019). Development of herbal milk using tulsi juice.
International Journal of Chemical Studies, 7(2).
Gaur, rani, G. k., Dharaiya, R., & khushel, C. N. (2019). Development of herbal milk using
tulsi juice, ginger juice and turmeric powder. International Journal of Chemical
Studies, 1150-1157.
52
Gibson, S., & Williams, S. (1998). Dental Caries in Pre-School Children:Associations with
Social Class, Toothbrushing Habit and Consumption of Sugars and
SugarContaining Foods. Caries Research, 101-113.
Gideon O. Okunlola, M. A. (2018). Proximate analysis, mineral composition, and

antioxidant properties of bitter leaf and scent leaf. International Journal of
Vegetable Science, 1-9.
Gopalakrishnan Ramakrishnan, S. J. (2009). Silymarin attenuated mast cell recruitment

thereby decreased the expressions of matrix metalloproteinases-2 and 9 in rat liver
carcinogenesis. Springer, 233–240 .
Hassan, M. D. (2015). Physico-chemical, sensory and microbial characteristics of fruit

flavoured milk based beverages during refrigerated storage. Advances in
Biomedicine and Pharmacy, 32-39.
Hossain, T. J., Alam, M. K., & Sikdar, D. (2011). Chemical and Microbiological Quality
Assessment of Raw and Processed Liquid Market Milks of Bangladesh. Continental
journal of food science and technology, , 6–17.
Huppertz T, K. A. (2009). Properties and constituents of cow’s milk. Tamime AY (ed) Milk
processing and quality management, 23-47.
Jennifer P. Taylor, S. E. (2005). Determinatiion of healthy eating in children and youth. Canadian
Journal of Public Health-Revue, 20-26.
Johnson, R. K., Frary, C., & Wang, M. Q. (2002). The nutritional consequences of
flavored-milk consumption by school-aged children and adolescents in the United
States. Journal of the American Dietetic Association, 853-856.
Johri S, C. G. (2014). Physicochemical and Organoleptic Evaluation of Misthi Doi

Prepared With Different Herbs. The International Journal of Agriculture Food
Science, 106-110.
Jost, R. (2012). Milk and Dairy Products. Switzerland: Nestle Product Technology, 316375.
Kadian, R., & Parle, M. (2012). Therapeutic potential and phytopharmacology of tulsi.
International Journal of Pharmacy &Life Sciences, 1858-1867.
53
Kailasapathy, K. (2015). Chemical Composition, Physical,. Adjunct Professor, School of Science
and Health, University of Western Sydney, 77-105.
Kailasapathy, K. (2016). Chemical Composition, Physical and Functional Properties of Milk and
MIlk Ingredients.
Kailasapathy, K. (2016). Chemical Composition, Physical,and Functional Properties of Milk and

Milk Ingredients. Dairy Processing and Quality Assurance, 77-105.
Kumar R, D. R. (2017). Development of healthy milk drink with incorporation of wheat grass
juice. The Pharma Innovation Journal, 27-29.
Kumar S, R. D. (2013). The functional, rheological and sensory attributes of tulsi (holy basil
(ocimum sanctum) extract based herbal ice-cream. The Bioscan, 77-80.
Kumar, T. P., & Shakeel, A. (2017). Diversification of flavored milk: A review. Internal Journal of
Clinical Sciences, 15-20.
Lalit Mohan, A. M. (2010). Ocimum Santcum Linn( Tulsi)- An overview. International Journal of
Pharmaceutical Sciences Review and Research, 51-53.
Lambros Sakkas, A. M. (2014). Assessment of heat treatment of various types of milk. Elsevier,
293-301.
Laura Sanz Ceballos, E. R. (2008). Composition of goat and cow milk produced under
similar conditions and analyzed by identical methodology. Journal of Food
Composition and Analysis, 322-329.
MacDonald, H. B. (2008). Dairy nutrition: What we knew then to what we know now.
International Dairy Journal, 774-777.
Marjan, S., Das, K. K., Munshi, S. K., & Noor, R. (2014). Drug-resistant bacterial pathogens in
milk and some milk products. Nutrition & Food Science- Emerald.
Md. Sarwar Hossin, S. D. (2021). Physico-chemical, Sensory and Microbiological

Characteristicsof Strawberry Flavored Milk under Refrigerated Storage. Asian
Journal of Dairy and Food Research, 1-6.
Mehta, B. M. (2015). Chemical Composition of Milk and Milk. India: Springer-Verlag Berlin
Heidelberg.
54
Mogla Achal Maharaj Kishore, D. C. (2020). Herbal Flavoured Milk and Sensory
Evaluation. International Journal of Current Microbiology and Applied Sciences,
1861-1870.
Mohd, M., Ahmed, K. T., & Firoz, M. (2011). Medicinal Plants of Rural India: A Review of
Use by Indian Folks. Indo Global Journal of Pharmaceutical Sciences, 286-304.
Negar Jamshidi, M. M. (2017). The Clinical Efficacy and Safety of Tulsi in Humans: A
Systematic Review of the Literature. Evidence-Based Complementary and
Alternative Medicine, 1-13.
Ozer B. H. & Kirmachi H. A. (2010). Functional milks and dairy beverages. International Journal
of Dairy Technology, 1-15.
P. Grayhurst. (2012). Glass packaging. UK: Woodhead Publishing Limited, 109-121.
Palthur S, A. C. (2014a). Research Journal of Agricultural and Environment Science, 54-

59.
Palthur S, D. N. (2014a). Development and evaluation of ginger flavored herbal milk. Research
Journal of Plant, Animal and Environmental Sciences, 54-59.
Palthur S, D. N. (2014b). Antioxidant and organoleptic properties of tulsi flavored herbal milk.
International Journal of Plant, Animal and Environmental Sciences, 35-40.
Peryam David R, P. F. (1957). Hedonic scale method of measuring food preferences. Science
Direct, 9-14.
Priyanka, H., & Chavan, S. (2021). Effect of incorporation of herbs on physico-chemical

properties of flavoured milk. Journal of Pharmacognosy and Phytochemistry,
270275.
Puteri, N. P. (2014). Effects of formulation on characteristics of probiotic yoghurt enriched by

Gac and Passion fruits. Khon Kaen Agric. J, 284-263.
Raiesi F, T. F. (2014). Production of a New Drink by Using Date Syrup and Milk. Journal of
of Food Bioscience Technology, 67-72.
Ranganna, S. (1992). Handbook of analysis and control fro fruits and vegetable products. New
Delhi: Tata McGraw-Hill.
55
Ranganna, S. (2005). Handbook Analysis and quality control fro fruits and vegetables products
(2nd ed). New Delhi: Tata Mc Graw-Hill.
Ranganna, S. (2007). Handbook of Analysis and Quality Control for Fruits and Vegetable Products
(2nd ed). New Delhi: Tata Mc Graw-Hill Publishing Co. Ltd.
Renu Kedian, M. P. (2012). Therapeutic potential and phytopharmacology of tulsi. International

Journal of Pharmacy & Life Sciences, 1858-1867.
Sara De Pelsmaeker, J. S. (2014). TheThe consumption of flavored milk among a children

population. The influence of beliefs and the association of brands with emotions.
Elsevier, 93-98.
Sarda Acharya, N. K. (2018). Bacterial Analysis of Different Types of Milk (Pasteurized,
Unpasteurized and Raw Milk) Consumed in Kathmandu Valley. Tribhuvan
University Journal of Microbiology, 32-38.
Sawale, P. D., Singh, R. R., & Arora, S. (2015). Stability and quality of herb (Pueraria
Tuberosa)-milk model system. Journal of Food Science and Technology,
10891095.
Singh, S. (2014). Dairy Products and Quality Assurance Volume 2. Delhi: New Inida Publishing
Agency.
Srinivasan Damodaran, K. L. (2007). Fennema's Food Chemistry. London: CRC press.
SS Sayyad, B. P. (2022). Evaluation of physico-chemical properties of Lass iprepared with

optimized level of Tulsi or Basil(Ocimum sanctum L.). The Pharma Innovation
Journal , 285-288.
SS Sayyad, B. P. (2022). Evaluation of physico-chemical properties of Lassi prepared with

optimized level of Tulsi or Basil (Ocimum sanctum L.). The Pharma Innovation
Journal, 285-288.
T. Juntachote, E. B. (2005). Antioxidative properties and stability of ethanolic extracts of Holy

basil and Galangal. Food Chemistry, 193-202.
Tanim Jabid Hossain. Md. Khorshed Alam, D. S. (2011). Chemical and Microbiological
Quality Assessment of Raw and Processed Liquid Market Milks of Bangladesh.
Food science and technology, 5(2), 6-17.
56
Trivedi V.B, P. J. (2014). Use Of Basil (Tulsi) As Flavouring Ingredient In The Manufacture Of
Ice Cream. American IJ, 28-43.
Trivedi VB, P. J. (2014). Use of basil (Tulsi) as flavoring ingredient in the manufacture of
ice cream. American International Journal of Contemporary Scientific Research,
28-43.
Walstra.P, G. J. (1999). Dairy Technology Principles of Milk. New York.
Walter H. Glinsmann, H. I. (1986). Evaluation of health aspects of sugars contained in

carbohydrate sweetners. The Journal of nutrition, 116.
Watts, S. (2016). A mini review on technique of milk pasteurization. Journal of Pharmacognosy

and Phytochemistry, 99-101.
Wijesinha Bettoni, R. B. (2013). Milk and dairy product composition. Milk and dairy products in
human nutrition, 41-102.
Wiliam Horwitz, G. W. (2010). Official methods of analysis of AOAC International.

Maryland: AOAC International.
Wilson, G. S. (1943). The pasteruization of milk. British Medical Journal, 261.
57
Appendixes
Appendix A Equipment and chemicals
The equipment and chemicals used were available in campus. The list of chemicals and
equipment used for the analysis is shown in table A.1 and table A.2 respectively. TableA.1
List of chemicals used Chemicals
Chemicals Supplier/Manufacturer Other specifications
Sodium hydroxide (NaOH) Thermo fisher Scientific Pellets, AR grade, 98%
India Pvt. Ltd.
Hydrochloric acid (HCL) Themo Electron LLS India 30.6%, LR grade
Pvt. Ltd
Sulphuric acid (𝐻2𝑆04) Thermo fisher Scientific 90.7%, LR grade
India Pvt. Ltd
Boric acid Merck (India) Limited Amorphous
Petroleum Ether Merck life Pvt. Ltd BP 60°C-80°C
Sodium Carbonate Qualligens fine chemicals 99.0.5%, LR grade
(𝑁𝑎2𝐶𝑂3)
Sodium bicarbonate - -
(NaH𝐶𝑂3)
Methanol Merck life sciences Pvt. Ltd 99% liquid
Ethanol Mt. Everest Industrial
𝐻2𝑂2 solution Thermo electron LLS India 30%
Sodium nitrate (Na𝑁𝑂2) Thermo fisher Scientific
India Pvt. Ltd
Ferric chloride (Fe𝐶𝑙3) Thermo fisher Scientific 90.6% anhydrous
India Pvt. Ltd
Acetic acid Thermo fisher Scientific 99% liquid
India Pvt. Ltd
Aluminum chloride (Al𝐶𝑙3) S.D fine-chem Ltd. 98% hydroscopic
Chloroform Merck life sciences Pvt. Ltd 99% liquid
Table A.2 List of Physical equipment

SN Equipment Supplier/ manufacturer
1 Electric balance Phoenix instrument, 620g
58
2 Soxhlet Apparatus Y.P Scientific Industries
3 Hot air oven Victolab, India
4 Incubator Victolab, India
5 Muffle Furnace Accumax, India
6. Micropipette, pipette
7. Desiccator
8. Thermometer
9. Measuring cylinder
10. Refrigerator
11. Mortar and pestle, grinder etc.
12. Autoclave
13. Glassware (beaker, volumetric flask, conical flask, burette, Petri dish, porcelain basin, crucible
etc.)
14. Kjeldhal digestion and distillation set
Appendix B
Sensory Evaluation
Hedonic rating test
Name of the panelist….
Date……….
Name of the product: Herbal milk
Dear panelist, you have 4 sample of milk. Please taste the given Sample And check how
much you prefer the each one. Please give points for your degree of preference for each
parameter as shown below using the scale given. Write any of the defects present, if any.
An honest expression of your personal feeling will help me.
SN Quality Parameter A B C D
1 Mouth feel
59
2 Color
3 Aroma
4 Taste
5 Overall acceptability
Give points as below
Like extremely 9 Like very much 8 Like moderately 7
Like slightly 6 Neither like nor dislike 5 Dislike slightly 4
Dislike moderately 3 Dislike very much 2 Dislike extremely 1
Comment (If any) ………….
Signature
Appendix C
Table C.1 One-way ANNOVA for the proximate analysis of herbal milk
ANNOVA
Sum of
Squares df Mean Square F Sig.
Specific Between Groups .004 3 .001 9.089 .006

Gravity
Within Groups .001 8 .000
Total .005 11
Fat Between Groups .155 3 .052 17.983 .001
Total .178 11
60
Protein Between Groups .758 3 .253 51.125 .000
Total .797 11
Moisture Between Groups 2.434 3 .811 540.993 .000
Total 2.446 11
Ash Between Groups .034 3 .011 15.458 .001
Total .040 11
Lactose Between Groups .043 3 .014 .778 .538
Total .189 11
Multiple Comparisons
95% Confidence Interval
(I) (J) Mean
Lower Upper
Sampl Sampl Difference Std. Bound Bound
Dependent Variable e e (I-J) Error Sig.
Specific LSD A B .01200 .00954 .244 -.0100 .0340
Gravity
.0070
C .02900* .00954 .016 .0510
.0247
D .04667* .00954 .001 .0687
B A -.01200 .00954 .244 -.0340 .0100
C .01700 .00954 .113 -.0050 .0390
D .03467* .00954 .007 .0127 .0567
61
C A -.02900* .00954 .016 -.0510 -.0070
B -.01700 .00954 .113 -.0390 .0050
D .01767 .00954 .101 -.0043 .0397
D A -.04667* .00954 .001 -.0687 -.0247
B -.03467* .00954 .007 -.0567 -.0127
C -.01767 .00954 .101 -.0397 .0043
Fat LSD A B .09000 .04378 .074 -.0110 .1910
C .16667* .04378 .005 .0657 .2676
B D .31000* .04378 .000 .2090 .4110
A -.09000 .04378 .074 -.1910 .0110
C .07667 .04378 .118 -.0243 .1776
D .22000* .04378 .001 .3210
C A -.16667* .04378 .005 -.2676 -.0657
B -.07667 .04378 .118 -.1776 .0243
D .14333* .04378 .011 .0424 .2443
D A -.31000* .04378 .000 -.4110 -.2090
B -.22000* .04378 .001 -.3210 -.1190
C -.14333* .04378 .011 -.2443 -.0424
Protein LSD A B .52333* .05740 .000 .3910 .6557
C .53333* .05740 .000 .4010 .6657
D .65000* .05740 .000 .5176 .7824
B A -.52333* .05740 .000 -.6557 -.3910
C .01000 .05740 .866 -.1224 .1424

D .12667 .05740 .058 -.0057 .2590
62
C A -.53333* .05740 .000 -.6657 -.4010
B -.01000 .05740 .866 -.1424 .1224
D .11667 .05740 .077 -.0157 .2490
D A -.65000* .05740 .000 -.7824 -.5176
B -.12667 .05740 .058 -.2590 .0057
C -.11667 .05740 .077 -.2490 .0157
Moisture LSD A B -.62667* .03162 .000 -.6996 -.5537
C -.85000* .03162 .000 -.9229 -.7771

D -1.24333* .03162 .000 -1.3163 -1.1704
B A .62667* .03162 .000 .5537 .6996
C -.22333* .03162 .000 -.2963 -.1504
D -.61667* .03162 .000 -.6896 -.5437
C A .85000* .03162 .000 .7771 .9229
B .22333* .03162 .000 .1504 .2963
D -.39333* .03162 .000 -.4663 -.3204
D A 1.24333* .03162 .000 1.1704 1.3163
B .61667* .03162 .000 .5437 .6896
C .39333* .03162 .000 .3204 .4663
Ash LSD A B .05633* .02209 .034 .0054 .1073
C .07667* .02209 .008 .0257 .1276

D .14867* .02209 .000 .0977 .1996
B A -.05633* .02209 .034 -.1073 -.0054
C .02033 .02209 .384 -.0306 .0713
D .09233* .02209 .003 .0414 .1433
63
C A -.07667* .02209 .008 -.1276 -.0257
B -.02033 .02209 .384 -.0713 .0306

D .07200* .02209 .012 .0211 .1229
D A -.14867* .02209 .000 -.1996 -.0977
B -.09233* .02209 .003 -.1433 -.0414
C -.07200* .02209 .012 -.1229 -.0211
Lactose LSD A B .05333 .11033 .642 -.2011 .3077
C .09333 .11033 .422 -.1611 .3477
D .16333 .11033 .177 -.0911 .4177
B A -.05333 .11033 .642 -.3077 .2011
C .04000 .11033 .726 -.2144 .2944
D .11000 .11033 .348 -.1444 .3644
C A -.09333 .11033 .422 -.3477 .1611
B -.04000 .11033 .726 -.2944 .2144
D .07000 .11033 .543 -.1844 .3244
A -.16333 .11033 .177 -.4177 .0911
B -.11000 .11033 .348 -.3644 .1444

D
C -.07000 .11033 .543 -.3244 .1844
*The mean difference is significant at the 0.05

level.
Table C.2: One-way ANNOVA for sensory evaluation of herbal milk
ANOVA
Sum of
Squares df Mean F Sig.

Square
64
Mouthfeel Between 50.750 3 16.917 9.902 .000
Groups
Within Groups 75.167 44 1.708
Total 125.917 47
Colour Between 52.729 3 17.576 7.090 .001

Groups
Total 161.812 47
Odour Between 22.396 3 7.465 2.970 .042

Groups
Total 132.979 47
Taste Between 38.500 3 12.833 6.239 .001

Groups
Total 129.000 47
Overall Between 36.167 3 12.056 10.104 .000

acceptability Groups
Total 88.667 47
(I) (J) Mean Std. Sig. 90.5% Confidence
Dependent Variable Sample Sample Difference Error Interval
65
Lower Upper
(I-J) Bound Bound
Mouthfeel LSD Plain Sample -2.00000* .53359 .001 -3.0754 -.9246

Milk A
Sample -.91667 .53359 .093 -1.9921 .1587

B
Sample .75000 .53359 .167 -.3254 1.8254

C
Sample
Plain 2.00000* .53359 .001 .9246 3.0754
A
Milk
Sample 1.08333* .53359 .048 .0079 2.1587

B
Sample 2.75000* .53359 .000 1.6746 3.8254

C
Sample Plain .91667 .53359 .093 -.1587 1.9921

B Milk
Sample -1.08333* .53359 .048 -2.1587 -.0079

A
Sample 1.66667* .53359 .003 .5913 2.7421

C
Sample Plain -.75000 .53359 .167 -1.8254 .3254

C Milk
Sample -2.75000* .53359 .000 -3.8254 -1.6746

A
66
Sample -1.66667* .53359 .003 -2.7421 -.5913
B
Colour LSD Plain Sample -1.58333* .64280 .018 -2.8788 -.2879

Milk A
Sample -1.91667* .64280 .005 -3.2121 -.6212

B
Sample -2.91667* .64280 .000 -4.2121 -1.6212

C
Sample Plain 1.58333* .64280 .018 .2879 2.8788

A Milk
Sample -.33333 .64280 .607 -1.6288 .9621

B
Sample -1.33333* .64280 .044 -2.6288 -.0379

C
Sample Plain 1.91667* .64280 .005 .6212 3.2121

B Milk
Sample .33333 .64280 .607 -.9621 1.6288

A
Sample -1.00000 .64280 .127 -2.2955 .2955

C
67
Sample Plain 2.91667* .64280 .000 1.6212 4.2121
C Milk
Sample 1.33333* .64280 .044 .0379 2.6288

A
Sample 1.00000 .64280 .127 -.2955 2.2955

B
Odour LSD Plain Sample -.66667 .64721 .309 -1.9710 .6377

Milk A
Sample -1.75000* .64721 .010 -3.0544 -.4456

B
Sample -.16667 .64721 .798 -1.4710 1.1377

C
Sample Plain .66667 .64721 .309 -.6377 1.9710

A Milk
Sample -1.08333 .64721 .101 -2.3877 .2210

B
Sample .50000 .64721 .444 -.8044 1.8044

C
Sample Plain 1.75000* .64721 .010 .4456 3.0544

B Milk
Sample 1.08333 .64721 .101 -.2210 2.3877

A
Sample 1.58333* .64721 .018 .2790 2.8877

C
68
Sample Plain .16667 .64721 .798 -1.1377 1.4710
C Milk
Sample -.50000 .64721 .444 -1.8044 .8044

A
Sample -1.58333* .64721 .018 -2.8877 -.2790

B
Taste LSD Plain Sample -1.66667* .58549 .007 -2.8467 -.4867

Milk A
Sample -.75000 .58549 .207 -1.9300 .4300

B
Sample .75000 .58549 .207 -.4300 1.9300

C
Sample Plain 1.66667* .58549 .007 .4867 2.8467

A Milk
Sample .91667 .58549 .125 -.2633 2.0967

B
Sample 2.41667* .58549 .000 1.2367 3.5967

C
Sample Plain .75000 .58549 .207 -.4300 1.9300

B Milk
Sample -.91667 .58549 .125 -2.0967 .2633

A
Sample 1.50000* .58549 .014 .3200 2.6800

C
69
Sample Plain -.75000 .58549 .207 -1.9300 .4300
C Milk
Sample -2.41667* .58549 .000 -3.5967 -1.2367

A
Sample -1.50000* .58549 .014 -2.6800 -.3200

B
Overall LSD Plain Sample -1.83333* .44594 .000 -2.7321 -.9346

acceptability Milk A
Sample -.91667* .44594 .046 -1.8154 -.0179

B
Sample .41667 .44594 .355 -.4821 1.3154

C
Sample Plain 1.83333* .44594 .000 .9346 2.7321

A Milk
Sample .91667* .44594 .046 .0179 1.8154

B
Sample 2.25000* .44594 .000 1.3513 3.1487

C
Sample Plain .91667* .44594 .046 .0179 1.8154

B Milk
Sample -.91667* .44594 .046 -1.8154 -.0179

A
Sample 1.33333* .44594 .005 .4346 2.2321

C
Sample Plain -.41667 .44594 .355 -1.3154 .4821

C Milk
70
Sample -2.25000* .44594 .000 -3.1487 -1.3513
A
Sample -1.33333* .44594 .005 -2.2321 -.4346

B
*. The mean difference is significant at the 0.05 level.
C.2: One-way ANNOVA for acidity of herbal milk
ANOVA
Sum of Squares df Mean Square F Sig.
Day 0 Between Groups .004 3 .001 12.053 .002
Total .005 11
Total .003 11
Total .001 11
Total .001 11
71
.001 8 .000
Within Groups .004 11
Total
(I) (J) Mean 95% Confidence Interval
Dependent Sampl Sampl Difference (IJ)
Variable e e Std. Error Sig. Lower Bound Upper Bound
Day 0 LSD A B -.04500* .00851 .001 -.0646 -.0254
C -.04100* .00851 .001 -.0606 -.0214
D -.03833* .00851 .002 -.0580 -.0187
B A .04500* .00851 .001 .0254 .0646
C .00400 .00851 .651 -.0156 .0236
D .00667 .00851 .456 -.0130 .0263
C A .04100* .00851 .001 .0214 .0606
B -.00400 .00851 .651 -.0236 .0156
D .00267 .00851 .762 -.0170 .0223
D A .03833* .00851 .002 .0187 .0580
B -.00667 .00851 .456 -.0263 .0130
C -.00267 .00851 .762 -.0223 .0170
Day 3 LSD A B -.02833 .01280 .058 -.0579 .0012
C -.01933 .01280 .170 -.0489 .0102
72
D -.02600 .01280 .077 -.0555 .0035
B A .02833 .01280 .058 -.0012 .0579
C .00900 .01280 .502 -.0205 .0385
D .00233 .01280 .860 -.0272 .0319
C A .01933 .01280 .170 -.0102 .0489
B -.00900 .01280 .502 -.0385 .0205
D -.00667 .01280 .617 -.0362 .0229
D A .02600 .01280 .077 -.0035 .0555
B -.00233 .01280 .860 -.0319 .0272
C .00667 .01280 .617 -.0229 .0362
Day 6 LSD A B -.01500 .00863 .121 -.0349 .0049
C -.00700 .00863 .441 -.0269 .0129
D .00333 .00863 .710 -.0166 .0232
B A .01500 .00863 .121 -.0049 .0349
C .00800 .00863 .381 -.0119 .0279
D .01833 .00863 .066 -.0016 .0382
C A .00700 .00863 .441 -.0129 .0269
B -.00800 .00863 .381 -.0279 .0119
D .01033 .00863 .266 -.0096 .0302
D A -.00333 .00863 .710 -.0232 .0166
B -.01833 .00863 .066 -.0382 .0016
C -.01033 .00863 .266 -.0302 .0096
Day 9 LSD A B .00167 .00552 .770 -.0111 .0144
C .01033 .00552 .098 -.0024 .0231
D .02033* .00552 .006 .0076 .0331
73
B A -.00167 .00552 .770 -.0144 .0111
C .00867 .00552 .155 -.0041 .0214
D .01867* .00552 .010 .0059 .0314
C A -.01033 .00552 .098 -.0231 .0024
B -.00867 .00552 .155 -.0214 .0041
D .01000 .00552 .108 -.0027 .0227
D A -.02033* .00552 .006 -.0331 -.0076
B -.01867* .00552 .010 -.0314 -.0059
C -.01000 .00552 .108 -.0227 .0027
Day 12 LSD A B .03867* .01074 .007 .0139 .0634
C .02967* .01074 .025 .0049 .0544
D .02000 .01074 .100 -.0048 .0448
B A -.03867* .01074 .007 -.0634 -.0139
C -.00900 .01074 .426 -.0338 .0158
D -.01867 .01074 .120 -.0434 .0061
C A -.02967* .01074 .025 -.0544 -.0049
B .00900 .01074 .426 -.0158 .0338
D -.00967 .01074 .394 -.0344 .0151
D A -.02000 .01074 .100 -.0448 .0048
B .01867 .01074 .120 -.0061 .0434
C .00967 .01074 .394 -.0151 .0344
*. The mean difference is significant at the 0.05 level.
C.3: One-way ANNOVA for pH of herbal milk
ANOVA
74
Sum of Squares df Mean Square F Sig.
Total .021 11
Total .189 11
Day 12 Between Groups 3.837 3 1.279 2.020E3 .000
Total 3.842 11
(I) (J) 90.5% Confidence Interval
Dependent Sampl Sampl Mean
Lower Bound Upper Bound
Variable e e Difference (I-J)
Std. Error Sig.
*
Da LSD A B .10333 .00943 .000 .0816 .1251
y0
C .05000* .00943 .001 .0283 .0717
D .01000 .00943 .320 -.0117 .0317
B A -.10333* .00943 .000 -.1251 -.0816
-.05333* .00943 .000 -.0751 -.0316
75
C -.09333* .00943 .000 -.1151 -.0716
D
C A -.05000* .00943 .001 -.0717 -.0283
B .05333* .00943 .000 .0316 .0751
D -.04000* .00943 .003 -.0617 -.0183
D A -.01000 .00943 .320 -.0317 .0117
B .09333* .00943 .000 .0716 .1151
C .04000* .00943 .003 .0183 .0617
Da LSD A B -.33000* .01414 .000 -.3626 -.2974

y6
C -.27000* .01414 .000 -.3026 -.2374
D -.18000* .01414 .000 -.2126 -.1474
B A .33000* .01414 .000 .2974 .3626
C .06000* .01414 .003 .0274 .0926
D .15000* .01414 .000 .1174 .1826
C A .27000* .01414 .000 .2374 .3026
B -.06000* .01414 .003 -.0926 -.0274
D .09000* .01414 .000 .0574 .1226
D A .18000* .01414 .000 .1474 .2126
B -.15000* .01414 .000 -.1826 -.1174
C -.09000* .01414 .000 -.1226 -.0574
Da LSD A B -1.39000* .02055 .000 -1.4374 -1.3426
76
y 12 C -1.30000* .02055 .000 -1.3474 -1.2526
D -1.20000* .02055 .000 -1.2474 -1.1526
B A 1.39000* .02055 .000 1.3426 1.4374
C .09000* .02055 .002 .0426 .1374
D .19000* .02055 .000 .1426 .2374
C A 1.30000* .02055 .000 1.2526 1.3474
B -.09000* .02055 .002 -.1374 -.0426
D .10000* .02055 .001 .0526 .1474
D A 1.20000* .02055 .000 1.1526 1.2474
B -.19000* .02055 .000 -.2374 -.1426
C -.10000* .02055 .001 -.1474 -.0526
Table 13:Mean sensory score for different concentration of Tulsi incorporated milk
Parameters Sample A Sample B Sample C Sample D
Color 4.83±4.8𝑎 6.41±1.56𝑏 6.75±1.54𝑏𝑐 7.75±1.13𝑐
Mouthfeel 6.0±1.2𝑎𝑏 8.0±1.27𝑐 6.91±1.37𝑏 5.25±1.28𝑎
Odor 6.0±2.0𝑎 6.67±1.15𝑎𝑏 7.75±1.48𝑏 6.16±1.58𝑎
Taste 6.33±1.23𝑎𝑏 8.0±0.85𝑐 7.08±1.44𝑏𝑐 5.58±1.97𝑎
Overall 6.08±1.08𝑎 7.91±0.79𝑐 7.0±1.27𝑏 5.67±1.15𝑎
acceptability
Table 14: Proximate analysis of Tulsi incorporated milk

Composition Sample A Sample B Sample C Sample D
77
Moisture 86.21±0.04𝑎 86.84±0.04𝑏 87.06±0.02𝑐 87.45±0.03𝑑
Ash content 0.72±0.01𝑐 0.66±0.04𝑏 0.64±0.01𝑏 0.57±0.03𝑎
Protein content 3.93±0.07𝑏 3.37±0.01𝑎 3.36±0.02𝑎 3.25±0.06𝑎
Fat content 2.93±0.06𝑐 2.84±0.05𝑏𝑐 2.77±0.04𝑏 2.62±0.04𝑎
Specific gravity 1.07±0.01𝑐 1.06±0.01𝑏𝑐 1.04±0.01𝑎𝑏 1.02±0.01𝑎
Lactose 5.20±0.02𝑎 5.15±0.01𝑏 5.11±0.04𝑎𝑏 5.04±0.03𝑎
Appendix D
Photo Gallery
Photograph 1: Standardized milk Photograph 2: Test for caustic soda
78
Photograph 3: Herbal milk samples Photograph 4: Samples for sensory
Photograph 5: Microbial analysis
79

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EFFECT OF INCORPORATION OF HOLY BASIL ON PHYSICO

CHEMICAL PROPERTIES OF MILK

Department of Science and Technology

Lalitpur Valley College

Institute of Science and Technology

Tribhuvan University, Nepal

A dissertation submitted to the Department of Food Technology, Lalitpur Valley

Department of Food Technology

Lalitpur Valley College

Affiliated to Tribhuvan University

Department of Food Technology

Lalitpur Valley College, Lalitpur

This dissertation entitled Effect of incorporation of holy basil on physico chemical

Dr. Sitaram Joshi

Mr. Sandeep Raj Pradhan

Mr. Abhitosh Dhungel

The proximate composition of herbal milk indicated a significantly higher content of

Table 1: Typical Chemical Composition of Milk of Different Species ............................... 9

Figure 1: Chart of preparation of flavored milk ..................................................................

Photograph 1 Standardized milk 79

Photograph 2 Test for caustic soda 79

Photograph 3 Herbal milk samples 79

Photograph 4 Samples for sensory evaluation 79

ANNOVA Analysis of variance

et.al And others

AOAC Association of Official Analytical Chemist

DDC Dairy Development Corporation

DFTQC Department of Food Technology and quality control

FAO Food and Agriculture Organization

FDA Food and Drug Administration

IDF International Dairy Federation

SNF Solid Non-Fat

SPSS Statistical Package for Social Science

WHO World Health Organization

FDA Food and Drugs Administration

List of abbreviations………………………………………………………...……….… viii

1.2 Statement of problem ...................................................................................................

1.3 Objectives of study ......................................................................................................

1.3.1Specific objectives ..................................................................................................

1.4 Significance of work ....................................................................................................

1.5 Limitations: ..................................................................................................................

2. Literature review .............................................................................................................

2.1. Background .................................................................................................................

2.1.1 Milk ...........................................................................................................................

2.1.2 Basil .......................................................................................................................

2.3 Varieties in Flavored milk ...........................................................................................

2.3.1 Chocolate flavored milk ........................................................................................

2.3.2. Sterilized flavored milk ........................................................................................

2.3.3. Fruit flavored milk ...............................................................................................

2.3.4. Herbal flavored milk ............................................................................................

2.4 Chemical composition of milk .....................................................................................

2.5. Constituents of milk ....................................................................................................

2.5.1 Water .....................................................................................................................

2.5.2 Milk Fat .................................................................................................................

2.5.3. Milk Protein ........................................................................................................

2.5.4. Lactose ...............................................................................................................

2.5.5. Enzymes in milk .................................................................................................

2.5.6. Gases in milk ......................................................................................................

2.6. Flavor Component ....................................................................................................

2.7. Minor component ......................................................................................................

2.8. Effect of Heat on Milk Proteins ................................................................................

2.9. Flavored milk among children ..................................................................................

2.11. Nutrient contribution of flavored milk ....................................................................