UTILIZATION OF WHEY PROTEIN CONCENTRATE IN THE DEVELOPMENT

OF HIGH PROTEIN BAKERY AND CONFECTIONERY PRODUCTS

By

P. NARENDER RAJU
B.Tech (Dairy Technology)

THESIS SUBMITTED TO THE

ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF

MASTER OF SCIENCE IN
FOOD SCIENCE AND TECHNOLOGY

POST GRADUATE AND RESEARCH CENTER

INTERFACULTY PG PROGRAMME IN FOOD SCIENCE & TECHNOLOGY
ACHARYA N. G. RANGA AGRICULTURAL UNIVERSITY
RAJENDRANAGAR, HYDERABAD – 500 030.

OCTOBER, 2004
 CERTIFICATE

Mr. P. NARENDER RAJU has satisfactorily prosecuted the course of research

and that the thesis entitled, “UTILIZATION OF WHEY PROTEIN

CONCENTRATE IN THE DEVELOPMENT OF HIGH PROTEIN BAKERY AND

CONFECTIONERY PRODUCTS” submitted, is the result of original research work

and is of sufficiently high standard to warrant its presentation to the examination, I also

certify that the thesis or part thereof has not been previously submitted by him for a

degree of any university.

Date: (Dr. (Mrs.) N. LAKSHMI DEVI)

Place: Hyderabad Major Advisor

 CERTIFICATE

This is to certify that the thesis entitled “UTILIZATION OF WHEY PROTEIN

CONCENTRATE IN THE DEVELOPMENT OF HIGH PROTEIN BAKERY AND

CONFECTIONERY PRODUCTS” submitted in partial fulfillment of the requirement

for the degree of MASTER OF SCIENCE IN FOOD SCIENCE AND TECHOLOGY

of the Acharya N. G. Ranga Agricultural University, Hyderabad is record of the bonafide

research work carried out by Mr. P. NARENDER RAJU under my guidance and

supervision. The subject of the thesis has been approved by the student’s Advisory

Committee.

No part of the thesis has been submitted for any degree or diploma. The published

part has been fully acknowledged by the author of the thesis.

(Dr. (Mrs.) N. LAKSHMI DEVI)

Chairman of the Advisory Committee.
Thesis approved by the Student’s advisory committee.

Chairman : Dr. (Mrs.) N. LAKSHMI DEVI

Associate Professor
Department of Foods and Nutrition
Post Graduate and Research Center
Acharya N.G.Ranga Agricultural University
Rajendranagar, Hyderabad. ________________________

Member : Dr. (Mrs.) S. SUMATHI

Associate Professor
Department of Foods and Nutrition
Post Graduate and Research Center
Acharya N.G.Ranga Agricultural University
Rajendranagar, Hyderabad. ________________________

Member : Dr. K. KONDAL REDDY

Associate Professor
Department of Livestock Products Technology
College of Veterinary Science
Rajendranagar, Hyderabad. _______________________
 DECLARATION

I, Mr. P. NARENDER RAJU, hereby declare that the thesis entitled

“UTILIZATION OF WHEY PROTEIN CONCENTRATE IN THE

DEVELOPMENT OF HIGH PROTEIN BAKERY AND CONFECTIONERY

PRODUCTS” submitted to Acharya N. G. Ranga Agricultural University for the degree

of MASTER OF SCIENCE IN FOOD SCIENCE AND TECHNOLOGY, is a result

of the original research work done by me. I also declare that the thesis or part thereof has

not been published earlier elsewhere in any manner.

Date: (P.NARENDER RAJU)

Place: Hyderabad
 LIST OF CONTENTS

Chapter Title Page No.

No.

I INTRODUCTION

II REVIEW OF LITERATURE

III MATERIALS AND METHODS

IV RESULTS AND DISCUSSION

V SUMMARY AND CONCLUSIONS

LITERATURE CITED

APPENDICES
 LIST OF TABLES

Table Title Page No.

No.

1. Composition of whey from different sources.

2. Chemical composition of commercial WPC and WPI.

3. Physico chemical characteristics of whey proteins of bovine milk.

4. Nutritive value of some food proteins.

5. Concentration of essential amino acids in FAO’s reference protein

and some dietary proteins.

6. Composition of whey components in human and bovine milk.

7. Mean scores of sensory evaluation of biscuits.

8. Mean scores of sensory evaluation of cakes.

9. Mean scores of sensory evaluation of chocolates.

10. Mean scores of objective evaluation of biscuits, cakes and chocolates.

11. Nutrient composition of control and experimental biscuits.

12. Nutrient composition of control and experimental cakes.

13. Nutrient composition of control and experimental chocolates.

14. Mean sensory scores of biscuits during storage.

15. ANOVA of taste panel scores for overall acceptability of biscuits.

16. Mean sensory scores of cakes during storage.

17. ANOVA of taste panel scores for overall acceptability of cakes.

18. Mean sensory scores of chocolates during storage.

19. ANOVA of taste panel scores for overall acceptability of chocolates.

20. Moisture and acidity of extracted fat of biscuits during storage.

21. Moisture and acidity of extracted fat of cakes during storage.

22. Moisture and acidity of extracted fat of chocolates during storage.

23. Microbial counts in biscuits during storage.

24. Microbial counts in biscuits during storage.

25. Microbial counts in biscuits during storage.

 LIST OF ILLUSTRATIONS

Figure Title Page No.

No.
1. Protein content of control and experimental biscuits.
2. Protein content of control and experimental cakes.
3. Protein content of control and experimental chocolates.
 LIST OF PLATES

Plate No. Title Page No.

1. WPC used in the present investigation.

2. Prepared control and experimental biscuits.

3. Prepared control and experimental cakes.

4. Prepared Experimental chocolates.

5. Sensory evaluation of developed products by judges.

6. Metallized polypropylene pouches used in the study.

 LIST OF APPENDICES

S.No Title Page No.

1 Method of preparation of products

2 Score cards for sensory evaluation

3 Estimation of moisture

4 Protein estimation

5 Estimation of fat

6 Estimation of crude fiber

7 Estimation of ash

8 Estimation of acid insoluble ash

9 Estimation of acidity of extracted fat.

10 Methods for enumerating microbial colonies.

 ACKNOWLEDGEMENTS

It is by the profuse love of my parents Sri P. Yellaiah and Smt. P. Rajya

Lakshmi and the benediction of the almity I have been able to complete my studies
successfully hitherto and present this piece of work uninterruptedly, for which I am
eternally indebted to them.

I deem it my privilege in expressing my deep sense of reverence and gratitude to

erudite chairman of my advisory committee, Dr. (Mrs) N. Lakshmi Devi, Associate
Professor, Dept. of Foods and Nutrition, Post Graduate and Research Center for her
perspicuous nature, impeccable logic, dexterous, meticulous and secretarial guidance
during the advancement of my work. Ineffable is my gratitude for her keen interest,
wholehearted co – operation and unflagging encouragement, constructive criticism and
constant help rendered during the preparation of the thesis.

I express my immense gratitude to Dr. (Mrs) S. Sumathi, Associate Professor,

Dept. of Foods and Nutrition, Post Graduate and Research Center and member of my
advisory committee for her sumptuous suggestions, constant co – operation and
unconditional help throughout the tenure of my post graduate studies.

A nice gentleman and sharer of the beacon light of wisdom was Dr. K. Kondal
Reddy, Associate Professor, Dept. of Livestock Products Technology, College of
Veterinary Sciences, Rajendranagar and member of my advisory committee, whose
professional approach and advise has shown the right path in pursuit of this degree. I
thank him for his wise counseling.

Equally revered are the kind words of encouragement by our beloved Professor,
Programme Director of Food Science and Technology, Associate Dean of College of
Home Science, Hyderabad and the Director of Center of Advanced Studies in Food and
Nutrition (ICAR), Dr. V. Vimala, whose strong support provided me with the diligent
sense of achievement.

I am running short of vocabulary to express my gratitude to Dr. K. Hanumantha

Rao, Senior Scientist, National Academy of Agricultural Research Management,
Rajendranagar, Hyderabad for sparing his valuable time and for helping me in all respects
in completing my post graduate studies and in bringing this thesis to a shape. I could not
have accomplished the task, of presenting my findings, but for the transcendent
suggestions offered by him. From the bottom of my heart I thank him for his
munificence.

I acknowledge my sincere thanks for the all round co – operation and help offered
by Dr. (Mrs) K. Uma Maheshwari, Associate Professor, Dept. of Foods and Nutrition,
Post Graduate and Research Center during my research work.
 I acknowledge my sincere thanks for the all round co – operation and help offered
by Dr. K. Dharma Reddy, Associate Professor, Dept. of Entomology, College of
Agriculture and former Warden of Prashantha Nilayam (Hostel - D) during my stay in the
hostel.

I am obliged to thank Dr. P. Jayaram, Dr. K.Ranga Rao, Sri S. Sripad, Sri K.
Rajagopal, Sri K. S. Umapathy and other staff of the Dept. of Dairy Technology,
Government College, Kamareddy for their guidance and encouragement during my
graduation.

Diction is not enough to express my deep feelings to my beloved DT seniors V.

Sridhar Reddy, K. Venkat Rao, K. Balasubrahmanyam and A. Venkat Rao for their
unparallel affection and persistent encouragement throughout my graduation and post
graduation.

I express my sincere thanks to my beloved friends Pawan Vuppala, Pawan

Varma, Arvind, Vijay, Rupesh, Jai prakash, and Rajashekar for their constant moral
support and continuous help since my plus two days.

I affectionately acknowledge the help and encouragement received from my FST

seniors Yellamanda, Ramana Prakash, Sudheer, Ramesh, Farheen, Radha,
Soujanya, Vijaya Bhanu and Sravanthi.

With great sense of affection I place it on my record the co – operation and

encouragement extended by my beloved friends Vijay Bhasker Reddy, Vijay (DT),
Vijay (FST), Krishna Mohan and Bindu, Rajanikanth, Padmaja and Vishnu,
Srilakshmi, Ranjeetha, Vinodini, and my beloved juniors Pradeep, Balesham,
Siddeshwar, and Sampath.

A word of appreciation and thanks goes to Veera Kumari, Babu Rao,

Venkataiah, Yedukondalu, Uma Maheshwar Reddy, Narasinga Rao, Ravi, Nazer,
and Sarojinamma the staff of Post Graduate and Research Center for rendering their
help during my course and research work.

Finally, I thank my beloved sisters Kavitha, Naveena, Varalakshmi and beloved

brother Chandra Shekar, Aunt Varalakshmi, and grand parents for their understanding,
patience, help and inspiration throughout my studies.

(P. Narender Raju)

Author : P. NARENDER RAJU
Title of the Thesis : UTILIZATION OF WHEY PROTEIN CONCENTRATE
IN THE DEVELOPMENT OF HIGH PROTEIN
BAKERY AND CONFECTIONERY PRODUCTS
Degree : MASTER OF SCIENCE IN FOOD SCIENCE AND
TECHNOLOGY
Faculty : POST GRADUATE FACULTY
Discipline : FOOD SCIENCE AND TECHNOLOGY
Major Advisor : Dr. (Mrs.) N. LAKSHMI DEVI
University : ACHARYA N.G. RANGA AGRICULTURAL
UNIVERSITY
Year of Submission : 2004

ABSTRACT

Diet is one of the important factors that affects the well being and health of
human beings. Health foods are those, which are nutritious, prevent diseases and
maintain health. Health foods are also known as designer foods, therapeutic foods,
functional foods, etc. These foods contain ingredients that aid specific bodily functions in
addition to being nutritious. Whey proteins are potential functional ingredients for food
applications.

Whey proteins are the proteins that remain in the whey. By virtue of their content
of essential amino acids, the biological value of whey proteins is very high compared
with that of other dietary proteins. They are known to have high nutritional value and
good digestibility. Whey proteins contains a mixture of secreted proteins, some being
bioactive in their own right while others containing bioactive sequences, which are
released during digestive process. These proteins have been implicated in a number of
biological effects observed in human and animal studies including anticancer activity,
anti thrombotic activity, HIV treating ability, influencing digestive function etc.
 Whey proteins also possess excellent functional properties like water holding
capacity, gelation, solubility, emulsification, foaming, etc., which affect the final quality
of the foods. Bakery and confectionery products in India have become increasingly
popular due to an increased demand for convenience foods, shift in eating habits, etc.
Hence, an attempt was made in the present study to develop and evaluate the
acceptability of some bakery and confectionery products incorporated with whey protein
concentrate (WPC), to analyze the proximates content of the developed products and to
study the shelf life of the products.

Common bakery products like biscuit and cake were developed by incorporating
WPC at 10, 20, and 30 percent levels. The confectionery item chocolate was developed
by replacing skimmed milk powder (SMP) with WPC in the standard recipe at 25, 50, 75
and 100 percent levels. The developed control and experimental products were evaluated
by subjective and objective methods. The control and experimental products were
analyzed for proximate content by standard analytical procedures. Biscuits, cakes and
chocolates were stored in metallized polypropylene pouches at room temperature for 60,
15, and 60 days respectively. Sensory, chemical and microbiological analysis of the
stored products was conducted intermittently fresh, after 30 and 60 days for biscuit, and
chocolates while fresh, after 5, 10, and 15 days for cakes.

The overall acceptability of the experimental biscuits and cakes recorded similar
scores to that of the control samples. The overall acceptability of experimental chocolate
prepared by replacing upto 75% of SMP in the standard recipe with WPC received the
highest score. The cutting and compression strength values increased with the levels of
incorporation of WPC.

Carbohydrate and energy values were computed by appropriate calculations.

Protein and ash content was higher in all the experimental products. Fat and energy
content decreased with the increase in the levels of WPC incorporation in the
experimental products. The overall acceptability of the stored products recorded less
scores than the fresh, but are all accepted.
 The moisture and the acidity of the extracted fat of the stored products increased
over storage period. No microbial counts were observed in all the fresh products.
However, microbial colonies were observed with storage period. All the products were
within the safe permissible limit of microbial count.

From the present study, it can be inferred that upto 30 percent of WPC can be
added to the flour to improve the quality and quantity of protein in bakery products and in
chocolates skim milk powder can be replaced with WPC to improve the quality of the
proteins and also imparting these products the nutraceuticals property. Hence, these
products can be explored for commercialization to gain the attraction of health conscious
consumers.
 CHAPTER – I

INTRODUCTION

Presently, increased awareness of the diet – health relationship in many countries

has stimulated a trend in nutrition science, where by more attention is given to the health

effects of individual foods. The role of diet and specific foods in the prevention and

treatment of diseases and improving body functions has become more prominent and

active (Steijns, 2001).

Functional foods, in addition to their basic nutritive value being, will contain

proper balance of ingredients which will help us to function better and more effectively in

many aspects of our lives, including helping us directly in the prevention and treatment of

illness and disease (Goldberg, 1994). Japan is currently the world leader in the

development of functional foods also called as foods for specified health use.

The ingestion of amino acids, peptides and proteins from a variety of food sources

is essential for maintaining health. Humans, as do other animals, use protein chiefly for

its amino acid content. Proteins are converted to large and small peptides and individual

amino acids by gastric and duodenal proteases. Research has suggested that food

proteins, peptides and amino acids may be useful in the treatment of a number of

pathological conditions arising from disease or injury (Marshall, 1994).

Nutritionally, only the essential amino acids (Lysine, Isoleucine, Leucine,

Methionine, Phenylalanine, Threonine, Tryptophane, Valine) are required from

exogenous sources. The others can be synthesized in vivo. Inadequate levels of essential

amino acids result in depression of food intake and retardation of growth (Marshall,

1994). These consequences may be seen among the world’s poor, where protein deficient

diets are common.

Traditionally, whey was defined as a byproduct of cheese making and

regarded by cheese producers as waste with little or no commercial value. This view

changed radically as increasing numbers of technical and nutritional applications were

discovered for whey or whey components, and whey is now considered as a co – product

of cheese making (Walzem et.al. 2002). On an average, whey contains about 65 g/kg

solids comprising 50g lactose, 6g proteins, 6g ash, 2g non – protein nitrogenous

substances and 0.5g fat (Zadow, 1994). Whey solids for human nutrition are being

produced in a variety of forms such as dried whey, condensed whey, partially delactosed

whey, partially demineralized whey, whey protein concentrate, whey protein isolate.

Whey proteins are known to have nutritional value due to their varied amino acid

composition and good digestibility (De Wit, 1998). Whey proteins have proportionately

more sulfur – containing amino acids (cysteine, methionine) than caseins, which

contributes to the higher protein efficiency ratio (PER) of whey proteins than of casein.

As whey proteins have a relative surplus of some essential amino acids (lysine, threonine,

methionine, isoleucine), they are effective supplements to vegetable proteins, which often

are limiting in those amino acids.

Branched chain amino acids must be present in the muscle cells to promote

protein synthesis (Walzem, et.al. 2002). Among all protein sources, whey proteins
contain the highest concentration of the branched amino acids L – isoleucine, L – leucine,

and L – valine. These branched chain amino acids are proposed to provide safe

nutritional support for athletes and individuals seeking optimal lean muscle.

Whey proteins possess excellent functional properties like solubility, water

binding capacity, gelation, emulsification etc., which affect the final quality of the foods.

Recent research reveals that whey proteins also possess bioactivity, which can affect

biological processes or substrates and hence have an impact on body function or

condition and ultimately health (Warner et.al. (2001), Bounous et.al. (1989), Bounous

et.al. (1993).

Bakery products in India have become increasingly popular due to an increased

demand for convenience foods. The demand for bakery products is bound to increase

further in the country due to an increasing demand for convenience foods, shift in eating

habits and better transportation and distribution methods. Most wheat flour has a protein

content of only 12 to 16 %, and is too deficient in lysine and essential amino acids, and so

the quality and content of bakery items is far below that of proteins in the milk – meat

class (Saxena, 2003). The addition of whey solids to biscuits, cakes or bread, which will

not only increase the protein content, but also help to substantiate claims like increased

branched chain amino acids content and others (Parchure, 2002).

Confectionery is eaten all over the world quite simply because people find

pleasure in it. Chocolates and sweets bring color and excitement into everyday lives, and

throughout adult life continue to symbolize gaiety, festivity and goodwill. People eat

chocolate and sweets because of the frequent need for one person to transmit to another

by way of a physical object some element of feeling, such as love and affection,
gratitude, hospitality, even remorse. In such circumstances, confectionery makes an

acceptable gift.

Dairy products have been used as valued ingredients by the confectionery

industry for many years as they help achieve the required flavor, color, and texture in

many products including chocolate coatings, caramels, aerated confections, and toffee.

Dairy ingredients like skimmed milk powder, whole milk powder, butter milk powder,

whey powder, etc. are used in confectionery. Whey powder is generally used as replacer

for skim milk powder in chocolate manufacturing (Haylock and Dodds, 1999). Whey

protein concentrates or isolates can be added to chocolates to improve the quality and

quantity of protein of the chocolates.

Hence, in view of the above benefits of whey proteins for food applications a

study was planned with the following objectives.

Objectives

General:

 To formulate bakery and confectionery products by incorporating whey

protein concentrate.

Specific:

 To develop bakery and confectionery products using whey protein

concentrate at different levels of incorporation.

 To test the acceptability of the developed products by subjective and

objective evaluation.

 To analyze the proximates of the developed products.

 To study the shelf – life of the developed products.

 CHAPTER – II

REVIEW OF LITERATURE

Milk and milk by – products improve the nutritional quality, taste and keeping

quality in bakery and confectionery products. Milk by – products especially whey

proteins could be used as functional ingredient in a variety of food products. Whey

proteins possess very good functional properties such as solubility, foaming,

emulsifying, gelling and water binding. Whey protein concentrate (WPC) in a

predominantly undenatured form, with minimal lactose and lipid contents, thus serve

as a functional protein source for food industry.

There has been a continuous increase in WPC production since the

introduction of the latest ultrafiltration process about three decades ago. Ultrafiltered

milk protein concentrates are employed for protein fortification in bakery products

mainly for improving the consistency and controlling the texture of foods. Ever

increase in WPC production may be attributed mainly to the greater efforts on the part

of whey producers to abide by pollution regulations and to the emergence of reliable,

hygienic ultrafiltration plants and the refinement of continuous operations using

multistage recycle loops and diafiltration.

Increased production of WPC paved its greater application in food products.

Though soluble WPC have been found to be technically suited to a wide range of

products, its use is not cost effective in all cases. Presently, WPC constitutes a very

small proportion (10%) of protein utilization in food industry. More product

formulation work, specially in the food industry is needed to move WPC into the

general market place. The available literature is reviewed here under the following

heads.

2.1 Whey production.

2.2 Composition of whey.

2.3 Isolation of whey proteins from whey.

2.4 Fractions of whey proteins.

2.5 Functional properties of whey proteins.

2.6 Health benefits and bioactivity of whey proteins

2.7 Application of whey proteins in food industry.

2.1 WHEY PRODUCTION

Whey is the largest by-product of the dairy industry. It is obtained during the

manufacture of cheese, casein, paneer, channa, and shrikhand. In India, milk products

like paneer, shrikhand, and channa are very popular and are in great market demand.

With the increase in their production levels, there is a corresponding increase in the

whey as a by-product. In general the manufacture of 1 tonne of cheese or casein

results in the production of 8 or 25 tonnes of liquid whey respectively (Marshall,

1982). Little information is available on worldwide production of whey. However, as

per the FAO (2004) statistics, 2,070,502 MT of dried whey is produced all over the

world. India’s estimated whey production is 1000 million kg and the production is

likely to increase as multi national companies are setting up their cheese plants in

India.
2.2 COMPOSITION OF WHEY

Whey may be defined, broadly, as the serum or watery part of milk remaining

after separation of the curd that results from the coagulation of milk by acid or

proteolytic enzymes (Zadow, 1994). There are wide variations in composition

depending on milk supply, and the process involved in the production of whey. Its

composition will vary substantially, depending on the variety of cheese produced or

the method of casein manufacture employed (Zadow, 1994). The composition of whey

from different sources is given in Table – 1. On an average, whey contains about 65g

per kg of solids comprising about 50g lactose, 6g proteins, 6g ash, 2g non – protein

nitrogenous substances and 0.5g fat. Presence of several nutritionally important

constituents having excellent functional characteristics enhances opportunities for a

wide range of application of whey and whey constituents in food industry.

Whey can be conveniently classified into groups such as sweet wheys having a

titratable acidity of 0.10 – 0.20% and pH about 5.8 – 6.6, medium acid wheys having a

titratable acidity of 0.20 – 0.40% and pH about 5.0 – 5.8, and acid wheys having a

titratable acidity of more than 0.4% and pH below 5.0 (Zadow, 1994). In general,

wheys produced from rennet-coagulated cheeses develop low acidity, whereas the

production of fresh acid cheeses, such as Ricotta, or Cottage cheese yields medium

acid or acid wheys. Whey from casein produced by acid addition is classified as high

acid whey, whereas whey from rennet casein is sweet whey.

 Table – 1. Composition of whey from different sources (g / lt).

Component Rennet Lactic Mineral Cheddar Feta Channa

Casein a Casein a Acid cheese a Cheese b Whey c
Casein a
Total 66 64 63 67 69.8 65.3
Solids
Protein 6.57 6.20 6.10 6.47 13 5.3
(N x 6.38)
Lactose 52.3 44.3 46.9 52.4 50.7 49.9
Ash 5 7.5 7.9 5 5.2 5.2
Fat 0.2 0.3 0.3 0.3 0.2 5.0
Calcium 0.5 1.6 1.4 0.4 0.356 --
(mg /lt)
Phosphorus 1.0 2.0 2.0 0.5 0.385 --
Magnesium 0.07 0.10 0.11 0.08 0.093 --
Potassium 1.45 1.40 1.40 1.50 1.154 --
Sodium 0.53 0.51 0.50 0.50 0.434 --
Chloride 1.02 0.9 0.9 1.0 1.246 --
PH 6.4 4.6 4.7 5.9 6.32 --
Titrtatble - 6.4 - 2.0 1.45 3.1
Acidity
Source: a - Marshall (1982); b - Charalabos and Marios (2001); c – Mandal et.al.
(1997);

2.3 ISOLATION AND COMPOSITION OF WHEY PROTEINS

Milk contains a large variety of proteins that typically can be broken into two

classes, the caseins and the whey proteins. From a processing perspective, the caseins

are those proteins that aggregate into curds during cheese production, and whey

proteins are those dissolved in the aqueous portion and not retained with in the curd

(Walzem, et.al. 2002). Normal bovine milk contains about 3.5% of protein, of which

casein constitutes 80% and whey proteins 20%. The concentration changes

significantly during lactation, especially during the first few days post partum, and the

greatest change occurs in the whey protein fractions (Pihlanto and Korhonen, 2003).
 The major constituents of cheese whey (water, lactose, ash, protein, and fat)

can be separated from one another by modern processes to give liquid or dried

products tailored to the needs of various users (Young, 1985). Whey solids contain

about 11% protein. Many of the most popular methods of whey treatment aim to

increase this level, with end products containing between 35% and virtually 100%

protein. The terms whey protein concentrate (WPC) and whey protein isolate (WPI)

refer to whey protein powders containing 35 – 85% and above 85% protein on a dry

basis respectively (Mulvihill, 1994). These products are usually prepared on a

commercial scale by such processes as ultrafiltration, diafiltration, and ion – exchange

method. Whey proteins are very susceptible to denaturation. During preparation of

whey protein concentrate, maximum care must be taken to minimize denaturation

during preheating, evaporation, drying, and subsequent processing (Kinsella and

Whitehead, 1989).

The various methods of recovering protein from whey are reviewed by

Kinsella and Whitehead (1989); Gupta (1997 a); Marshall (1982) and Mulvihill

(1994). They include i) conventional method, ii) heat precipitation process, iii)

precipitation by complexing agents, iv) gelfiltration process, v) adsorption method, vi)

reverse osmosis, and vii) ultrafiltration.

COMPOSITION OF WHEY PROTEIN CONCENTRATES

The chemical composition of some of the commercial WPC and WPI has been

reported by Morr and Ha (1993) and is presented in Table –2.

 Mann and Malik (1996) studied the composition and properties of WPC

isolated from cheese whey using CM – Sephadex C – 50, a cation exchange medium

and reported that the average composition was 77.3% protein, 0.29% milk fat, 7.45%

moisture, 0.36% carbohydrates and 13.2% ash. Harper (1984) reported that with the

increase in the protein content the composition of other constituents is markedly

changed.

Table –2. Chemical composition of commercial WPC and WPI (%).

Parameter WPC WPI
Moisture 4.14 – 6.01 2.4 – 5.57
Protein 72.0 – 76.6 88.6 – 92.7
NPN Compounds 0.93 – 4.56 0.29 – 0.34
Lactose 2.13 – 5.75 0.42 – 0.46
Total Lipids 3.30 – 7.38 0.39 – 0.67
Phospholipids 0.80 – 1.54 0.11 – 0.31
Ash 2.52 – 6.04 1.37 – 2.15
Sodium 0.15 – 1.71 0.36 – 0.42
Potassium 0.07 – 0.46 0.10 – 0.16
Calcium 0.23 – 1.05 0.20 – 0.24
Magnesium 0.02 – 0.40 0.02 – 0.03
Phosphorus 0.20 – 1.30 0.05
Source: Morr and Ha (1993).

2.4 FRACTIONS OF WHEY PROTEINS

Whey proteins are universally defined as those proteins that remain in milk

serum after coagulation of the caseins at pH 4.6 and 200 C (Eigel, et.al. 1984). Liquid

whey, contains approximately 20% of the original protein of milk ranging from 4 to

7g/lt of which 3.7g is β – Lactoglobulin, 0.6g is α – Lactalbumin, 0.3g is Bovine

Serum Albumin, and 1.4g is proteose – peptone fractions (Marshall, 1982). In

addition, it contains other proteins such as lactoferrin, immunoglobulins,

ceruloplasmin, and milk enzymes such as lysozyme, lipase, and xanthine oxidase,
which present in low concentrations. The physico – chemical characteristics of whey

proteins of bovine milk are given in Table – 3.

Table – 3. Physico – Chemical characteristics of whey proteins of Bovine Milk

Protein Component Molecular Approx. Isoelectric Cystine

Mass Content in pH Groups
Skim Milk
Protein (g/lt)
 - Lactoglobulin 18,600 7 – 12 5.3 2 (1 – SH)
 - Lactalbumin 14,200 2–5 4.8 4
Bovine Serum 66,000 0.7 – 1.3 5.1 17 (1 - SH)
Albumin
Immunoglobulin 15 – 96 x 104 1.9 – 3.3 5.5 – 6.8 32
Protesoe – Peptone
 - CN – 5P(1 - 105) 11,500 -- 3.7 0
 - CN – 5P(1 - 107) 13,000 -- 4.5 0
 - CN – 4P(1 – 28) 4,100 -- 3.0 0
Lysozyme 18,000 0.13 – 0.32 9.5 3
Lactoferrin 76,500 0.02 – 0.35 N/A 19
Source: Kinsella and Whitehead. (1989).

β – LACTOGLOBULIN

β – Lactoglobulin is the most abundant of the whey proteins, which constitute

about 50 % of the total whey proteins (Morr and Ha, 1993). It consists of 162 amino

acid residues in its amino acid sequence. (Kinsella and Whitehead, 1989; Swaisgood,

1982). It exists in at least five different genetic variants (Eigel, et.al. 1984). The two

most common genetic variants, known as A and B, differ at positions 63 and 118,

where an Asparatic acid and a Valine in the A variant are substituted by a Glycine and

an Alanine in the B variant (Kinsella and Whitehead, 1989). The native conformation

is sensitive to heat and pH; at temperatures below 250 C and pH values above 7.0, the

protein forms octamers.

 Native β – Lactoglobulin possesses two disulfide bonds (cys66 – cys160 and

cys106 – cys119), and a free thiol group, which is inaccessible to solvent at or below

neutral pH (Swaisgood, 1982). β – Lactoglobulin has about 15, 43 and 47 % α – helix,

β – sheet, and unordered structure respectively (Kinsella, 1984), that is pH and

temperature sensitive. This protein undergoes time – and temperature – dependent

denaturation at above 650 C, which is accompanied by extensive conformational

transition that expose highly reactive – SH and ξ – NH2 groups (Kinsella, 1984).

β – Lactoglobulin is resistant to peptic and chymotryptic digestibility because

of its stable conformation. Because this protein is thermolabile, heat processing may

alter its digestibility and render it biologically available (Reddy, et.al. 1988).

α – LACTALBUMIN

α – Lactalbumin accounts for 25% whey protein. The ratio of α – lactalbumin

to β – lactoglobulin in bovine milk is approximately 1:3 (Kinsella and Whitehead,

1989). This protein consists of 123 amino acid residues in its molecular disulfide

bonds, linking amino acid residues 6 and 120, 28 and 111, 61 and 77, 73 and 91. It is

nearly spherical and has a highly compact, globular structure with four disulfide bonds

(Swaisgood, 1982). Ruegg et.al. (1977) showed that α – lactalbumin is denatured at

65.20 C and pH 6.7 and that 80 to 90% of the denaturation is reversed upon cooling.

α – lactalbumin binds Ca+2, Zn+2, and other metal ions (Fox, 1989). The

biological function of α – lactalbumin is to modulate the substrate specificity of

galactosyltransferase in the lactose synthetase complex, which is responsible for

lactose synthesis in lactating mammary tissue (Kinsella and Whitehead, 1989).

BOVINE SERUM ALBUMIN

Bovine serum albumin (BSA), present in cow’s milk is identical to that

isolated from blood serum (Eigel, et.al. 1984). This protein consists of a single

polypeptide chain consisting about 580 amino acid residues with 17 intra chain

disulfide bonds and one free thiol group at residue 34. BSA is well – known transport

protein for insoluble fatty acids in the blood circulating system. The binding of fatty

acids stabilizes the protein molecule against heat denaturation (De Wit, 1989). There

is some evidence that between 400 and 500 C, BSA partially unfolds (Kinsella and

Whitehead, 1989).

IMMUNOGLOBULINS

The immunogobulins refer to a heterogeneous family of glycoproteins ranging

in size from 15 to 1000 kDa that share common anti – activity (Eigel, et.al. 1984).

Although the concentration in colostrum is high. IgG, IgA, IgM, and IgE have been

identified in milk and enter it from blood serum (Eigel, et.al. 1984). Although the

concentration in colostrum is high. IgG is the principal type in bovine milk and

comprises about 80% of the total content of immunoglobulins. All classes of

immunoglobulins exist as either polymers or monomers of a basic unit composed of

four polypeptide chains linked covalently by disulfide bridges. The monomeric form

consists of two identical light and two identical heavy polypeptide chains. Each of the

light and heavy chains contains constant (C – terminal half) and variable (N – terminal

half) regions of amino acid residues responsible for various functions, e.g. membrane

transport, and antigen binding, etc. (Kinsella and Whitehead, 1989). These proteins are

very thermolabile.
PROTEOSE – PEPTONES

The proteose – peptone (PP) fraction of bovine milk has been characterized as

a heterogeneous mixture of heat – stable, acid soluble (pH 4.6) polypeptides

precipitated by 12% trichloroacetic acid (Eigel, et.al, 1984). Whey preparations

contain highly variable amounts (from 2 – 20g/lt) of PP (Kinsella and Whitehead,

1989). The PP fraction is actually a heterogeneous group of phospho – glycoproteins

formed following proteolysis of the N – terminal region in the sequence of β – casein

by plasmin (Eigel, 1981). The three PP components have been classified as β – casein

fragments 1P (29 - 107), 4P (1 - 28), 5P (1 - 105) and 5P (1 - 107) (Eigel, et.al. 1984),

this has been demonstrated by Andrews (1978).

LACTOFERRIN

Lactoferrin is present in bovine milk at 0.02 to 0.35g/lt, while there are

considerably larger quantities (approximately 2 – 5 g/lt) in human milk (Kinsella and

Whitehead, 1989). Lactoferrin is a single glycoprotein with an approximate molecular

mass of 80,000 possessing two attached carbohydrate groups (Kinsella and

Whitehead, 1989). Lactoferrin is an iron – binding protein, containing two ferric – ion

(Fe3+) binding sites per protein. It is structurally and functionally homologus with two

other non – heme, iron binding proteins, serum transferring and ovotransferrin.

Because of their ability to bind free iron, all three apo – transferrins exert a

bacteriostatic effect in vitro. They may function as iron transport proteins, and

lactoferrins may also possess a mitogenic function and stimulate the development of

intestinal mucosa in the neonate (Reiter, 1985).

LYSOZYME

Bovine milk contains between 13 and 32μg lysozyme per100 ml, depending on

the stage of lactation, while human milk contains 10μg per 100 ml. The enzyme is

synthesized in the mammary gland. Lysozyme isolated from milk is apparently

different both in molecular mass and amino acid composition than lysozymes from

human milk or egg white sources. It has an isoelectric pH of 9.5. Lysozyme is

considered to be an important component of the antibacterial system of milk possibly

affecting the general immune system as well (Reiter, 1985).

2.5 FUNCTIONAL PROPERTIES OF WHEY PROTEINS

Functional properties of proteins are those physico – chemical properties

which govern the performance and behavior of proteins in food systems during their

preparation, processing, storage, and consumption, i.e., properties affecting the final

quality of foods (Kinsella and Whitehead, 1989). Today, the food industries are

looking for ingredients, which can provide good functional and nutritional properties

for the formulation of value added food products, and they has come to realize that

milk proteins in general and whey proteins in particular have potential to improve the

quality of food products (Jayaprakasha and Brueckner, 1999).

Most of the key protein functional properties may be classified into two main

groups: hydration – related and surface – related properties (Morr and Ha, 1993).

Hydration related functional properties include dispersibility, solubility, swelling,

viscosity, and gelation. Surface related properties include emulsification, foaming, and

adsorption at air – water interfaces. Other functional properties include diffusion,

molecular unfolding (denaturation); and protein – protein, protein – ion, and protein –
ligand binding. Jayaprakasha and Brueckner, (1999), De Wit (1989), Kinsella (1984),

Kinsella and Whitehead (1989), Sharma and Bhatia (1999), Rathour, et.al. (2004), and

Morr (1982) reviewed the functional properties of whey proteins extensively.

HYDRATION AND SOLUBILITY

The solubility of a whey protein can be defined as the percentage of nitrogen in

the protein product that is soluble under specified condition (De Wit, 1989). The

solubility of proteins depends upon the fact that protein / solvent interactions have a

lower free energy than the sum of the protein / protein and solvent / protein

interaction. However, near the isoelectric point when the net charge on protein is

minimal, protein / protein interactions are favored, the solubility is minimum

(Mangino, 1992). In contrast to other food proteins, undenatured whey proteins are

soluble at their isoelectric region (Kinsella and Whitehead, 1989).

Several environmental conditions affect the solubility of whey proteins: pH,

solute concentration, ionic strength, ion valency, surface / shear effects and, most

acutely heat treatment. Heat treatments alter the conformation of whey proteins and

often result in denaturation, loss, and aggregation (Kinsella and Whitehead, 1989).

WPCs and WPIs are valuable as food ingredients, not only for their ability to

aggregate and provide structure to foods, but because they are highly soluble over a

wide pH range.

Grandison and Jindal (1994) reported that protein solubilities of channa whey

powders varied from 57 – 100% depending on pH. Protein solubility was lower in the
isoelectric region (pH 4 - 5) but was quite high out side this range. In general, the

results for channa and cheese whey products were very similar.

Mann and Malik (1996) reported that Ion – exchange whey protein concentrate

(IE – WPC) prepared from cheese whey had the average solubility 62.20% at pH 3.5

and 100% at pH 7.0 respectively at 300 C. While the ultrafiltered whey protein

concentrate (UF – WPC) had the average solubility 74.18% at pH 3.5 and 81.94% at

pH 7.0 respectively at 300 C.

Vijaykumar and Sangwan (2001) reported that the heat coagulated whey

proteins, precipitated at 900 C for 30 min from cheddar cheese whey, had a low

solubility of 15.6% at pH 7.0, which was attributed to protein denaturation by severe

heat treatment during its preparation. Trypsin hydrolysates had shown an increase in

solubility with increase in time of hydrolysis.

GELATION

Gelation, i.e., the property of forming a structural network which maintains a

shape, has mechanical strength, viscoelasticity, and retains entrapped water with

minimum syneresis, is an important functional attribute of functional proteins in many

food applications (Kinsella and Whitehead, 1989). Gelation is a two – stage process

involving an initial unfolding of a protein molecule followed by subsequent

aggregation (Sharma and Bhatia, 1999). Whey proteins can form gels that range in

properties from viscous fluid soft, smooth pastes or curds to stiff, rubbery gels. WPC

gels also vary in visual appearance from firm elastic transparent gels to opalescent

curd like coagula. The ability of whey proteins in WPC solutions to form stable gels
upon heating to 700 to 900 C is an important functional property. Recently, Rathour,

et.al. (2004) reviewed the gelling properties of WPC.

At low protein concentrations and low ionic strength, weak gels with gray or

translucent appearances are obtained (Kinsella and Whitehead, 1989). Protein

solutions (1 – 10g/dl), when heated above a critical temperature, undergo

conformational changes and on cooling may set to viscous, soft, opaque coagula or

clear viscoelastic gels depending on type of protein, concentration, heating rate, and

environmental conditions, especially pH and calcium (Mulvihill and Kinsella, 1988).

Whey proteins have excellent gelling characteristics, particularly above pH

7.0. Exposure to alkaline pH conditions accelerates heat – induced gelation and

reduces the temperature treatment required for gelation (Kinsella and Whitehead,

1989). The gel structure is affected by salt concentration. In presence of sodium

chloride, a coarse gel of large aggregates is formed compared to finer gels developed

in the absence of salt (Schmidt, et.al. 1979). Addition of cysteine (upto 10000M)

prior to heat treatment enhances gelling opportunities, where as, at higher

concentrations, gelation is impaired (Mc Kenzie, 1971).

Grandison and Jindal (1994) reported that channa whey products formed weak

gels on heating to 800 C at acid pH, and did not form gels at all in the range of 6.0 –

9.0. Mann and Malik (1996) reported that the gel formed from IE – WPCs were

comparatively less strong than that of UF – WPCs, which was attributed to high

concentration of minerals in the former concentrates.

EMULSIFICATION

Emulsions are heterogeneous systems consisting of one or more phases

dispersed in a continuous phase. The major function of emulsifiers is to reduce the

interfacial energy and facilitate dispersion of the discontinuous phase (Kinsella and

Whitehead, 1989). In protein – stabilized emulsions, the role of the protein is to form

an interfacial membrane rapidly around the oil droplet to prevent coalescence,

flocculation, creaming, and oiling – off (Kinsella and Whitehead, 1989). The

adsorption of whey proteins onto the surface of a fat globule is selective and

influenced by pH, presence of salts, protein concentration, and temperature

(Yamauchi, et.al. 1980).

Whey protein concentrates have good emulsifying capacity, which has been

proved useful in emulsifying the oils in many food systems (Hood, 1985). Purified β –

lactoglobulin has been shown to be more efficient in emulsion formation than the

other whey proteins (Pearce and Kinsella, 1978). The emulsion property of channa

whey products has been shown to be comparable to cheese whey products (Grandison

and Jindal, 1994).

Vijayakumar and Sangwan (2001) reported that the heat coagulated whey

proteins had very low emulsifying activity index, and trypsin hydrolysis increased the

emulsification activity index of hydrolysates, and the higher EAI of trypsin

hydrolysates showed a linear relationship with their activity. Mann and Malik (1996)

reported that the emulsion stability of IE – WPCs is more than that of UF – WPC.
FOAMING

Proteins contribute to foam film formation by lowering interfacial tension

(Morr and Ha, 1993). A typical foam is composed of millions of bubbles each

encapsulated by a protein film and separated by thin water – filled canals (lamella)

(Kinsella and Whitehead, 1989). Whey protein concentrate is capable of forming a

tough, resilent structure surrounding the foam cells and also acts as an excellent

surfactant and film forming agent and hence has to be considered as a potential

ingredient for formulating products like bread, rolls, biscuits, cakes (Jayaprakasha and

Brueckner, 1999), whipped toppings, quinches, meringues and chiffon desserts

(Sharma and Bhatia, 1999). Commercial whey protein concentrate preparations vary

immensely in whipping properties because of variability in extent of denaturation of

proteins, high ash content, the presence of lipids and possibly proteose – peptone

fractions (Kinsella and Whitehead, 1989).

Foaming properties of WPC are commonly determined as maximum overrun

or maximum foam expansion and foam stability (Morr and Ha, 1993). Kim and

Kinsella (1985) have elucidated relationships between film – forming behavior and

foam properties of BSA and β – casein. β – casein is very surface active and rapidly

forms foams, but because of limited protein – protein interactions, the foam collapses

easily. In contrast, globular proteins such as BSA, which retain considerable tertiary

structure at the interface, form stable foams because of more extensive intermolecular

network formation.

Mann and Malik (1996) reported that the percent overrun and foam stability of

IE – WPC is 115.35% and 6.34 while that of UF – WPC is 40% and 1.40 respectively.
 Grandison and Jindal (1994) reported that the foaming properties of channa

whey powders were inferior to commercial cheese whey products, which was

attributed to higher fat levels in former.

Jayaprakasha and Brueckner (1999) reported that WPC – 80 may be used as

foaming agent i.e., as an egg white substitute or extender.

WATER BINDING AND VISCOSITY

Water holding capacity refers to the properties involving interaction between

the protein product and water, as a result of which some of the water remains with

protein (Kumar et.al. 2000). Denatured proteins are essentially insoluble, but have

very high water binding capacity. On the other hand, undenatured whey proteins are

generally very soluble, but they exhibit limited water binding capacity. Many of the

functional food applications of dairy proteins depend on the ability to hydrate and bind

or entrap water (Jayaprakasha and Brueckner, 1999).

In a food system, some of the important factors affecting the water binding

capacity are: amino acid composition, protein conformation, surface polarity /

hydrophobicity, ion strength, ion species, pH and temperature (Kinsella, 1982). Some

amino acids bind more water than do others and hence, proteins that contain large

amounts of charged amino acids will tend to bind large amounts of water

(Jayaprakasha and Brueckner, 1999). An increase of about 110% in the water holding

capacity of the trypsin modified milk proteins compared to 107% of the neutrase

modified milk proteins has been reported by Venkatesh (1995).

 Change of pH can also alter conformation of the protein, which may either

expose or bury the potential water binding sites (Mangino, 1984). The formation of

protein aggregates increases the volume occupied by the proteins and thus increases

viscosity (Jayaprakasha and Brueckner, 1999). Grandison and Jindal (1994) reported

that viscosity of solutions of channa whey powders was similar to equivalent cheese

whey products above pH 4.0, but was very high at pH 2.5 – 4.0.

Resch and Daubert (2002) reported that, upon cold – set gelation of whey

proteins, the derivatized WPC powders exhibited a dramatic increase in thickening

and water holding capacity.

2.6 HEALTH BENEFITS AND BIOACTIVITY OF WHEY PROTEINS.

The nutritive value of dietary protein is essentially related to its amino acid

composition as well as to the availability of these amino acids. In this respect it is well

known that milk proteins have a high content of essential amino acids. Thus egg and

milk proteins are often used as references for the evaluation of the nutritive value of

food proteins mainly because they seem to be the only ones originally intended to

function as a single source of nutrients for the off spring (Hambraeus, 1982). The

amino acid composition of the casein fractions and whey protein fractions are

reviewed by Swaisgood (1982) and Renner (1983). Whey proteins are known to have

high nutritional value due to their varied amino acid composition and good

digestibility (De Wit, 1998).

 A number of indices of protein quality, which have been obtained by means of

chemical, biological and microbiological methods, are used to compare the nutritional

properties of different proteins. Puranik and Rao (1996) reviewed the chemical and

biological methods. The biological value (BV), net protein utilization (NPU), and

protein efficiency ratio (PER) are the most commonly used methods.

The biological value of the milk proteins as well as their PER and NPU values

with those of other dietary proteins are given in Table – 4. The concentration of

essential amino acids in the FAO’s reference protein, egg protein and milk protein are

given in table – 5. The higher nutritional value of whey proteins is based upon its

higher concentrations of essential amino acids such as lysine, tryptophan, isoleucine,

threonine, etc (Renner, 1983). Whey proteins have proportionately more sulfur

containing amino acids (cysteine and methionine) than caseins, which contributes to

the higher PER of whey proteins (3.2) than casein (2.6). Any protein with a PER of

2.5 is considered as good quality (Walzem, et.al. 2002).

Table –4. Nutritive value of some food proteins.

Food Protein BV PER NPU
Whole egg 100 3.8 94
Cow’s milk 91 3.1 82
Casein 77 2.9 76
 - Lactalbumin 104 3.6 92
Beef 80 2.9 73
Soy protein 74 2.1 61
Rice 59 2.0 57
Wheat 54 1.5 41
Beans 49 1.4 39
Source: Renner (1983)
Table – 5. Concentration of essential amino acids in the FAO’s reference protein,
and some dietary proteins.

Essential FAO’s Whole Milk Whey

Amino Acid reference Egg protein Protein Protein
protein
Tryptophan 1.0 1.5 1.4 2.1
Phenylalanine 6.0 10.5 10.5 7.3
+ Tyrosine
Leucine 7.0 9.1 10.4 11.1
Isoleucine 4.0 6.7 6.4 6.8
Threonine 4.0 5.1 5.1 8.0
Methionine + 3.5 5.9 3.6 4.8
Cystine
Lysine 5.5 6.9 8.3 9.9
Valine 5.0 7.5 6.8 6.8
Total 36.0 53.2 52.5 56.8
Source: Renner (1983)

BIOACTIVITY OF WHEY PROTEINS.

Bioactivity of food refers to food components that can affect biological

processes or substrates and hence have an impact on body function or condition and

ultimately health. The role of proteins as physiologically active components in the diet

has been increasingly acknowledged in recent years. Such proteins or their precursors

may occur naturally in raw food materials, exerting their physiological action directly

or upon enzymatic hydrolysis in vitro or in vivo (Pihlanto and Korhonen, 2003).

Bioactive peptides usually contain 3 – 20 amino acid residues per molecule.

At present, milk proteins are considered the most important source of bioactive

peptides (Pihlanto and Korhonen, 2003). Milk contains components that provide

critical nutritive elements, immunological protection, and biologically active

substances to both neonates and adults. From these, bioactive peptides may be

generated in vivo through gastrointestinal processes. These peptides encoded with in

the sequences of native protein precursors, may also be generated in vitro by

enzymatic hydrolysis (Clare and Swaisgood, 2000). Peptides with biological activity

could be produced in and from foods in several ways. The most common methods are

(a) processing of foods using heat and / or acid / alkali conditions that hydrolyze

proteins, (b) enzymatic hydrolysis of food proteins during the course of digesting a

meal, (c) microbial activity in fermented foods (Marshall, 1994).

Apart from being a source of nitrogen, whey protein acts as carriers for ligands

and trace elements and have various biological function (Pihlanto and Korhonen,

2003). Recently, Walzem, et.al. (2002), Pihlanto and Korhonen, (2003), Clare and

Swaisgood (2000), Bajaj and Sangwan (2002) and Steijns (2001) reviewed the

bioactivity of whey proteins.

OPIOID ACTIVITY

Opiates are drugs containing opium, whose basic substance is morphine. They

have been used since ancient times in medicine to relieve pain and induce sleep.

Opioid peptides are defined as peptides having an affinity for an opiate receptor and

opiate – like effects inhibited by naloxone (Pihlanto and Korhonen, 2003). Opioid

receptors are located in the nervous, endocrine, and immune systems as in the

intestinal tract of the mammalian organism and can interact with their endogenous

ligands as well as with exogenous opioids and opioid antagonists (Sabikhi, 2000).

Whey proteins contain opioid –like sequences, namely α – lactalbumin f (50 -

53) and  - lactalbumin f (102 - 105), in their primary structure. These peptides have
been termed as - and  - lactorphins (Chiba and Yoshikawa, 1986). Antila, et.al.

(1991) indicated that proteolysis of  - lactorphin, and that digestion of  -

lactoglobulin with pepsin and then with trypsin and chymotypsin, yields  -

lactorphin.

ANTI – HYPERTENSIVE ACTIVITY

Angiotensin, a blood polypeptide exists in two forms, the physiologically

inactive angiotensin – I and the active angiotensin – II (Sabikhi, 2000). The inactive

form is converted into the active one, by angiotensin – I converting enzyme (ACE),

which is a key enzyme in the regulation of peripheral blood pressure. It has been

classically associated with the rennin – angiotensin system, which converts

angiotensin – I into a potent vasoconstrictor, angiotensin – II (Pihlanto and Korhonen,

2003).

Angiotensin – I converting enzyme is a multifunctional enzyme located in

different tissues including plasma, kidney, lung and brain (Gerdes, et.al. 2001). Its

inhibition results in an anti – hypertensive effect and may influence different

regulatory systems of the host involved in modulating blood pressure, immune defense

and nervous system activity. The first ACE – inhibiting peptides derived from whey

proteins were synthetic peptides corresponding to the known bioactive sequence of  -

lactoglobulin ( - lactorphin and  - lactotensin) and  - lactalbumin ( - lactorphin)

(Pihlanto and Korhonen, 2003). The casein-derived peptides are called as casokinins.
ANTI – THROMBOTIC ACTIVITY

Thrombosis is defined as the formation or presence of a blood clot

within a blood vessel (Gerdes, et.al. 2001). Fibrinogen is a plasma protein that is

produced in the liver and is converted into fibrinogen to the platelets is necessary for

platelet aggregation. Milk peptides are believed to inhibit this platelet fixation

(Gerdes, et.al. 2001).

Hydrolysis of bovine  - casein by chymosin constitutes the first stage

of milk clotting. In this reaction, one bond (Phe105 – Met106) of  - casein is rapidly

hydrolyzed, leading to the release of an insoluble N – terminal fragment (Para -  -

casein, residues 1 – 105) and a soluble C – terminal fragment (caseinomacropeptide,

residues 106 – 169) from which a series of tryptic peptides active in platelet function

has been characterized. These peptides are referred to as casoplatelins (Pihlanto and

Korhonen, 2003).

IMMUNE SYSTEM STIMULATION

The immune system plays a central role in protection against bacterial, viral,

parasitic and fungal infection and also cancers. Deficiencies in any aspect of the

immune system can predispose an individual to a greater risk of infection and may

enhance the severity of disease (Bajaj and Sangwan, 2002).

Milk protein derived peptides are known to have an effect on the cells of the

immune system, as well as on down stream immunological responses and cellular

functions (Pihlanto and Korhonen, 2003). The immune system employs both non –
specific and specific responses to confer protection against the disease. Non – specific

components of the host defense include physico – chemical barriers such as skin,

mucus, lysozyme, complement and interferons, as well as natural killer cells and from

phagocytic cells (cellular immunity) such as neutrophils and monocytes /

macrophages. Specific immune responses are mediated by anti bodies (Ig G, Ig A, Ig

M, Ig E, and Ig D) produced by  - lymphocytes (humoral immunity), while T –

lymphocytes give rise to T – helper, T – successor and cytotoxic lymphocytes (cell

mediated immunity) (Bounous and Kongshavan, 1985).

Whey protein has higher concentration of cysteine, which is thought to be rate

limiting for glutathione (GSH) synthesis. The GSH – modulating effects of whey

proteins is believed to underline both immuno – enhancing and some antioxidant

actions of whey proteins (Walzem, 1999). The concentration is very high in

colostrums, and it is lower in milk. Immunoglobulin content constitutes part of the

passive immunity conferred to the neonate via colsotrum (Walzem, et.al. 2002).

Colostral immunoglobulins currently are being developed as food – grade

antimicrobials.

APPETITE SUPPRESSION

Glycomacropeptide (GMP) is a powerful stimulator of cholecystokinin (CCK),

which is an appetite-suppressing hormone that plays many essential roles relating to

gastrointestinal function, including the regulation of food intake (Beucher, et.al.

1994). In addition to being a regulator of food intake, CCK stimulates gall bladder

contraction and bowel motility, regulates gastric emptying and stimulates the release
of enzymes from pancreas. High protein intakes lead to increases in CCK release until

pancreatic protease release (primarily trypsin) matches protein intake. Casein has a

higher concentration of GMP than whey, but whey appears to have an effect on CCK

release.

ANTIOXIDANT ACTIVITY

Consumption of dietary antioxidants has been positively correlated with the

prevention of diseases such as cancer and atherosclerosis. While dietary antioxidants

including carotenoids and alpha tocopherol are found in milk, dairy products are

generally not considered a rich source of antioxidants (Bajaj and Sangwan, 2002).

Decker et.al. (1997) showed that whey is a rich source of antioxidants in the

form of low molecular weight protein fractions or peptides with molecular weight

range 500 – 3000 Da. It has also been shown that these antioxidants are stable in raw

or pasteurized milk stored at 40 C for upto 5 days. These water-soluble low molecular

weight antioxidants are capable of inhibiting a wide array of lipid oxidation catalysts

including iron, lipoxygenase, singlet oxygen, ferryl radicals, and hydroxyl radicals.

These antioxidants are responsible for over 90% of the antioxidant activity of milk

(Decker, et.al. 1997).

ANTICARIOGENIC ACTIVITY

Dental caries involves the demineralization (solubilization of the calcium and

phosphorus) of tooth enamel, which consists mainly of crystalline calcium phosphate

embedded within a protein matrix. The demineralization is brought by the action of

acids, either directly (because of the consumption of acidic foods) or indirectly (as a
result of fermentation by plaque bacteria of residual food particles either between teeth

or adhering to the plaque) (Rosen, et.al. 1984). After this initial demineralization,

which creates small cavities, tooth decay takes place by the action of the microflora

present in the plaque.

HUMAN IMMUNO DEFICIENCY VIRUS (HIV) TREATMENT ABILITY

Whey protein isolate is rated so high by medical professional that it is used to

treat HIV patients. Bounous, et.al. (1988) reported that whey elevates deficiency

levels of glutathione (GSH) and so provides an extremely important antioxidant

involved in the maintenance of functional and structural integrity of muscular tissues

undergoing oxidation damage during exercise and ageing.

It has been shown that HIV needs low GSH levels to replicate and that HIV

has an antagonistic relationship to GSH, that is, low cellular GSH allows HIV to

multiply and high GSH dramatically slows viral replication. In cells with an improved

GSH status after the ingestion of WPC, there was a substantial reduction in virus

activity and increased survival expectancy (Bounous, et.al. 1989; Bounous, et.al.

1993).

HYPOCHOLESTEROLEMIC PROPERTY

Several studies of the effects of whey proteins on cholesterol concentrations in

rats and pigs have been reported. The effects of dietary whey protein and casein on

plasma and live cholesterol concentrations were investigated in female, weanling rats
-1
fed 10 g cholesterol kg feed for three weeks by Zhang and Beynen (1993). They
-1
reported that after three week feeding of a low dietary protein (150 g kg feed),
compared with casein, whey protein did not affect plasma total cholesterol, but

lowered the concentration of liver cholesterol, where as at high dietary protein (300 g

protein kg –1 feed) whey protein significantly lowered plasma and liver cholesterol and

also plasma triacylglycerols compared with casein. The hypocholestrolemic effect of

whey protein was associated with decreased very – low – density – lipoprotein

cholesterol. The cholesterol lowering effect of whey proteins in rats was possibly

caused by an inhibition of hepatic cholesterol synthesis.

2.7 APPLICATION OF WHEY PROTEINS IN FOOD INDUSTRY

Functional foods, in addition to their basic nutritive value being, will contain

the proper balance of ingredients which will help us to function better and more

effectively in many aspects of our lives, including helping us directly in the prevention

and treatment of illness and disease (Goldberg, 1994). Human nutrition science has

moved from a focus on the prevention of nutrient deficiencies to an emphasis on

health – maintenance and reduced risk of chronic diseases. A variety of foods and their

components are emerging as factors capable of modifying growth, development,

performance and disease resistance.

The increasing awareness of nutrition, health and quality food consciousness of

consumers and the keen competition in the market, compel the food industry to search

for those ingredients which impart specific functionalities to food products, while

reserving or enhancing the nutritional quality of food stuffs in order to sell their

products profitably. In this context, the food manufacturing industry has come to

realize that milk proteins in general and whey proteins in particular have potential to
improve the quality of food products (Jayaprakasha and Brueckner, 1999). Gupta and

Thapa (1991) reviewed the WPC applications in food industry. In a healthy individual,

eating a varied diet, the presence of bioactive peptides may help keep the nervous,

immune, and digestive systems in a well – maintained state (Marshall, 1994).

WHEY PROTEINS IN INFANT FOOD FORMULATION

Infants are born with relatively under developed organ functions, especially of

the kidneys and intestines. This requires that certain special nutritional demands be

met, especially during the first 3 months of life. Research work in this area has shown

that feeding infants excessively on cow’s milk leads to the development of altered

intestinal physiology (Mathur and Shahani, 1979). This has been attributed to not only

gross compositional characteristics but also to the chemical make up of various

constituents of cow’s milk, which differ appreciably from that of human milk. Table –

6 shows the comparison of whey components in human and bovine milk.

Table – 6. The comparison of whey components in human and bovine milk.

Human Milk Bovine Milk

g / dl % g / dl %
Total Proteins 0.89 100 3.30 100

Caseins 0.25 35 2.60 79

Whey Proteins 0.64 65 0.70 21
 - Lactalbumin 0.25 17 0.12 2.5
 - Lactoglobulin -- -- 0.12 9.0
Lactoferrin 0.17 17 Trace
Bovine Serum Albumin 0.05 6 0.03 1.0
Lysozyme 0.05 6 Trace
Ig A 0.10 11 0.03 3.0
Ig G 0.003 0.06
Ig M 0.002 0.003
Others 0.07 8 0.15 4.5
Source: Renner (1983).
 Due to various reasons, buffalo and cow milks are being humanized and used

partly or excessively for feeding human infants throughout the world. For

humanization, apart from making other modifications, whey proteins proportion needs

to be increased in these milks. For this, a great potential lies in the application of WPC

(Gupta, 1997).

WHEY PROTEIN IN BAKERY PRODUCTS.

Bakery products available on Indian shelves and a study of their ingredients

will reveal that the country is not far behind the world as far as application of

nutraceuticals is concerned (Ashlesha Parchure, 2002). Whey proteins have been

considered as a potential ingredient for the bakery industry in view of their desirable

functional characteristics and nutritive value (Gupta and Thapa, 1991).

The physical structure of bread reflects the unique properties of the major

proteins of wheat flour. Upon hydration, gluten forms a stretchable viscoelastic

network that can entrap gas produced by yeasts. The structure stabilizes during baking.

Bread, with milk proteins added in one form or another shows a good crumb structure,

bread yield, flavor and keeping quality (Puranik, 2003). In bread making, some

denaturation of whey proteins is necessary to avoid adverse reactions between whey

proteins and other components of the system. Addition of denatured WPC results in

weaker, less elastic doughs, which after baking yields loaves of reduced volume

(Gupta and Puranik, 1997).

Awasthi and Yadav (2000) studied the effect of incorporation of liquid dairy

by – products on chemical characteristics of soy – fortified biscuits and found that the
incorporation of dairy by – products would further enhance the protein, ash and crude

fiber contents of soy – fortified biscuits, and minerals especially calcium, phosphorus

and iron could also be substantially improved.

In the manufacture of high protein biscuits, milk proteins play an important

role as they increase the nutritive value and also the texture. The unbalanced amino

acid composition of wheat proteins, especially the deficit of lysine, can be improved

by supplementation (Puranik, 2003).

Milk proteins from by – products are often incorporated into the base flour for

pasta manufacture for the purpose of enhancing nutritional quality and to improve

texture. Products fortified by addition of sodium or calcium caseinate, low calcium co

– precipitates or WPC prior to extrusion include macaroni and pasta (Puranik, 2003).

Towler (1982) reported that when a 5% replacement level of undenatured

WPC to the flour, little change was noted apart from some weakening of the cooked

noodles, the addition of 10% WPC gave a very sticky dough, the noodles were

significantly harder to dry, but the color was enhanced.

A number of researches have investigated the possibility of replacing egg

white with whey proteins in the manufacture of cakes with varying degree of success.

A situation quite different from that in bread exists in some cakes, where the main

functions of egg proteins are encapsulation during the mixing process, stabilization of

the aqueous foam in the intermediate baking stage and coagulation of the egg proteins
in the heat – setting stage of the cake batter. Mathur (1975) reported that WPC can

also substitute egg white for the manufacture of meringues and macaroons.

Arunepanlop, et.al. (1996) studied the effects of partial replacement of egg

white proteins with whey protein isolate on the appearance, structure, texture, and

sensory properties of angel food cakes baked in conventional and microwave ovens

and found that upto 25% of the egg white protein (EWP) could be replaced with WPI

without adversely affecting physical and sensory properties of angel food cakes, and

also reported that EWP / WPI blend cakes baked in the conventional oven were given

slightly higher acceptability scores than cake baked in the microwave oven.

Singh, et.al. (2003) reported that the values of all physical properties of cake

decreased with the increase in the level of WPC, and cake volume decreased with 0 to

100% egg replacement.

Puranik (1997) developed a technology for the manufacture of eggless cake

mixes using milk by – products, in lieu of egg in cake formulation produced cake with

excellent physical and sensory characteristics, and the ready – to – use eggless cake

mixes could be stored up to six months at ambient temperature.

WHEY PROTEINS IN DAIRY PRODUCTS

The cheese manufacturing industry has considerable interest in developing

applications to use WPC. Processed cheese foods provide a good opportunity for the

utilization of non – cheese dairy ingredients like cream, skim milk, non – fat dry milk,

whey and whey products etc. Addition of WPC to processed cheese foods would
restore to it most of the proteins lost during cheese manufacture and increase its

nutritive value and yield. Thapa and Gupta (1996) conducted studies on the

manufacture of processed cheese by replacing 15 or 20% of cheese solids of WPC

obtained by Ultrafiltration of cheddar cheese whey and found that processed cheese

foods with 20% WPC solids, containing 43.32 – 46% moisture and 2.5% trisodium

citrate as emulsifier were judged to be best in sensory qualities.

Thapa and Gupta (1992) studied the rheology of processed cheese foods with

added WPC using Instron and reported that increased levels of WPC and emulsifier

and decreased moisture level generally gave higher hardness, springiness,

adhesiveness, gumminess and chewiness, while cohesiveness of the product did not

show any definite trend.

New types of cream cheese spreads have been developed from 59% fat

cultured cream blended with up to 60% whey proteins (on a protein basis) with no

differences in firmness or smoothness from a commercial cheese spread (Modler,

et.al. 1985).

Khoa is one of the most important heat desiccated products in India. Buffalo

milk khoa serves as a base and a filler for the preparation of various milk – based

sweets. Cow milk khoa is generally not favored due to its excessive smooth and pasty

body, slight sandy texture and salty taste (Rajorhia, et.al. 1991). In this context, Patel,

et.al. (1993) studied the effect of addition of WPC to cow milk for preparing khoa and

reported that addition of WPC at 5% produced a desired grainy texture in the WPC

incorporated khoa and also reported that additional browning was also observed in the
WPC incorporated khoa which partly masked the undesirable, yellowish color of cow

milk khoa.

Fermented milk products play a significant role in human nutrition all over the

world. To increase the utility of fermented products like yogurt, kefir, etc. the milk

supplemented with whey proteins is used (Gupta, 1997 b). The formulation of yogurt

products with optimum consistency and stability to synersis (whey separation) is of

primary concern to dairy industry. The viscosity and stability of yogurt is almost

wholly dependent on the protein content of the milk. Jayaprakasha, et.al. (2000 a)

studied the preparation of frozen yogurt by replacing skim milk solids with WPC and

found that with the increase in incorporation of WPC upto 50% level, there was

appreciable decrease in the whey separation.

The WPC containing 35% protein can be used commonly as replacement for

milk solids – not – fat (MSNF) because of the cost advantage. Tirumalesha and

Jayaprakasha (1997 and 1998) conducted considerable studies by incorporating WPC

in ice cream and related products.

Jayaprakasha, et.al. (2000 b) studied the formulation of ready – to – use kulfi

mix by utilizing whey solids and reported that among various levels of admixture was

superior in all aspects compared to other blends of admixture.

WHEY PROTEINS IN MEAT INDUSTRY

Whey protein concentrates may find better applications as ingredients in

minced and ground meat products such as Weiner, Frankfurter, Hotdogs, Bologna
(large sausages) and meat loaves or luncheon meat. Minced meat products often

contain large amounts of fat and the mince is considered to be an emulsion. To

increase the stability of the emulsion during processing and cooking, a protein

product, which is less expensive than meat is often added. Rao, et.al. (1999) reviewed

the role of milk proteins as emulsion stabilizers in comminuted meat products. In

comminuted meat proteins, WPC contributes to fat emulsification, water binding and

improved consistency, as it releases meat proteins for gel formation and water binding.

Egg white powder can be easily replaced with WPC – 80 in fish minced

products and a traditional Japanese product such as Surimi. Because of the excellent

gelling properties of WPC, the protein reinforces the gel, which is formed by the fish

protein; it binds water and makes the product whiter and glossier (Jayaprakasha and

Brueckner, 1999).

Rao, et.al. (1999 b) studied the effect of WPC on the quality of smoked

chicken sausages from broiler spent hens and reported that frying loss of experimental

smoked chicken sausage samples significantly reduced on addition of WPC and it was

only 1.23% in sausages with 3.5% WPC as against the control sample with 4.9%.

Texturized protein of high sensory quality can be produced by extrusion

cooking of WPC – 80. This texturization by extrusion cooking is based on gelling and

the thermoplastic properties of the proteins (Jayaprakasha and Brueckner, 1999). Hale,

et.al. (2002) conducted studies on the preparation of beef patties extended with

extrusion – textured whey proteins and reported that beef patties made with 40%
textured whey proteins were liked by consumer panel as much as the 100% beef

patties in tenderness, juiciness, texture, flavor, and overall acceptability.

Taylor and Walsh (2002) conducted studies on the development of a textured

whey protein meatless patty using WPC – 80 and reported that mushroom flavored

and vegetable flavored textured whey protein patties were as acceptable as commercial

soy patty.

In frankfurters and luncheon rolls, upto 20% of the meat proteins may be

replaced by whey proteins (Jayaprakasha and Brueckner, 1999). The use of whey

proteins can also be considered as partial replacement of meat proteins, partial or total

replacement of soy proteins and other meat binders / fillers, modified starches, and

hydro colloid gums (alginates, gum arabica, and others) in processed meats.

WHEY PROTEINS IN TRADITIONAL FOODS

Malnutrition is one of the biggest concerns of the developing countries. Diets

of different states of Indian sub – continent are marked by the preponderance, of

cereals and millets, but these cereals and millets fail to provide some of the important

nutrients like essential amino acids. Hence, deficiency of almost all the nutrients in the

diets is commonly experienced. The solution to overcome this situation is by

supplementing the average diet with a source of high quality protein from less

expensive sources.

Tripathy, et.al. (2003) studied the effect of incorporation of WPC in some of

the ragi – based (Finger Millet) products such as ragi – malt and ragi – dosa and
reported that incorporation of WPC upto 30% does not have any adverse effect on the

sensory attributes when compared to control. Further, they concluded that ragi dosa

prepared with incorporation of WPC was better than the one prepared without WPC.

Roasted peanuts are highly susceptible to oxidation due to a high content of

lipids that mainly contain unsaturated fatty acids combined with the roasting process.

Lee, et.al. (2002 a) studied the effect of coating the peanuts with whey – protein –

based coating and found that the rancidity was significantly lower for whey proteins –

coated peanuts than for uncoated peanuts.

WHEY PROTEINS IN CONFECTIONERY

Dairy products have been used as valued ingredients by the confectionery

industry for many years as they help achieve the required flavor, color, and texture in

many products including chocolate coatings, caramels, aerated confections, and toffee.

Commercially, three major categories of products are used by the confectionery

industry: sweet whey and modified whey products, WPC and WPI. The milk proteins

in sweetened condensed milk contribute significantly to the emulsification of fats and

give body, texture, and mouth feel to the final product (Hancock, et.al. 1990). In

chocolate manufacture whey powders are generally used as replacers for skim milk

powder to reduce cost (Haylock and Dodds, 1999). Both the emulsifying properties

and dispersibilities of WPC impart good spreadability to chocolate (Edwards, 1984).

Demineralized whey, WPC, and blends are used as total or partial replacement

for milk powders in coating formulations. Polishing and glazing provides a brilliant

surface and a moisture – barrier coating to high quality confectionaries. Lee, et.al.
(2002 b) studied the gloss stability of whey – protein / plasticizer coating formulations

on chocolate surface and found that WPI and sucrose, in the ratio of 1:1, coatings

provided the highest and most stable gloss.

Lee, et.al. (2002 c) reported that water based WPI – lipid coatings can be used

as an alternative glaze, with higher consumer acceptance than alcohol – based shellac.

Protein bars have become popular in the United States of America with the

onset of protein – rich, low – glycemic index diets. Ultra Whey – 99, a powder

typically having 94% protein on dry basis, is currently used as the protein of choice in

many nutrition bars because it provides good – quality protein in a concentrated form

with little associated fat and lactose (Neville, et.al. 2001).

WHEY PROTEINS IN LOW – FAT APPLICATIONS.

Whey protein concentrates are considered as fat mimetics and they have found

extensive use in reduced – fat foods, either alone or in combination with other

mimetics (Johnson, 2000). “Simplesse” is a microparticultated protein that provides

fat – like mouth feel and allows consumers to lower dietary fat without sacrificing

sensory quality. Simplesse is derived from egg whites, skim milk, and whey protein.

This protein based fat – substitute is approved by FDA (Sandrou and Arvanitoyannis,

2000).

“Dairy Lo”, a dried whey product, when used at 2 to 5% in dairy products,

contribute to desirable mouth feel attributes (Sharma, et.al, 1998). The various

commercial protein – based fat replacers is given by Kilara (1998).

 Whey protein concentrates and whey protein isolates are important dairy

proteins used as fat replacers in meat products (Anandh et.al. 2003)

WHEY PROTEINS IN ACID FOODS AND BEVERAGES

Solubility of WPC at low pH, a unique property among proteins, allows it to

function in acid foods and beverages, where non – fat dry milk and casein cannot. This

opens the door to use of WPC as a source of protein in such products as fruit jams,

jellies, fruit juices, carbonated beverages and other soft drinks (Hoogstraten, 1987).

Utilization of Ultrafiltered WPC in drink manufacture ensures the more rational usage

of undenatured whey proteins in food (Kravchenko, 1988). Beverages based on

Ultrafiltered WPC have high nutritive biological value as well. The unique solubility

of WPC enables them to be used in milk – based beverages, fruit juices, soft drinks,

cream liqueurs, wine aperitifs, etc. (Mulvihill, 1991). It is worth noting that WPC

based soft drinks such as strawberry, banana, or yogurt – flavored drinks are already

marketed in Japan (Jayaprakasha and Brueckner, 1999).

The above discussion clearly exhibits that WPC has suitable and significant

functional properties for food application. Therefore, the present study is undertaken.
 CHAPTER – III

MATERIALS AND METHODS

Whey protein concentrates are very desirable as nutritional ingredients due to

their high concentration of sulfur – containing amino acids and also possess

nutraceuticals properties. In the present study an attempt has been made to develop

common bakery products viz. biscuit and cake and a confectionery product, chocolate

by addition of functional ingredient namely whey protein concentrate. All the

experiments were carried out at the Post Graduate and Research Centre, Acharya N.

G. Ranga Agricultural University, Rajendranagar, Hyderabad. A detailed description

of the methodology followed in the development of above-mentioned products is

discussed in this chapter under the following heads.

3.1 Procurement of raw materials.

3.2 Product development.

3.3 Acceptability of the products.

3.3.1 By sensory evaluation.

3.3.2 By objective evaluation

3.4 Proximate analysis.

3.5 Storage studies.

3.5.1 Sensory evaluation.

3.5.2 Chemical analysis.

3.5.3 Microbiological analysis.

3.6 Statistical analysis.

3.1 PROCUREMENT OF RAW MATERIALS.

The materials required for the preparation of the products namely refined flour,

cocoa powder, sugar, baking powder, skimmed milk powder, vanilla essence,

hydrogenated fat, etc. were procured from the local supermarket. The whey protein

concentrate (WPC) (plate – 1) was supplied by M/s Mahaan Proteins Limited, New

Delhi, on request. The proximate composition of the WPC, as given by the

manufacturer, is given in appendix – I.

3.2 PRODUCT DEVELOPMENT.

Common bakery items like biscuit (plate – 2) and cake (plate – 3) and

confectionery like chocolate (plate – 4) were selected and prepared by incorporating

whey protein concentrate (WPC) replacing refined flour at 10, 20 and 30 percent

levels in the basic biscuit and cake recipes. Different trials were made to standardize

the chocolate in the laboratory. In the standardized chocolate recipe, WPC was

replaced with skim milk powder (SMP) at 25, 50, 75 and 100 percent levels since

SMP forms one of the major proportions in the product. The detailed method of

preparation of all the three products is given in appendix – I.

3.3 ACCEPTABILITY OF THE PRODUCTS.

3.3.1 BY SENSORY EVALUATION.

Scorecards were prepared separately, (appendix - II) keeping in view the

quality characteristics of the products, on a five point hedonic scale with trained panel

members (Plate – 5). Descriptive terms were given to various quality attributes like

color, appearance, taste, texture, flavor and overall acceptability. While scoring,
highest score (5) was assigned to most preferred characteristic and least score (1) to

the least desired characteristic.

3.3.2 BY OBJECTIVE EVALUATION.

The texture of the products was assessed by using Universal testing machine or

Instron (Shizmadzu E2 food tester) (Model No: SM – 500 N – 168) for compression

and cutting strength.

3.4 NUTRIENT ANALYSIS.

All the products were analyzed for proximate composition viz. moisture,

protein, fat, crude fiber and ash by following standard methods of AOAC (1984). The

methodology for the estimation of proximates is given in appendix – III to VII. Acid

insoluble ash of the products was estimated following the standard methods of Bureau

of Indian Standards (BIS) (IS: 12711 - 1989) and AOAC (1984) for bakery and

confectionery products respectively, given in appendix – VIII. Carbohydrate content

of the samples was calculated by difference i.e., 100 minus sum of the percentages of

moisture, protein, fat, crude fiber and ash. The calorie content of the products was

computed i.e., total calories = (carbohydrate content x 4) + (Protein content x 4) + (fat

content x 9) and rounded to the nearest value.

3.5 STORAGE STUDIES.

All the products (control and experimental) viz. biscuits, cakes and chocolates

were packed in metallized polypropylene pouches (plate - 6) having a thickness of 40

and stored at room temperature.

3.5.1. ACCEPTABILITY STUDIES.

Biscuit and chocolate were stored for up to 2 months from the date of

manufacture of the products, while cake was stored up to 15 days from the date of

preparation. The acceptability of the stored products was evaluated by 10 trained panel

members using the scorecards. The acceptability of biscuits and chocolates were

evaluated intermittently at fresh, after one and two months, while cake was evaluated

fresh, after five, ten and fifteen days from the date of preparation.

3.5.2 CHEMICAL ANALYSIS.

The products were analyzed for moisture and acidity of the extracted fat during

the storage periods using the standard procedures outlined in the bulletin of Bureau of

Indian Standards (IS 12711: 1989). The estimation of moisture and acidity of extracted

fat was carried out fresh, after one and two months for biscuit and chocolate while

cake was analyzed fresh, after five, ten and fifteen days from the date of preparation

(appendix - IX).

3.5.3 MICROBIOLOGICAL STUDIES.

The microbiological quality of fresh and stored products was evaluated by

determining the colony forming units of bacteria, yeast, and molds per gram of the

product by following the procedure given by Harrigan (1998). The detailed methods

for determining bacterial count and yeast and mold count are given in appendix – X.

For biscuit and chocolate samples, microbiological count was done initially and at

different intervals. While for cake samples it was done initially, after 5, 10, and 15

days of storage.
3.6 STATISTICAL ANALYSIS.

Statistical analysis was carried out by the procedures laid down by Snedecor

and Cochran (1989). The data was tabulated and subjected to statistical analysis at the

end of the study for Mean, Standard Deviation (SD) and Analysis of Variance

(ANOVA).
Plate – 1. Whey Protein Concentrate used in the present study.

1. Control 2. 10 % WPC 3. 20 % WPC 4. 30 % WPC

Plate 2. Prepared Control and Experimental Biscuits

1. Control. 2. 10 % WPC. 3. 20 % WPC. 4. 30 % WPC.

Plate 3. Prepared Control and Experimental Cakes.

1. 25 % WPC 2. 50 % WPC 3. 75 % WPC 4. 100% WPC.

Plate – 4. Prepared Experimental Chocolates.

Plate – 5. Sensory Evaluation of developed products by Judges.

Plate – 6. Metallized Polypropylene Pouches used in the study.

 CHAPTER – IV

RESULTS AND DISCUSSION

Whey protein concentrates are potential functional ingredients used in formulated

foods as they contain sulfur – containing amino acids, and possess nutraceutical

properties. In the present study two common bakery items namely biscuit and cake and a

confectionery item chocolate were prepared by incorporating the whey protein

concentrate at different levels. The acceptability of the products was carried out by

sensory and objective evaluation. The proximates analysis of developed products was

carried out. The sensory, chemical and microbiological quality of the stored products was

also assessed. The results obtained in the present study are presented and discussed under

the following heads.

4.1. Development and evaluation of the products.

4.1.1. Sensory evaluation of the products.

4.1.2. Objective evaluation of the products.

4.2. Estimation of nutrients in the developed products.

4.3. Storage studies.

4.3.1. Sensory analysis of the stored products.

4.3.2. Chemical analysis of the stored products.

4.3.3. Microbiological studies of the stored products.

4.1 DEVELOPMENT AND EVALUATION OF THE PRODUCTS.

4.1.1. SENSORY EVALUATION OF THE PRODUCTS.

The control and the experimental products were evaluated by selected panel of

trained judges using scorecard prepared separately for each product on a five point

hedonic scale.

BISCUIT.

The mean scores of sensory evaluation of the biscuit samples are presented in

Table – 7. The results revealed that the highest scores for sensory characteristics texture

(4.8) and flavour (4.9) were obtained for the control biscuit sample. The biscuit prepared

by incorporating 20 and 30% WPC received highest score for color and appearance (4.2).

The biscuit prepared by incorporating 20% WPC got highest score for taste (4.6). The

control biscuit secured highest score (4.6) for the overall acceptability.

Among the WPC incorporated biscuits, the biscuit prepared by incorporating 10%

WPC obtained highest score for texture (4.6) and overall acceptability (4.5). The biscuit

prepared by incorporating 20% WPC obtained highest scores for taste (4.6) and flavour

(4.8).

Lactose present in whey protein concentrate readily reacts with proteins

(Maillard’s reaction) giving baked products a highly flavored desirable golden brown

color (Burrington, 1999). The highest scores obtained for color and appearance of

biscuits prepared with 20 and 30% WPC than the control samples, could be attributed to

the Maillard’s reaction.

 Table – 7. Mean Scores of sensory evaluation of biscuits.
Level of
Color and Overall
WPC Texture Taste Flavor
Appearance Acceptability
(%)
Control 4.10 + 0.88 4.80 + 0.63 4.50 + 0.53 4.90 + 0.32 4.60 + 0.52
10 4.10 + 0.88 4.60 + 0.84 4.50 + 0.71 4.60 + 0.70 4.50 + 0.71
20 4.20 + 0.79 4.50 + 0.71 4.60 + 0.52 4.80 + 0.42 4.40 + 0.70
30 4.20 + 0.79 4.30 + 0.95 4.50 + 0.53 4.50 + 0.71 4.30 + 0.67

The low scores for flavor of biscuits prepared by incorporating WPC could be due

to the flavor binding capacity of WPC. Thapa and Gupta (1996) reported that higher

levels of WPC addition imparted milder flavor to the processed cheese foods. Similar

results were obtained by Jayaprakasha et.al. (2000 a) who reported that decreased flavor

scores in frozen yogurt were obtained when the extent of substitution of skim milk solids

increased from 10 to 50 percent.

In the present investigation, the texture of biscuits was not improved much

beyond 10% incorporation of WPC. The low scores for texture of WPC incorporated

biscuits could be due to increased moisture content. Moisture loss and gain is a serious

problem in many bakery products that can result in textural changes (James et.al. 2004).

Kim and Maga (1987) have observed crispy texture of cereal flour at 20% blend of WPC

besides improvement in flavor and appearance.

The overall acceptability of the experimental biscuits received an average score of

4.4, which indicates that WPC can be incorporated into wheat flour upto 30% without

much changes in sensory attributes. In another study conducted by Dogra et.al. (2004) it
was found that acceptable biscuits could be prepared by incorporating upto 30% soy bean

flour in wheat flour.

CAKE.

The mean scores of sensory evaluation of cake samples are presented in Table –

8. It was observed that the cakes prepared by incorporating WPC at 10, 20 and 30 percent

received highest scores for color and appearance (4.5). The cake prepared by

incorporating 20% WPC obtained highest scores for texture (4.7). The control cake

sample received highest score for taste (4.6), while all the experimental cakes received a

uniform score (4.5). Both the control and 20% WPC incorporated cake got highest score

for flavor (4.6). The 10% WPC incorporated cake was adjudged with highest score for

overall acceptability (4.6).

It is evident from the sensory analysis that all the cakes incorporated with WPC

are better in color and appearance and texture than the control sample. Improved color

and appearance could be due to Maillard’s reaction. Improved texture of WPC cakes

could be due to the emulsifying properties of WPC. Whey protein concentrate will give a

good fat distribution because of hydrophilic and lipophylic properties and thus aid in

good structure formation in the bread (Jayaprakasha and Brueckner, 1999).

From the scores for overall acceptability of the WPC incorporated cakes it can be

inferred that incorporation of WPC upto 30% does not have any adverse effect on the

sensory attributes when compared to control. This could be attributed to the functional

properties possessed by whey proteins.

 Table – 8. Mean Scores of sensory evaluation of cake.

Level of
Color and Overall
WPC Texture Taste Flavor
Appearance Acceptability
(%)
Control 4.40 + 0.52 4.50 + 0.53 4.60 + 0.52 4.60 + 0.52 4.50 + 0.53
10 4.50 + 0.53 4.60 + 0.52 4.50 + 0.53 4.50 + 0.53 4.60 + 0.52
20 4.50 + 0.53 4.70 + 0.48 4.50 + 0.53 4.60 + 0.52 4.50 + 0.53
30 4.50 + 0.53 4.60 + 0.52 4.50 + 0.53 4.50 + 0.53 4.50 + 0.53

Sinha and Kulkarni (2004) reported that cakes with 30% soy flour

supplementation were highly acceptable. Similarly, Tripathy et.al. (2003) reported that

ragi based foods prepared by incorporating WPC received high scores for over all

acceptability.

CHOCOLATE.

The mean scores of sensory evaluation of chocolate samples are presented in

Table – 9. It was observed that the chocolates prepared by replacing 50% of skim milk

powder (SMP) in the standardized recipe with WPC received highest scores for color and

appearance (4.4). The chocolates prepared by replacing 50 and 75% of SMP with WPC

got highest scores for texture (4.4).

The control chocolate received highest scores for taste (4.3). The highest score for

flavor was obtained by control chocolate, chocolate prepared by replacing 50 and 75% of

SMP with WPC (4.6). The highest score for overall acceptability of the chocolates was

obtained by the chocolate prepared by replacing 75% of SMP with WPC (5.0).
 The control, and the experimental samples prepared by replacing 25, 75 and 100%

SMP with WPC all received similar score for the color and appearance (4.2). This could

be due to uniform color of cocoa powder in all the control and experimental samples,

which the judges could not differentiate. The chocolate prepared by replacing 100% SMP

with WPC recorded least score for texture (3.6) and taste (3.8). The low scores for texture

of chocolate with 100% replacement of SMP could be due to higher moisture in the

product. The 100% SMP replaced chocolate recorded low score, which could be due to

the flavor binding capacity of WPC, which might have masked the flavor of coca powder

slightly.

Table – 9. Mean Scores of sensory evaluation of chocolate.

Levels
Color and Overall
of WPC Texture Taste Flavor
Appearance Acceptability
(%)
Control 4.20 + 0.75 3.90 + 0.70 4.30 + 0.64 4.60 + 0.49 4.60 + 0.49
25 4.20 + 0.75 4.00 + 0.63 4.00 + 0.63 4.40 + 0.49 4.20 + 0.75
50 4.40 + 0.80 4.40 + 0.80 4.00 + 0.63 4.60 + 0.80 4.40 + 0.49
75 4.20 + 0.75 4.40 + 0.49 4.00 + 0.63 4.60 + 0.49 5.00 + 0.00
100 4.20 + 0.75 3.60 + 0.49 3.80 + 0.40 4.40 + 0.49 4.40 + 0.80

The highest scores for texture of chocolates prepared by replacing 50 and 75% of

SMP with WPC may be due to the graininess of the samples, which might be due to

protein precipitation as opined by the judges. This improved the biting characteristics of

chocolate. The overall acceptability score of the 75% SMP replaced sample received

ultimate score of 5.0. Heating to more than 70% could cause partial loss of solubility

between pH 3 and 5 because some of the whey proteins might aggregate and precipitate

at their isoelectric points ranging from pH 4.5 to 5.3 (Jayaprakasha and Brueckner, 1999).
It was observed that beyond 75% replacement of SMP with WPC the overall

acceptability scores decreased.

4.1.2. OBJECTIVE EVALUATION OF THE PRODUCTS.

The Shimadzu E2 tester or Instron is a universal testing machine that can be used

to evaluate the physical properties for controlling the quality of food, medical supplies,

textiles, packaging materials, etc. A simple tensile test, compression test or any other can

be conducted easily by selecting the test jigs suited to the test from a wide variety of jigs

separately available and mounting them to the machine. In the present study for the

instrumental texture analysis of control and experimental products the Shimadzu E 2 tester

was used. The results obtained in the instrumental texture analysis of the prepared

products are presented in Table – 10.

BISCUIT.

The results revealed that the highest value for cutting strength was recorded by

the biscuit prepared by incorporating 30% WPC (33.775 N). The lowest value was

recorded by the control biscuit sample (21.363 N). While the cutting strength of the

biscuits prepared by incorporating 10 and 20% WPC are 21.625 and 33.488 N

respectively. The cutting strength of the samples increased as the level of WPC of the

biscuit increased. The increased values of cutting strength of WPC incorporated biscuits

imply that the hardness of the biscuits was increased.

Similar results were obtained by Gandhi et.al. (2001) who reported that the

cutting strength increased as the protein content increased in the defatted soy flour
fortified biscuits. The increased values of cutting strength could be due to increase in

protein content and decrease in fat and carbohydrates.

The highest compression strength value was recorded by the cake prepared by

incorporating 30% WPC (16.325 N). The lowest value was recorded by the control

biscuit (15.263 N). The compression strength values of the samples increased as the

protein content of the biscuit increased.

CAKE.

The results in Table – 10 reveal that the highest cutting strength value was

recorded by the cake prepared by incorporating 30% WPC (2.9 N). The lowest value was

obtained by the control cake sample. The highest compression strength value was

recorded by the cake prepared by incorporating 30% WPC (50.214 N) while the control

cake recorded the lowest value (50.175 N).

The results revealed that there is an increase in both the cutting strength and

compression strength values of the control and experimental cake samples. The increase

in the cutting strength values could be due to increase in the levels of WPC of the

experimental cakes.

CHOCOLATE.

It was observed that the highest cutting strength value was recorded by the

chocolate sample prepared by replacing 100 percent SMP with WPC in the standard

recipe (3.285 N). The lowest value was obtained by the control chocolate (2.375 N)

(Table – 10).
 Table – 10. Mean values of objective evaluation of developed products.

Levels of
Cutting Strength Compression Strength
Product Incorporation
(in Newton) (in Newton)
(%)
Control 21.363 15.263
10 21.625 15.383
Biscuit
20 33.488 15.633
30 33.775 16.325
Control 2.2 50.175
10 2.4 50.188
Cake
20 2.7 50.200
30 2.9 50.214
Control 2.375 15.138
25 2.510 15.413
Chocolate 50 2.846 15.525
75 2.984 15.588
100 3.285 15.675

The lowest compression strength value was recorded by the control chocolate

(15.138 N) while the highest value was obtained by the chocolate prepared by replacing

100% SMP with WPC in the standardized recipe (15.675 N).

4.2 ESTIMATION OF NUTRIENTS IN THE DEVELOPED PRODUCTS.

BISCUIT.

The nutrient composition of the control and experimental biscuits is presented in

Table – 11. The results reveal that the moisture content of the control and experimental

biscuits ranged from 2.71 to 2.91%. The control biscuit contained 2.71% of moisture. The

moisture content of the 10, 20 and 30 percent WPC incorporated biscuits was 2.77, 2.84
and 2.91 percent respectively. The moisture content in the samples increased with the

increase in protein content. The fat content of the control and experimental biscuits

ranged from 18.5 to 20.5%. The control biscuit contained 20.5% fat while the biscuits

containing 10, 20 and 30% WPC contained 20.0, 19.2 and 18.5 % respectively. The fat

content of the samples decreased as the protein content increased in the biscuits. This

could be due to binding of fat by whey proteins (Kinsella and Whitehead, 1989) and the

complete extraction of fat by petroleum ether could not be possible. Further investigation

is required for the modification of fat extraction process whereby the bound fat may be

freed when whey proteins are used in the formulation.

The protein content in the control and experimental biscuits ranged from 5.23 to

14.11% (Fig.1). The control biscuit contained 5.23% protein, while the experimental

biscuits prepared by incorporating 10, 20 and 30% WPC contained 8.06, 11.71, and

14.11% respectively. The protein content in the experimental biscuits increased with the

increase in levels of incorporation of WPC.

The carbohydrate content of the control and experimental biscuits ranged from

62.58 to 70.04%. While the biscuit containing 10, 20 and 30% WPC contained 67.47,

64.44, and 62.58% respectively. The carbohydrate content of the biscuits decreased with

the increase in the levels of incorporation of WPC. The crude fiber content in the control

and experimental biscuits ranged from 0.4 to 0.47%. The control biscuit contained 0.47

% crude fiber while the biscuit containing 10, 20 and 30% WPC contained 0.45, 0.42,

and 0.40 percent respectively. With the increase in levels of WPC incorporation, the

crude fiber content decreased in the biscuits.

 Table – 11. Nutrient composition of control and experimental biscuits (%).

Levels of Incorporation
Control 10 20 30
Moisture 2.71 2.77 2.84 2.91
Fat 20.50 20.00 19.20 18.50
Protein 5.23 8.06 11.71 14.11
Carbohydrates* 70.04 67.47 64.44 62.58
Crude Fiber 0.47 0.45 0.42 0.40
Ash 1.06 1.25 1.39 1.50
Acid insoluble Ash 0.23 0.20 0.24 0.23
Energy Content **
486 482 477 473
(Kcal)

* by difference ** computed value

The ash content in the control and experimental biscuits ranged from 1.06 to

1.50%. The control biscuit contained 1.06 % ash while the biscuits containing 10, 20 and

30% WPC contained 1.25, 1.39 and 1.50 % respectively. The percent of ash increased

with the increase in the levels of incorporation of WPC. The acid insoluble ash content of

the control and experimental biscuits ranged from 0.20 to 0.24 %. The control biscuit

contained 0.23 % of the acid insoluble ash while the biscuits prepared by incorporating

10, 20, and 30 % WPC contained 0.20, 0.24, and 0.23 % respectively acid insoluble ash.

There was no much difference observed between the acid insoluble ash content of control

and experimental biscuits with the levels of incorporation of WPC.

The water binding or holding capacity of native whey proteins can be enhanced

by heat treatments that particularly denature these proteins. Heating whey protein

solution causes a slight increase in viscosity and water holding capacity. The partial

unfolding of the protein by heat exposes additional water binding sites that are

unavailable in the native unheated proteins and increases the volume occupied by the

protein (Jayaprakasha and Brueckner, 1999). The increased moisture content of the
control and experimental biscuits in the present study may be due to denaturation of the

whey proteins during baking and increased water holding capacity of WPC.

With the incorporation of WPC in the biscuits an increase in protein and total ash,

and decrease in fat, carbohydrate and crude fiber contents was observed. The increase in

the concentration of protein and minerals could be mainly attributed to the composition

of WPC used in the investigation, which had higher percentage of these constituents

compared to carbohydrate content.

Similar results were obtained by Gandhi et.al (2001) who reported that the protein

and ash contents increased from 6.5 to 14.8% and 4.2 to 5.0% respectively with

increasing the defatted soy flour in the biscuit from 0 to 40%. The fat and carbohydrate

contents decreased from 26 to 21% and 61.5 to 55.2%, respectively with the

incorporation of defatted soy flour. Similar findings were also obtained by Awasthi and

Yadav (2000).

The moisture and crude fiber content of all the samples are within the standards

laid down by the Bureau of Indian Standards (BIS) for the protein-enriched biscuits (IS:

7487 - 1986) (Appendix - X). The fat content of the control and experimental biscuits is

above the minimum fat content of 12% for the protein-enriched biscuits. The

experimental biscuit prepared by incorporating 30% WPC meets the specifications of BIS

for protein-enriched biscuit.

The energy content of the control and experimental biscuits ranged from 473 to

486 Kcal. The control biscuit contained 486 Kcal while the biscuit prepared by
incorporating 10, 20, and 30 % WPC contained 482, 477, and 473 Kcal respectively. The

calculations were based on the extracted fat. But, the so-called bound fat also contribute

to the energy content. Considering the bound fat, the energy content of the biscuits

prepared by incorporating WPC ranges from 483 to 485 Kcal. The 10, 20 and 30 % WPC

containing biscuits would contain 485, 484 and 483 Kcal of energy respectively. Similar

results were observed by Shalini (2003) in the preparation of defatted soy flour enriched

biscuits.

CAKE.

The nutrient composition of control and experimental cake is presented in Table –

12. The moisture content of control and experimental cakes ranged from 18.76 to

20.55%. The control cake contained 18.76% of moisture while the cakes prepared by

incorporating 10, 20, and 30% WPC contained 18.86, 19.55, and 20.55% respectively.

The moisture content increased with the increase in the level of incorporation of WPC.

The increased moisture content could be due to the increased water holding capacity of

whey proteins due to denaturation of these proteins during baking.

The fat content of the control and experimental cakes ranged from 25.18 to

32.84%. The control cake contained 32.84%, while the cakes prepared by incorporating

10, 20, and 30% WPC contained 30.0, 28.66 and 25.18% respectively. With the increase

in the level of incorporation of WPC there was a decrease in the fat content of the

prepared cakes. This could be due to binding of fat by whey proteins and the complete

extraction of fat by petroleum ether could not be possible and also may be due to lower

amount of fat in the WPC used for the present investigation. Shalini (2003) reported

similar trend of decrease of fat upon increasing the levels of defatted soy flour in cakes.
 Table – 12. Nutrient composition of control and experimental cakes (%).

Levels of Incorporation
Control 10 % 20 % 30 %
Moisture 18.76 18.86 19.55 20.55
Fat 32.84 30.00 28.66 25.18
Protein 4.71 6.67 8.89 11.01
Carbohydrates* 42.61 43.27 41.65 41.82
Crude Fiber 0.48 0.45 0.40 0.38
Ash 0.60 0.75 0.85 1.06
Acid insoluble Ash 0.07 0.07 0.07 0.07
Energy Content **
485 467 460 438
(Kcal)

* by difference ** computed value

The protein content of the control and experimental cakes ranged from 4.71 to

11.01% (Fig. 2). The control cake contained 4.71% protein while the cakes prepared by

incorporating 10, 20, and 30% WPC contained 6.67, 8.89, and 11.01% respectively.

Obviously with the increase in levels of incorporation of WPC there was an increase in

the protein content of cakes. The carbohydrates content of the control and experimental

cakes ranged from 42.61 to 43.27%. The control sample contained carbohydrate content

of 42.61% while the cake prepared by incorporating 10, 20, and 30% WPC contained

43.27, 41.65, and 41.82% respectively.

The crude fiber content of the control and experimental cakes ranged from 0.38 to

0.48%. The control cake contained 0.48% of crude fiber while the cakes prepared by

incorporating 10, 20, and 30% WPC contained 0.45, 0.40 and 0.38% respectively. With

the increase in the levels of WPC there was a decrease in the levels of crude fiber. The

decreased carbohydrate and crude fiber content of the experimental biscuits could be due

to the lower amount of these compounds in the WPC used for the present investigation.
 The ash content of the cakes prepared ranged from 0.6 to 1.06%. The control cake

contained 0.6% ash and the cake prepared by incorporating 10, 20, and 30% WPC

contained 0.75, 0.85, and 1.06% respectively. With the increase in the level of WPC there

was an increase in the ash content. This could be due to higher amounts of ash in WPC.

The acid insoluble ash content was found to be same in all the control and experimental

cakes.

The moisture and acid insoluble ash contents of all the cake samples are within

the range of the specifications for plain cake laid down by the BIS (IS: 9712 - 1981)

(Appendix - X). Similar results were obtained by Awasthi and Pareek (2004) who

reported that 25% defatted soy flour incorporation in soy fortified egg less cake resulted

in 96.49% rise in protein and 50.23% rise in ash content.

The energy content of the control and experimental cakes ranged from 438 to 485

Kcal. The control cake contained 485 Kcal while the cake prepared by incorporating 10,

20, and 30% WPC contained 467, 460, and 438 Kcal respectively. With the increase in

the levels of WPC incorporation the energy values decreased in the cake samples. The

calculations were based on the extracted fat. But, the so-called bound fat also contribute

to the energy content. Considering the bound fat, the energy content of the cakes prepared

by incorporating WPC ranges from 476 to 484 Kcal. The 10, 20 and 30 % WPC

containing cakes would contain 484, 481 and 476 Kcal of energy respectively. Similar

decreased energy values in defatted soy flour fortified cake were observed by Shalini

(2003). Sinha and Kulkarni (2004) reported that 30% soy fortified cake contributed 380

Kcal of energy.
CHOCOLATE.

The nutrient composition of control and experimental chocolates is presented in

Table – 13. The moisture content of the control and experimental chocolates ranged from

7.40 to 11.32%. The control chocolate contained 7.40% moisture while the chocolate

prepared by replacing 25, 50, 75, and 100% SMP in the standard recipe with WPC

contained 8.35, 9.6, 10.82, and 11.32% respectively. With the increase in the levels of

replacement of SMP with WPC the moisture content increased. The increased moisture in

the present investigation could be attributed to the water holding capacity, a functional

property of the whey proteins. Water holding capacity of whey proteins enhances upon

denaturation (Jayaprakasha and Brueckner, 1999).

The total fat content of the control and experimental chocolates ranged from

31.80 to 34.26%. The control chocolate contained 34.26% fat while the chocolate

prepared by replacing 25, 50, 75, and 100% SMP with WPC contained 34.04, 33.23,

32.54, and 31.80% respectively. With the increase in the levels of replacement of SMP

with WPC the fat content decreased. This could be due to the increase in the moisture

content of the samples. The total fat content of all the control and experimental

chocolates was found to be far above the specifications for milk chocolate laid down by

the BIS (IS: 1163 - 1992) (Appendix - X).

The protein content of the control and experimental chocolates ranged from 4.25

to 7.69% (Fig.3). The control sample contained 4.25% protein while the chocolates made

by replacing 25, 50, 75, and 100% SMP with WPC contained 5.06, 5.94, 6.81, and 7.69%

respectively. Obviously there is an increase in protein content with the increased levels of

replacement of SMP with WPC.

 Table – 13. Nutrient composition of control and experimental chocolates (%).

Levels of Replacement of SMP with WPC

Control 25 % 50 % 75 % 100 %
Moisture 7.40 8.35 9.60 10.82 11.32
Fat 34.26 34.04 33.23 32.54 31.80
Protein 4.25 5.06 5.94 6.81 7.69
Carbohydrates* 53.07 51.43 49.92 48.35 47.56
Crude Fiber 0.25 0.25 0.24 0.24 0.24
Ash 0.77 0.87 1.07 1.24 1.39
Acid insoluble Ash 0.10 0.12 0.14 0.16 0.18
Energy Content **
538 532 522 514 507
(Kcal)

* by difference ** computed value

The carbohydrate content of the control and experimental chocolates

ranged from 47.56 to 53.07%. The control sample contained 53.07% while the chocolate

prepared by 25, 50, 75, and 100% replacement of SMP with WPC contained 51.43,

49.92, 48.35 and 47.56% respectively. It was found that there was decrease in the

carbohydrate content with increased levels of WPC incorporation in the samples. The

crude fiber content of control and experimental chocolates was almost same. The control

chocolate and the chocolate prepared by replacing 25% of SMP with WPC contained

0.25% of crude fiber while the chocolate prepared by replacing 50, 75, and 100% of SMP

with WPC in the standard recipe contained 0.24% each.

The ash content of the control and the experimental chocolates ranged from 0.77

to 1.39%. The control chocolate contained 0.77% ash while the chocolate prepared by

replacing 25, 50, 75, and 100% SMP with WPC contained 0.87, 1.07, 1.24 and 1.39%

respectively. The increase in ash content in the experimental samples could be due to

high ash content of WPC. The acid insoluble ash content of the control and experimental

chocolates ranged from 0.10 to 0.18%. The control sample contained 0.10% acid
insoluble ash while the chocolate prepared by replacing 25, 50, 75, and 100% SMP with

WPC contained 0.12, 0.14, 0.16, and 0.18% respectively. With the increase in the levels

of replacement of SMP with WPC the acid insoluble ash was increased. The acid

insoluble ash content of all the chocolate samples is within the standards laid down by the

BIS (IS: 1163 - 1992).

The energy values of the control and the experimental chocolates ranged from 508

to 538 Kcal. The control chocolate contained 538 Kcal while the chocolate made by

replacing 25, 50, 75, and 100% SMP with WPC had 532, 523, 514, and 507 Kcal

respectively. With the increased levels of replacement of SMP with WPC the energy

values decreased. The calculations were based on the extracted fat. But, the so-called

bound fat also contribute to the energy content. Considering the bound fat, the energy

content of the chocolates prepared by incorporating WPC ranges from 519 to 533 Kcal.

The chocolates made by replacing 25, 50, 75, and 100% SMP with WPC would contain

533, 526, 522 and 519 Kcal respectively.

4.3. STORAGE STUDIES.

4.3.1. SENSORY ANALYSIS OF STORED PRODUCTS.

All the control and experimental products viz. biscuits, cakes and chocolates were

packed in metallized polypropylene pouches and stored at room temperature. Biscuits,

and chocolates, were stored for 60 days while cake was stored only for 15 days.

BISCUIT.

The control and experimental biscuits were stored upto 60 days. The sensory

analysis of the prepared biscuits was done fresh, after a storage period of 30 and 60 days.

The mean sensory scores received for various sensory parameters like color and
appearance, texture, taste, flavor, and overall acceptability during these storage periods is

given in Table – 14.

Table – 14. Mean sensory scores obtained for biscuits during storage period.

Sensory Attributes
Storage
Period Color and Overall
Texture Taste Flavor
Appearance Acceptability
Control
Fresh 4.10 + 0.88 4.80 + 0.63 4.50 + 0.53 4.90 + 0.32 4.60 + 0.52
30 days 4.00 + 0.00 3.80 + 0.92 4.40 + 0.52 4.60 + 0.70 4.20 + 0.42
60 days 3.90 + 0.74 3.80 + 0.79 4.40 + 0.52 4.10 + 0.32 3.90 + 0.57
10 % Incorporation of WPC
Fresh 4.10 + 0.88 4.60 + 0.84 4.50 + 0.71 4.60 + 0.70 4.50 + 0.71
30 days 3.80 + 0.42 4.10 + 0.88 4.40 + 0.52 4.70 + 0.48 4.30 + 0.48
60 days 3.70 + 0.67 3.80 + 0.79 4.10 + 0.57 4.20 + 0.63 3.90 + 0.57
20 % Incorporation of WPC
Fresh 4.20 + 0.79 4.50 + 0.71 4.60 + 0.52 4.80 + 0.42 4.40 + 0.70
30 days 4.30 + 0.48 4.20 + 0.63 4.10 + 0.57 4.50 + 0.53 4.30 + 0.48
60 days 4.00 + 0.67 4.10 + 0.74 4.10 + 0.74 4.40 + 0.52 4.00 + 0.47
30 % Incorporation of WPC
Fresh 4.30 + 0.67 4.30 + 0.95 4.50 + 0.53 4.50 + 0.71 4.20 + 0.79
30 days 3.90 + 0.32 4.30 + 0.82 4.10 + 0.74 4.20 + 0.63 4.00 + 0.47
60 days 3.90 + 0.74 4.00 + 0.67 4.10 + 0.57 4.00 + 0.67 4.00 + 0.67

In the present study it was observed that the scores for the sensory attributes

like color and appearance, texture, taste and flavor of the control and experimental

biscuits decreased with storage, however no undesired changes were noticed in the

biscuits.

The analysis of variance values (ANOVA) for taste panel scores of the overall

acceptability of biscuits is presented in Table – 15. The perusal of the table reveals that

there were no significant changes observed between levels of incorporation of WPC. But

significant changes were recorded in the overall acceptability after 30 days of storage.
 The interaction effect between treatment and the storage period was also found

significant. The control sample at zero days of storage was at par with 30 days of storage

but recorded significantly superior scores over 60 days of storage. The experimental

biscuit containing 10 percent WPC at zero days storage was at par with 30 days storage

but recorded significantly higher scores over 60 days of storage.

Table – 15. ANOVA of taste panel scores for overall acceptability of biscuits during
storage.

Levels of
0 Day 30 Days 60 Days Mean
WPC (%)
Control 4.6 4.2 3.9 4.2333
10 4.5 4.3 3.9 4.2333
20 4.4 4.3 4.0 4.2333
30 4.2 3.9 4.0 4.0333
Mean 4.425 4.175* 3.95*
S.E + C.D. (0.05)
Levels of WPC 0.1519 NS
Storage Periods 0.1316 0.2579
Interactions 0.2632 0.5158

* Significant at 5% level of significance.

It can be inferred from the statistical analysis of the sensory scores that upto 30%

WPC can be incorporated into wheat flour to prepare protein enriched biscuit and stored

upto 60 days without any major sensory changes.

CAKE.

The control and experimental cakes were stored upto 15 days. The sensory

evaluation of the prepared cakes was done at fresh, after 5, 10 and 15 days. The mean

sensory scores obtained for various sensory parameters during these storage periods are

given in Table – 16. The sensory scores of the control and experimental cakes stored upto
15 days recorded lower scores than the zero day observations. It was observed that, there

was an increase in the scores for color and appearance of control cake stored for 5 days.

However, the scores decreased after 15 days of storage. During the storage no undesired

changes occurred.

Table –16. Mean sensory scores of cakes obtained during storage.

Sensory Attributes
Storage
Period Color and Overall
Texture Taste Flavor
Appearance Acceptability
Control
Fresh 4.40 + 0.52 4.50 + 0.53 4.60 + 0.52 4.60 + 0.52 4.50 + 0.53
5 days 4.80 + 0.42 4.30 + 0.48 4.70 + 0.48 4.70 + 0.48 4.70 + 0.48
10 days 4.90 + 0.32 4.40 + 0.52 4.60 + 0.52 4.60 + 0.52 4.40 + 0.52
15 days 4.30 + 0.67 4.40 + 0.70 4.50 + 0.53 4.30 + 0.82 4.30 + 0.48
10 % Incorporation of WPC
Fresh 4.50 + 0.53 4.60 + 0.52 4.50 + 0.53 4.50 + 0.53 4.60 + 0.52
5 days 4.40 + 0.52 4.30 + 0.48 4.80 + 0.42 4.70 + 0.48 4.60 + 0.52
10 days 4.40 + 0.52 4.20 + 0.42 4.80 + 0.42 4.60 + 0.52 4.60 + 0.52
15 days 4.60 + 0.52 3.80 + 0.42 4.20 + 0.42 4.30 + 0.48 4.30 + 0.48
20 % Incorporation of WPC
Fresh 4.50 + 0.53 4.70 + 0.48 4.50 + 0.53 4.60 + 0.52 4.50 + 0.53
5 days 4.40 + 0.52 4.10 + 0.32 4.50 + 0.53 4.60 + 0.52 4.60 + 0.52
10 days 4.40 + 0.52 4.20 + 0.42 4.50 + 0.53 4.60 + 0.52 4.60 + 0.52
15 days 4.00 + 0.67 3.50 + 0.53 4.10 + 0.32 4.30 + 0.48 4.00 + 0.47
30 % Incorporation of WPC
Fresh 4.50 + 0.53 4.60 + 0.52 4.50 + 0.53 4.50 + 0.53 4.50 + 0.53
5 days 4.40 + 0.52 4.20 + 0.42 4.20 + 0.42 4.60 + 0.52 4.60 + 0.52
10 days 4.40 + 0.52 4.20 + 0.42 4.20 + 0.42 4.60 + 0.52 4.60 + 0.52
15 days 4.10 + 0.57 3.90 + 0.57 4.40 + 0.70 4.30 + 0.67 4.10 + 0.74

The ANOVA of taste panel scores of the overall acceptability of cakes is

presented in Table – 17. No significant changes were observed between the levels of

incorporation of WPC. But significant changes were recorded in the storage periods. It

was observed that significant changes were recorded in the overall acceptability of the

control and experimental cakes after 15 days of storage.

 The interaction effect between level of incorporation and the storage period was

also found significant. The experimental cake containing 20 percent WPC at zero days

storage was at par with 5 and 10 days storage but recorded significantly higher scores

over 15 days of storage.

It can be inferred from the statistical analysis, that upto 30% WPC can be

incorporated to prepare protein enriched cake and stored upto 15 days without any major

sensory changes.

Table – 17. ANOVA of taste panel scores for overall acceptability of cakes.

0 Day 5 Days 10 Days 15 Days Mean

Control 4.5 4.6 4.4 4.3 4.45
10 % 4.6 4.6 4.6 4.3 4.525
20 % 4.5 4.6 4.6 4.0 4.425
30 % 4.5 4.6 4.6 4.3 4.5
Mean 4.525 4.6 4.55 4.225 *
S.E + C.D. (0.05)
Levels of WPC 0.1170 NS
Storage Periods 0.1170 0.2292
Interactions 0.2339 0.4585

* Significant at 5% level of significance.

CHOCOLATE.

The control and experimental chocolates were stored upto 60 days. The sensory

evaluation of the prepared chocolates was done at fresh, after 30 and 60 days. The mean

sensory scores received for the various sensory parameters during the storage period are

given in Table – 18. The results revealed that the sensory scores of chocolate decreased

with storage.
Table – 18. Mean sensory scores for overall acceptability chocolates during storage.

Sensory Attributes
Storage
Period Color and Overall
Texture Taste Flavor
Appearance Acceptability
Control
Fresh 4.20 + 0.75 3.90 + 0.70 4.30 + 0.64 4.60 + 0.49 4.60 + 0.49
30 days 3.90 + 0.70 3.30 + 0.46 4.00 + 0.45 4.30 + 0.46 4.10 + 0.70
60 days 3.70 + 0.48 3.30 + 0.48 3.90 + 0.57 4.10 + 0.32 4.10 + 0.32
25 % Incorporation of WPC
Fresh 4.20 + 0.75 4.00 + 0.63 4.00 + 0.63 4.40 + 0.49 4.20 + 0.75
30 days 3.90 + 0.94 4.10 + 0.54 4.20 + 0.60 4.20 + 0.60 4.10 + 0.83
60 days 3.90 + 0.74 3.90 + 0.32 4.00 + 0.47 4.20 + 0.42 3.70 + 0.67
50 % Incorporation of WPC
Fresh 4.40 + 0.80 4.40 + 0.80 4.00 + 0.63 4.60 + 0.80 4.40 + 0.49
30 days 4.20 + 0.75 4.60 + 0.49 4.30 + 0.64 4.00 + 0.89 4.20 + 0.75
60 days 4.20 + 0.75 4.10 + 0.74 4.00 + 0.67 4.10 + 0.74 4.20 + 0.63
75 % Incorporation of WPC
Fresh 4.20 + 0.75 4.40 + 0.49 4.00 + 0.63 4.60 + 0.49 5.00 + 0.00
30 days 4.50 + 0.50 4.80 + 0.40 4.50 + 0.50 4.10 + 0.70 4.20 + 0.98
60 days 4.10 + 0.74 4.30 + 0.48 3.90 + 0.57 4.20 + 0.42 3.90 + 0.57
100 % Incorporation of WPC
Fresh 4.20 + 0.75 3.60 + 0.49 3.80 + 0.40 4.40 + 0.49 4.40 + 0.80
30 days 4.30 + 0.64 3.80 + 0.87 4.10 + 0.70 4.30 + 0.46 4.20 + 0.98
60 days 3.80 + 0.42 3.50 + 0.53 3.80 + 0.42 4.10 + 0.57 3.70 + 0.48

The ANOVA of the overall acceptability of control and experimental chocolates

is given in Table – 19. The results revealed that significant changes were observed in the

overall acceptability between the chocolates prepared by replacing 25% of SMP and 75%

of SMP with WPC. The chocolate prepared by replacing 75% of SMP with WPC

recorded significantly higher scores over the 25% SMP replaced chocolates. The control

and experimental samples at zero days of storage recorded significantly higher scores

over 30 and 60 days of storage.

Table – 19. ANOVA of taste panel scores for overall acceptability of chocolates
during.
0 Day 30 Days 60 Days Mean
Control 4.6 4.1 4.1 4.2667
25 % 4.2 4.1 3.7 4.0*
50 % 4.4 4.2 4.2 4.2667
75 % 5.0 4.2 3.9 4.3667*
100 % 4.4 4.2 3.7 4.1
Mean 4.52 4.16* 3.92*
S.E + C.D. (0.05)
Levels of WPC 0.1819 NS
Storage Periods 0.1409 0.2762
Interactions 0.3151 0.6175

* Significant at 5% level of significance.

The interaction effect between treatment and the storage period was also found

significant. The experimental chocolate containing 75% WPC at zero days storage

recorded significantly superior score over 30 and 60 days of storage. The experimental

chocolate containing 100% WPC at zero days storage was at par with 30 days storage but

recorded significantly higher scores over 60 days of storage.

4.3.2. CHEMICAL ANALYSIS OF THE STORED PRODUCTS.

All the control and the experimental products viz., biscuit, cake and chocolate

were analyzed for moisture and the acidity of the extracted fat during the storage periods.

The control and the experimental products were packed in metallized polypropylene

covers and stored. The estimations for biscuit and chocolate were carried out fresh, after

30 and 60 days while cake was analyzed fresh, after 5, 10 and 15 days of storage.

BISCUIT.

The results obtained after estimating the moisture and acidity of the extracted fat

of the biscuit is presented in Table – 20. It was observed that the initial moisture content
of the control and experimental biscuits ranged from 2.71 to 2.91%. The control biscuit

contained 2.71 percent moisture while the experimental biscuits prepared by

incorporating WPC at 10, 20, and 30 percent levels contained 2.77, 2.84, and 2.91

percent respectively.

Table – 20. Moisture and Acidity of extracted fat of biscuits during storage.

Levels of Fresh 30 days 60 days

WPC (%) Moisture Acidity Moisture Acidity Moisture Acidity
Control 2.71 1.06 2.75 1.10 2.90 1.20
10 2.77 1.08 2.81 1.12 2.90 1.22
20 2.84 1.07 2.88 1.12 3.00 1.21
30 2.91 1.10 2.95 1.13 3.10 1.19

After 30 days of storage the moisture content ranged from 2.75 to 2.95%. The

control biscuit contained 2.75 percent while the experimental biscuits containing 10, 20,

and 30% WPC contained 2.81, 2.88 and 2.95% respectively. After 60 days of storage the

moisture content ranged from 2.90 to 3.10%. The control biscuit contained 2.90 percent,

the experimental biscuits prepared with 10 and 20% WPC contained 2.90% each and

30% WPC containing biscuit contained 3.10%. It was evident from the results that the

moisture content of all the control and experimental biscuits increased with the level of

incorporation of WPC over the storage period.

The moisture content of all the biscuits, even after storing for 60 days, was much

below the specifications for protein enriched biscuits of the BIS (IS: 7487 - 1986).

Increased moisture of the control and experimental biscuits could be due to the water

vapor transmission of the packaging material used in the present investigation. Similar

results were obtained by Singh et.al. (2000) who reported that the moisture content of the

soy fortified biscuits increased with the time of storage. Moisture gain by biscuits in
different packaging materials has been reported by Zabik et.al. (1979), Sathe et.al. (1981)

and Millwalla and Subrahmanyam (1985).

The acidity of the extracted fat of the control and experimental biscuits initially

ranged form 1.06 to 1.10%. The control biscuit contained 1.06% while the experimental

biscuits containing 10, 20, and 30% WPC contained 1.08, 1.07, and 1.10 percent

respectively. After 30 days of storage the acidity of the extracted fat of the prepared

biscuits ranged from 1.10 to 1.13%. The control biscuit contained 1.10%. The

experimental biscuits containing 10 and 20% WPC contained 1.12% each and the 30%

WPC containing biscuit contained 1.13%.

At the end of the study the acidity ranged from 1.19 to 1.22%. The control biscuit

contained 1.20%, the biscuits containing 10, 20 and 30% WPC contained 1.22, 1.21, and

1.19% respectively. It is evident from the results that the free fatty acids (FFA) contents

of all biscuits increased gradually with the increase in the storage period. The increase in

the FFA contents of WPC incorporated biscuits might be due to greater moisture content

which would have promoted fat oxidation during storage, there by increasing the acidity.

However, not much difference was found in the sensory scores in terms of flavor and

taste upon storage. Similar results were obtained by Sathe, et.al. (1981). However, the

FFA contents of all biscuits were within the range of ISI specification (IS: 7487 - 1986),

which specifies the acidity of fat as 1.5% (max) for high protein biscuits.

CAKE.

The moisture and acidity of the control and experimental cakes during the storage

periods is given in Table – 21. It was observed from the results that the initial moisture
and acidity of the extracted fat of the control and the experimental cakes remained

constant upto 5 days of storage, increased slightly after 10 days of storage and further

remained constant till the end of the study.

Table – 21. Moisture and Acidity of extracted fat of cakes during storage.

Storage Levels of Incorporation of WPC (%)

Parameters
Periods Control 10 20 30
Moisture 18.76 18.86 19.55 20.55
Fresh
Acidity 1.20 1.20 1.26 1.30
Moisture 18.76 18.86 19.55 20.55
5 Days
Acidity 1.20 1.20 1.26 1.30
Moisture 18.80 18.90 19.60 20.60
10 Days
Acidity 1.22 1.22 1.28 1.32
Moisture 18.80 18.90 19.60 20.60
15 Days
Acidity 1.22 1.22 1.28 1.32

The moisture content of control cake increased from an initial value of 18.76 to

18.80% after 10 days of storage and remained constant till the end of the study. The

moisture content of 10, 20, and 30% WPC incorporated cake increased from an initial

value of 18.86, 19.55 and 20.55% to 18.90, 19.60 and 20.60% respectively after 15 days

of storage. It was evident that the moisture content in the control and experimental cakes

increased slightly upon storage upto 15 days. This could be due to the water vapor

transmission of the metallized polypropylene used in the present study. However, the

moisture content of cakes were with in the range of ISI standards (IS: 9712 - 1981).

The acidity of the extracted fat of the control and experimental cakes was slightly

increased during the storage. The perusal of the Table – 21 reveals that with the increase

in the level of incorporation of WPC in cakes the initial acidity of the extracted fat

increased. The acidity of both the control cake and cake prepared by incorporating 10%

WPC increased from an initial value of 1.2 to 1.22% after 5 days and remained constant
till the end of the study. The initial acidity of 20% WPC incorporated experimental cake

was 1.26%, which remained same after 5 days of storage and at the end of the study it

increased to 1.28%. The cake prepared by incorporating 30% WPC had an initial acidity

of 1.30% and after 15 days of storage it increased to 1.32%. However, not much

difference was found in the sensory scores in terms of flavor and taste upon storage.

The higher amounts of acidity in the control and experimental cakes could be due

to greater amounts of moisture and fat content, which promoted oxidation of fat during

storage, there by increasing the acidity. Similar results of acidity of extracted fat were

obtained by Narain and Subashini (2004) in milk bread prepared by incorporating WPC.

CHOCOLATE.

The moisture content and the acidity of the extracted fat of the chocolate is given

in Table – 22. The perusal of the table reveals that there is a gradual increase in the

moisture and acidity of the control and experimental chocolates upon storage.

Table – 22. Moisture and Acidity of extracted fat of chocolates during storage.

Levels of Fresh 30 days 60 days

WPC (%) Moisture Acidity Moisture Acidity Moisture Acidity
Control 7.40 0.90 7.60 1.00 7.85 1.10
25 8.35 0.95 8.58 1.06 8.80 1.21
50 9.60 1.12 9.84 1.25 10.03 1.41
75 10.82 1.19 11.00 1.30 11.20 1.48
100 11.32 1.21 11.45 1.34 11.60 1.51

The moisture content of control chocolates initially was 7.40% and after 30 and

60 days of storage it was 7.60 and 7.85% respectively. The chocolate prepared by

replacing 25% SMP with WPC initially had moisture of 8.35% and after 30 and 60 days
of storage it had 8.58 and 8.80% respectively. The 50% SMP replaced chocolate had

initial moisture of 9.60% and after 30 and 60 days of storage it was 9.84 and 10.03%

respectively.

The chocolate prepared by replacing 75% SMP with WPC contained 10.82%

initially, 11.0% after 30 days and 11.20% of moisture at the end of the study. The 100%

SMP replaced chocolate had 11.32% moisture initially, 11.45% after 30 days and at the

end of the study had 11.60%.

The acidity of the control chocolate initially was 0.90%, after 30 days it was 1.0%

and after 60 days it was 1.10%. The 25% SMP replaced chocolate had an initial acidity of

0.95%, after 30 days it was 1.06% and after 60 days it was 1.21%. The 50% SMP

replaced chocolate had an initial acidity of 1.12%, after 30 days it was 1.25% and after 60

days it was 1.41%. The 75% SMP replaced chocolate had an initial acidity of 1.19%,

after 30 days it was 1.30% and after 60 days it was 1.48%. The 100% SMP replaced

chocolate had an initial acidity of 1.21%, after 30 days it was 1.34% and after 60 days it

was 1.51%. However, not much difference was found in the sensory scores in terms of

flavor and taste upon storage.

4.3.3. MICROBIOLOGICAL ANALYSIS OF THE STORED PRODUCTS.

The microbiological quality of fresh and stored products was evaluated by

determining the colony forming units per gram (cfu /g) of bacteria and yeast and molds.

The nutrient agar medium and potato dextrose agar were used for enumerating bacterial

and yeast and mold colonies respectively.

BISCUIT.

The microbiological quality was determined on the day of preparation, after 30

and 60 days. The results are presented in Table – 23. There were no bacterial and yeast

and mold colonies initially in the control and experimental biscuits. After 30 days of

storage the control biscuit had 10 cfu/g each of bacteria and yeast and mold. The biscuit

prepared by incorporating 10% WPC had 20 bacterial cfu/g and 10 yeast and mold cfu/g.

The biscuit prepared by incorporating 20% WPC had 40 bacterial cfu/g and 20 yeast and

mold cfu/g. The biscuit prepared by incorporating 30% WPC had 30 bacterial cfu/g and

10 yeast and mold cfu/g.

Table – 23. Microbiological counts (cfu /g) in biscuits during storage.

Fresh 30 days 60 days

Levels of
Yeast & Yeast & Yeast &
WPC (%) Bacteria Bacteria Bacteria
Molds Molds Molds
Control - - 10 10 20 40
10 - - 20 10 35 25
20 - - 40 20 45 25
30 - - 30 10 35 20

After 60 days of storage, the control biscuit contained 20 bacterial cfu/g and 40

yeast and mold cfu/g. The biscuit prepared by incorporating 10% WPC had 35 bacterial

cfu/g and 25 yeast and mold cfu/g. The biscuit prepared by incorporating 20% WPC had

45 bacterial cfu/g and 25 yeast and mold cfu/g. The biscuit prepared by incorporating

30% WPC had 35 bacterial cfu/g and 20 yeast and mold cfu/g.

The bacterial and yeast and mold colony forming units increased with storage

period. Moisture loss and gain in many low and intermediate moisture promotes chemical
and microbiological spoilage (James et.al. 2004). However, the bacterial colony forming

units of the control and experimental biscuits were within the range of ISI specifications

(IS: 7487 - 1986). There is no specification for the limit of yeast and mold colony

forming units in the ISI specification for cake. Similar observations were made by Shalini

(2003) who reported that the bacterial count of biscuits stored for 15 days was negligible.

CAKE.

The microbiological quality of the control and experimental cakes were

determined fresh, after 5, 10 and 15 days of storage. The results are presented in Table –

24. There were no bacterial and yeast and mold counts initially.

Table – 24. Microbiological counts (cfu /g) in cakes during storage.

Storage Levels of WPC (%)

Parameters
Periods Control 10 20 30
Bacteria - - - -
Fresh
Yeast & Molds - - - -
Bacteria 1 1 1 2
5 Days
Yeast & Molds 2 4 2 3
Bacteria 1 1 1 2
10 Days
Yeast & Molds 4 7 5 8
Bacteria 3 5 4 7
15 Days
Yeast & Molds 5 9 9 11

After 5 days of storage, the control and the experimental cakes prepared by

incorporating 10 and 20% WPC had 1 cfu /g of bacterial and yeast and mold each. The

30% WPC incorporated cake had 2 cfu /g each of bacteria and yeast and mold.

After 10 days of storage, the control and the experimental cakes prepared by

incorporating 10 and 20% WPC had 1 cfu /g of bacteria. The 30% WPC incorporated
biscuit had 2 cfu /g of bacteria. The control biscuit had 4 cfu /g, the 10% WPC

incorporated biscuit had 7 cfu /g, the 20% WPC incorporated biscuit had 5 cfu /g and the

30% WPC incorporated WPC incorporated biscuit had 8 cfu /g.

After 15 days of storage, the control biscuit had 3 cfu /g of bacteria, the 10%

WPC incorporated biscuit had 5 cfu /g of bacteria, the 20% WPC incorporated biscuit

had 4 cfu /g of bacteria and the 30% WPC incorporated biscuit had 8 cfu /g of bacteria.

The control biscuit had 5 cfu /g of yeast and molds, the 10 and 20% WPC incorporated

biscuit had 11 cfu /g of yeast and molds. The increased bacterial and yeast and mold

counts in the cake samples during storage could be due to the absence of preservatives in

the standard recipe.

Shalini (2003) reported negligible bacterial count in cakes stored upto two weeks.

Narain and Subashini (2004) reported no microbial growth on zero days in 50% WPC

incorporated yeast leavened bread but on fourth day of storage 21 cfu /g was observed.

CHOCOLATE.

The microbiological counts of the control and experimental chocolates were

determined fresh, after 30 and 60 days. The results are presented in Table – 25. There

were no bacterial and yeast and mold counts initially.

The control chocolate had 9 and 12 cfu /g of bacteria after 30 and 60 days

respectively. It had 5 cfu /g and 35 cfu /g of yeast and molds after 30 and 60 days

respectively. The chocolate prepared by replacing 25% of SMP in the standard recipe
with WPC, contained 13 cfu /g and 15 cfu /g of bacteria after 30 and 60 days

respectively. It had 6 cfu /g and 48 cfu /g of yeast and molds after 30 and 60 days

respectively.

Table – 25. Microbiological counts (cfu /g) in chocolates during storage.

Fresh 30 days 60 days

Levels of
Yeast & Yeast & Yeast &
WPC (%) Bacteria Bacteria Bacteria
Molds Molds Molds
Control - - 9 5 12 35
25 - - 13 6 15 48
50 - - 14 8 15 43
75 - - 15 11 18 51
100 - - 15 13 20 49

The chocolate prepared by replacing 50% SMP with WPC contained 14 and 15

cfu /g after 30 and 60 days of storage respectively. It contained 8 and 43 cfu /g of yeast

and molds after 30 and 60 days of storage respectively. The chocolate prepared by

replacing 75% SMP with WPC contained 15 and 18 cfu /g of bacteria after 30 and 60

days of storage respectively. It contained 11 and 51 cfu /g of yeast and molds after 30 and

60 days of storage respectively. The chocolate prepared by replacing 100% SMP with

WPC contained 15 and 20 cfu /g of bacteria after 30 and 60 days of storage respectively.

It contained 13 and 49 cfu /g of yeast and molds after 30 and 60 days of storage

respectively.

The increased microbiological counts in chocolates could be due to low amount of

carbohydrates with increased WPC incorporation, there by low preservative effect of

sugars.
 Protein Content of Control and Experimental Biscuits

16 14.11
14 11.71
12
Percentage

10 8.06
8
6 5.23

4 Protein Content
2
0
Control 10% 20% 30%

Levels of WPC incorporation

Fig.1 Protein content of Control and Experimental Biscuits

Protein Content of Control and Experimental Cakes

12
11.01

10
8.89

8
Percentage

6.67

6
4.71

Protein Content
2

0
Control 10% 20% 30%
Levels of WPC incorporation

Fig. 2 Protein content of Control and Experimental Cakes.

 Protein Content of Control and Experim ental Chocolates

8 7.69
7 6.81
5.94
6 5.06
Percentage

5 4.25
4
3
2
Protein Content
1
0
Control 25% 50% 75% 100%
Levels of WPC incorporation

Fig. 3 Protein Content of Control and Experimental Chocolates

 CHAPTER – V

SUMMARY AND CONCLUSION

The present study was carried out to develop and evaluate the acceptability of

some common bakery and confectionery products viz. biscuit, cake, and chocolate by

incorporating the whey proteins in the form of whey protein concentrate. The whey

protein concentrate (WPC) for the present study was supplied by M/s Mahaan Proteins

Limited, New Delhi at free of cost on request. Other raw materials required for the

preparation of the products were procured from local market. The experiments were

carried out at Post Graduate and Research Center, Acharya N.G.Ranga Agricultural

University, Hyderabad. The results drawn from the present study are summarized here

under.

 Whey protein concentrate was incorporated at 10, 20, and 30 percent levels in basic

biscuit and cake recipes. In standardized chocolate recipe, skimmed milk powder

(SMP) was replaced with WPC at 25, 50, 75 and 100 percent levels.

 Sensory evaluation of the developed products by the trained panel members

revealed that the color and appearance of the biscuits and cakes incorporated with

WPC was superior to the control samples. However, the color and appearance of the

chocolate remained the same. The texture of the 10 and 20 percent WPC

incorporated biscuits scored higher than the control. The texture of the experimental

cakes and chocolates received better scores than the control ones. The flavor of the

cakes and chocolates developed by incorporating WPC received scores on par with
 the control ones, whereas the flavor of WPC incorporated biscuits received lower

scores.

 The overall acceptability of the WPC incorporated biscuits and cakes was similar to

that of the control biscuits. Among the various replacements of SMP with WPC in

chocolate, the 75% replacement obtained highest score for the overall acceptability.

 The objective evaluation of the control and the experimental products was carried

out using the universal testing machine or the Instron. Cutting and compression

tests were performed to evaluate the effect of functional ingredients on the textural

quality of experimental products and compared with the control. It was observed

that the textural attributes of compression and cutting tests of the experimental

products recorded slightly increased values than the control.

 The control and experimental products were estimated for moisture, protein, fat,

crude fiber, ash and acid insoluble ash content by standard analytical procedures.

Carbohydrate and energy content of the samples were computed by appropriate

calculations. The results of the analysis revealed that with increase in WPC

incorporation levels, protein and ash content increased in the products where as

carbohydrate and energy value decreased.

 The control and the experimental biscuits, cakes and chocolates were packed in

metallized polypropylene pouches and stored at room temperature. Biscuits and

chocolates were stored upto 60 days while cakes were stored upto 15 days. Storage

studies were carried out fresh, after 30 and 60 days for biscuits and chocolates while
 for cake, fresh, after 5, 10, and 15 days of storage. Sensory evaluation, chemical

analysis and microbial analysis were carried out in the stored products.

 The sensory evaluation of the stored biscuits revealed that no significant differences

were observed among the WPC level of incorporation for color and appearance,

texture, flavor and overall acceptability. But significant differences were recorded

in color and appearance after 60 days, in overall acceptability and texture 30 days

and 60 days, in flavor after 60 days of storage.

 The sensory evaluation of the stored cakes revealed that no significant differences

were found among the WPC level of incorporation for color and appearance and the

overall acceptability. However, significantly low scores were noticed for the texture

of 10 and 20 percent WPC incorporated cakes and for the taste of 30 percent WPC

incorporated. Significant differences were recorded in the storage periods for

texture and overall acceptability of the cakes.

 The sensory evaluation of the stored chocolates revealed that no significant

differences were recorded among the level of WPC incorporation for color and

appearance, flavor and taste. However, significantly increased scores were obtained

for the overall acceptability of the 75 percent SMP replaced chocolate. Significantly

low scores were observed by the chocolates for overall acceptability over 30 and 60

days of storage.

 Moisture and acidity of the extracted fat was estimated during the storage period for

all the control and experimental products. The results revealed that there was slight
 increase in the moisture and acidity of the extracted fat of all the control and

experimental samples.

 The microbiological analysis during the storage period for all the control and

experimental products was carried out. The results revealed that the fresh samples

did not show any bacterial or yeast and mold colony counts. The bacterial and yeast

and mold colonies increased over the storage period, but found to be acceptable

range for the products.

The present study conducted is a preliminary step towards development of

products incorporated with the functional ingredient WPC. The study gives scope for

future research, wherein the suitability of WPC can be tested in variety of food

applications, not restricting to bakery and confectionery products alone. It is therefore

imperative that regular consumption of whey proteins, as a part of daily diet should be

advocated to people without changing the dietary patterns. To accomplish this, it would

be important to develop and commercialize products that contain whey proteins, which

has the much-required bioactivity.

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 APPENDIX – I

METHOD OF PREPARATION OF PRODUCTS

1. RECIPES OF BISCUIT.

Recipes of Biscuits used in different treatments

Treatments
S. No Ingredients
Control 10% WPC 20% WPC 30% WPC
1 Refined Flour 100 g 90 g 80 g 70 g
2 WPC -- 10 g 20 g 30 g
3 Powdered Sugar 45 g 45 g 45 g 45 g
4 Hydrogenated Fat 35 g 35 g 35 g 35 g
5 Baking Powder 0.4 g 0.4 g 0.4 g 0.4 g
Ammonium
6 0.85 g 0.85 g 0.85 g 0.85 g
Bicarbonate
7 Skim Milk Powder 6g 6g 6g 6g
8 Salt 1g 1g 1g 1g
9 Soy Lecithin 0.35 g 0.35 g 0.35 g 0.35 g
10 Vanilla Flavor 0.5 ml 0.5 ml 0.5 ml 0.5 ml
11 Water 16.25 g 16.25 g 16.25 g 16.25 g

Method of preparation

 Hydrogenated fat and sugar were creamed till light and fluffy.

 Soy Lecithin and vanilla Flavor were added to the fat and sugar mixture.

 Refined flour and baking powder were sieved together for proper blending.

 Ammonium bicarbonate and salt were mixed separately in water meant for dough
making.

 Refined flour was mixed with the cream along with water to make dough.

 The prepared dough was made into thin sheet.

 Rectangular biscuit cutter was used to cut the sheet into uniform shape.

 The shaped biscuit were placed on metal mesh sheet, simulating industrial metal
conveyor, and baked at 2250 C for 6 – 8 minutes.

 After baking the biscuits were kept for cooling for about 5 minutes and packed in
metallized polypropylene pouches.
 2. RECIPES OF CAKE.

Recipes of cakes used in different treatments

Treatments
S. No Ingredients
Control 10% WPC 20% WPC 30% WPC
1 Refined Flour 115 g 103.5 g 92 g 80.5 g
2 WPC -- 11.5 23 34.5 g
3 Powdered Sugar 115 g 115 g 115 g 115 g
4 Hydrogenated Fat 70 g 70 g 70 g 70 g
5 Eggs (in number) 1½ 1½ 1½ 1½
6 Baking Powder 0.8 g 0.8 g 0.8 g 0.8 g
7 Vanilla Flavor 2 ml 2 ml 2 ml 2 ml

Method of preparation

 Flour and Baking Powder were sieved together enabling proper blending.

 Hydrogenated fat and grounded sugar were creamed together till light and fluffy.

 Eggs were beaten using an electric eggbeater and vanilla flavor was added to this.

 Beaten eggs were added to the creamed fat and sugar mixture and mixed well into
a smooth batter.

 The batter was poured into greased cake pans and baked at 2250 C for 25 – 30
minutes.

 The baked cake was kept for cooling for about 10 minutes and cut into uniform
pieces and packed in metallized polypropylene pouches.

3. THE STANDARDIZED RECIPE OF CHOCOLATE.

Recipes of cakes used in different treatments

Treatments
S. No Ingredients 25% 50% 75% 100 %
Control
WPC WPC WPC WPC
1 Sugar 250 g 250 g 250 g 250 g 250 g
2 Water 100 ml 100 ml 100 ml 100 ml 100 ml
Hydrogenated 60 g 60 g 60 g 60 g 60 g
3
Fat
4 SMP 50 g 37.5 g 25 g 12.5 g --
5 WPC -- 12.5 g 25 g 37.5 g 50 g
6 Cocoa Powder 25 g 25 g 25 g 25 g 25 g
7 Soy Lecithin 0.6 g 0.6 g 0.6 g 0.6 g 0.6 g
 Method of preparation

 Cocoa Powder and skim milk powder were sieved together for ensuring proper
blending.

 Sugar syrup was made into syrup of one string consistency.

 To the sugar syrup, hydrogenated fat was added and allowed it to dissolve.

 Soy lecithin, cocoa powder and skim milk powder mixture was added to the syrup
and mixed over while still heating till the temperature reached to 800 C.

 Then the mixture was poured into a thin slab onto a previously greased tray and
allowed to cool at room temperature cut into uniform sized pieces.

 The pieces were then packed in metallized polypropylene pouches.

4. PROXIMATE COMPOSITION OF WHEY PROTEIN CONCENTRATE AS

PROVIDED BY MANUFACTURER (M/s MAHAAN PROTEINS LTD.)

Product: Whey Protein Concentrate 70 % (PROCON 3700)

Date of Manufacture: 29 – 03 – 2004.
Production Code: C1EMK.N1

S. No Characteristics Result
Physico – Chemical Characteristics
Color Creamy
1
White
2 Taste Bland
3 Sediment, ADPI Disc B
4 PH (10 % w/v solution) 6.41
5 Bulk Density (g/ml) 0.42
6 Insolubility index, ml 1.60
7 Moisture, % 3.80
8 Fat, % as such 4.99
9 Protein, % (on dry basis) 70.00
10 Total Minerals, % as such 4.50
Microbiological Characteristics
11 Total plate count, per g 500
12 Coliforms, per 0.1 g - ve
13 Salmonella, per 100 g - ve
14 Yeast & Mold count, per g 10
 APPENDIX – III

ESTIMATION OF MOISTURE

The moisture content of the samples was determined by using the method of
AOAC (1984).

Procedure

 10 gm of the sample was taken in to a petri dish with lid and the exact weight was
noted down (W1)
 The sample was dried in an oven at 100 to 105 degrees till constant weight was
obtained.
 The sample was cooled in a dessicator and the final weight was taken (W2).

Calculation

Moisture % = Initial weight (W1) – Final weight (W2) x 100

Initial weight (W1)
 APPENDIX – IV

PROTEIN ESTIMATION

Protein content was estimated by Microkjeldhal method by the procedure of

AOAC (1984).

Principle: The nitrogenous compounds of the sample are converted in to ammonium

sulphate by boiling with concentrated sulphuric acid. It was subsequently
decomposed by addition of excess of alkali and the liberated ammonia was
absorbed in to boric acid solution containing bromocresol green indicator by
stream distillation. Ammonia formed a loose compound, ammonium borate,
with boric acid, which was titrated directly against standardized
hydrochloric acid.

Chemical reagents:

1. 40 % NaOH: 400 gm of NaOH was dissolved in 1000 ml of distilled water by

steeping the beaker in running water in a sink.
2. 2 % Boric acid: 10 gm of reagent grade boric acid was dissolved in 500 ml of hot
distilled water.
3. Mixed indicator :
a) 0.2 % Bromocresol green in alcohol: 20 mg of bromocresol green was
dissolved in 10 ml alcohol.
b) 0.2 % methyl red: 20 mg of methyl red powder was dissolved in 10 ml
alcohol.

5 ml of (a) and 2 ml of (b) are mixed together and used as mixed indicator.

4. 0.5 N HCl: 100 ml of 0.1 N HCl (accurately standardized against 0.1 N NaOH) was
dissolved in 500 ml in a volumetric flask.
5. Digestion mixture: 98 gm of potassium sulfate and 2 gm of copper sulfate are
ground together.

Procedure:

Digestion: 500 mg of food sample was weighed in to boiling tube. 5 gm of digestion

mixture and 10 ml of concentrated sulfuric acid were carefully added and
the samples were digested in the digestion block for 1 hour at 375 degrees
centigrade. The tubes were removed and distilled water (approximately 50
ml) was added carefully from the sides while still hot.

Distillation: In a 100 ml conical flask, 50 ml of 4 % boric acid was added with few drops
of mixed indicator. The condenser outlet of the steam distillation apparatus
should dip below the surface of the boric acid solution. The digested sample
was completely transferred by means of repeated washings to the chamber
 of the distillation apparatus. The chamber should be previously cleared off
any contaminated ammonia by repeated washings and steam generation
should be started. 50 ml of NaOH solution was slowly added, followed by
one more rinsing of the digested sample. Sample should be distilled for
exactly 3 minutes after solution changed to a blue color, the receiving flask
was lowered and steam generation was stopped. The condenser outlet should
be washed in to the receiving flask. The contents of the flask were titrated
with 0.5 N HCl till the color changed to original pink. The blank was also
run simultaneously.

Calculations:

1 ml of 0.5 N HCl = 14.007 gm of nitrogen.

1 gm of nitrogen = 100/16 = 6.25 gm of protein.

Protein % = Titre value x 14.007 x 0.5 x 6.25 x 100

Wt. of sample (mg)
 APPENDIX – V

ESTIMATION OF FAT

The fat content of the sample was estimated as crude ether extract of the dry
material.

Method:

The dry sample (5 – 10 gm) was weighed accurately in to a thimble (made with
Whatman no. 1 filter paper – AOAC technique) and was placed in the Soxhlet apparatus.
It was extracted with petroleum ether (60 – 80 degrees B.P.) for about 16 hours. The
ether extract was filled in to a weighed beaker. The flask was rinsed four to five times
with small quantities of petroleum ether added to the beaker. Petroleum ether was
removed by evaporation and the flask with the residue was dried in an oven at 80 – 100
degrees centigrade and later cooled in a dessicator and weighed.

Calculation:

Fat content of the sample % (gm / 100 gm) = Weight of ether extract x 100.
Weight of the sample
 APPENDIX – VI

ESTIMATION OF CRUDE FIBER

The crude fiber content of the samples was estimated by using the method of
AOAC (1984).

Principle:
The sample was allowed to boil with dilute sulphuric acid (0.255 N) and
dilute sodium hydroxide (1.25 %) and the remaining residue after the
digestion was taken as crude fiber.

Reagents:

1. Sulphuric acid (0.255 N)

2. Sodium hydroxide (1.25 % W/V).

Procedure:

 2 – 5 gm of moisture and fat free sample was weighed in to a 500 ml beaker.

 200 ml of boiled sulphuric acid (0.255 N) was added and the mixture was boiled
for 30 minutes, keeping the volume constant by the addition of water at frequent
intervals.
 After boiling, the mixture was filtered through a muslin cloth and the residue was
washed with hot water till free from acid.
 Then the material was transferred in to the same beaker and 200 ml of boiled
sodium hydroxide (1.25 % W/V) was added and boiled for 30 minutes.
 Later the mixture was filtered and washed with hot water.
 Finally the mixture was washed with few ml of alcohol and ether.
 The residue was transferred to a crucible and dried over night at 80 – 100 degrees
centigrade and the weight of the crucible was noted down.
 The crucible was heated in a muffle furnace at 600 degrees centigrade for 2 – 3
hours and cooled in a dessicator and weighed.

Observation :

Weight of the sample (W1) = gm.

Weight of the crucible + sample
Before heating at 600 degrees. C. (W2) = gm.
Weight of the crucible + sample
After heating at 600 degrees. C. (W3) = gm.
Weight of the crude fiber (W2 – W3) = gm. (A).

Calculation :
Crude fiber (gm %) = 100 – (moisture + fat) A
W1
 APPENDIX – VII

ESTIMATION OF ASH

The ash content of the sample was estimated by AOAC (1984) method.

Procedure:

 The temperature of the muffle furnace was set at 600 degrees centigrade and the
crucible was heated for 1 hour and transferred in to a dessicator. After cooling to
room temperature the crucible was weighed (W1).

 2 gm of sample (defatted) was weighed in to the crucible of known weight (W2).

 The sample was incinerated at 600 degrees centigrade for 2 hours.

 The crucible was transferred in to the dessicator and cooled to room temperature
and weighed (W3).

Calculation:

Weight of the sample taken = W2 – W1 g.

Weight of the ash obtained = W3 – W1 g.

% Ash = Weight of Ash x 100 i.e., (W3 - W1) x 100

Weight of sample taken W2 – W1
 APPENDIX – VIII

ESTIMATAION OF ACID INSOLUBLE ASH

The acid insoluble ash of bakery products was estimated by IS: 12711 – 1989
method.

Reagents

 Dilute HCl: 5 N prepared by diluting 1 volume of concentrated hydrochloric acid

to 2.5 volumes with water.

Method

To the ash contained in the silica dish, add 25 ml of dilute hydrochloric acid,
cover with a watch glass and heat on a water bath for 10 minutes. Allow to cool and filter
the contents of the dish through Whatman filter paper No.42 or its equivalent. Wash the
filter paper with water until the washings are free from the acid. Keep it in an electric air
– oven and heat it till it gets dried. Subsequently, ignite the contents of the ash over a
burner till the contents get charred. Complete the ignition by transferring the dish to a
muffle furnace 550 + 100 C until grey or white ash results. Cool the dish in a desiccator
and weigh. Heat the dish again at 550 + 100 C for 30 minutes. Cool in a desiccator and
weigh. Repeat the process of heating, cooling, and weighing until the difference between
two successive weighings is less than 1 mg. Record the lowest mass.

Calculation

Mass of the dish with the acid insoluble ash = M2 g.

Mass of the empty dish = M g.

Mass of the dish with the material taken for the test = M1 g.

W = percent of moisture content.

Acid insoluble ash (% by mass) = 10000 x (M2 - M)

(M1 - M) (100 - W)
 APPENDIX – IX

ESTIMATION OF ACIDITY OF EXTRACTED FAT

The acidity of extracted fat of bakery products was estimated by IS: 12711 – 1989
method.

Reagents

Pertroleum ether; Benzene – Alcohol – Phenolphthalein Stock solution; Standard

KOH.S

Method

 Weigh accurately about 10 – 30 g of prepared sample and transfer it to the

thimble and plug it from the top with extracted cotton and filter paper. Dry the
thimble with the contents for two hours at 1000 C in an oven. Take the weight of
empty dry Soxhlet flask. Extract the fat in the Soxhlet apparatus for 8 hours and
evaporate the solvent in the flask on a water bath. Remove the traces of the
residual solvent by keeping the flask in the hot air oven at 1000 C for about half
an hour and weigh.

 Cool the flask and add 50 ml of mixed benzene – alcohol –phenolphthalein

reagent and titrate the contents to a pink color with KOH solution taken in a 10 ml
micro burette. If the contents of the flask become cloudy during titration, add
another 50ml of the reagent and continue titration. Make a blank titration of the
50 ml reagent. Subtract from the titre of the fat, the blank titre.

Calculation

Acidity of extracted fat (as Oleic acid), percent by mass = 1.41 x V

M1 – M.

Volume of 0.05 N Potassium hydroxide solution

used in titration after subtracting the blank. = V ml

Mass of Soxhlet flask containing fat = M1 ml

Mass of empty Soxhlet flask = M g.

 APPENDIX – X

INDIAN STANDARD SPECIFICATIONS

1. Indian Standard Specification for Biscuits (IS 1011: 1992)

S. No Characteristics Requirements
1 Moisture, percent by mass, Max. 5.0
Acid insoluble ash (on dry basis), percent by
2 0.05
mass, Max.
Acidity of extracted fat (oleic acid), percent
3 1.2
by mass, Max.

2. Indian Standard Specification for Protein Enriched Biscuits (IS 7487: 1986)

S.No Characteristics Requirement

1 Protein, percent by mass, Min 12
2 Moisture, percent by mass, Max 6
3 Fat, percent by mass, Min 12
4 Acid insoluble ash, percent by mass, Max 0.08
5 Crude fiber (on dry basis), percent mass, Max 3.0
Acidity of extracted fat (oleic acid), percent by
6 1.5
mass, Max
7 Total bacterial count / g 50,000
8 Coliform bacterial count 10
9 Salmonella bacteria Nil
10 Vitamin A, g /100g, Min 250
11 Vitamin D, g /100g, Min 5
12 Folic acid, g /100g, Min 34
13 Thiamine (as hydrochloride), mg/100g, Min 0.4
14 Riboflavin, mg /100g, Min 0.50
15 Nicotinic acid, mg /100g, Min 5.0
16 Calcium, mg /100g, Min 200
17 Iron, mg /100g, Min 7
 3. Indian Standard Specification for Cakes (IS: 9712 - 1981)

Requirement
S. No Characteristic
Plain Fruit Sponge
Moisture, percent by mass,
1 15 – 25 15 – 25 20 – 27
Max
Acid insoluble ash (on as is
2 0.1 0.1 0.1
basis), percent mass, Max
Acidity of extracted fat
3 (oleic acid), percent by 1.0 1.5 1.0
mass, Max

4. Indian Standard Specification for Chocolates (IS: 1163 – 1992)

Requirements for
S. Characteristics Milk Plain
Milk Plain White Blended
No (%) Covering Covering
Chocolate Chocolate Chocolate Chocolate
Chocolate Chocolate
1 Total Fat, Min 25 29 25 29 25 25
2 Milk Fat, Min 2 2 -- -- 2 --
Cocoa Solids
(on moisture
3 2.5 2.5 12 12 -- 3.0
free and fat free
basis), Min
Milk Solids (on
moisture free
4 and fat free
basis), Min 10.5 10.5 -- -- 10 1
Max -- -- -- -- -- 9
5 Sugar, Max 55 55 60 60 55 60
Acid insoluble
6 0.2 0.2 0.2 0.2 0.2 0.2
ash
 APPENDIX – II

SCORE CARD FOR SENSORY EVALUATION OF BISCUITS

Name of the Evaluator: Date:

 Please evaluate the following samples using the 5-point hedonic scale.
 Write the preferred score in the columns as per evaluation.
 Rinse your mouth in between evaluating each sample.

Samples
Sensory Attributes
1 2 3 4
Colour & Appearance
Highly Appealing 5
Appealing 4
Moderately Appealing 3
Slightly Appealing 2
Not Appealing 1
Texture
Crisp and Crunchy 5
Hard and Brittle 4
Soft and Brittle 3
Hard 2
Not Acceptable 1
Taste
Highly Acceptable 5
Acceptable 4
Moderately Acceptable 3
Fairly Acceptable 2
Not Acceptable 1
Flavour
Pleasant Characteristic Flavour 5
Moderately Characteristic Flavour 4
Mild Characteristic Flavour 3
Predominant Flavour of any ingredient 2
Undesirable 1
Overall Acceptability
Highly Acceptable 5
Acceptable 4
Moderately Acceptable 3
Fairly Acceptable 2
Not Acceptable 1

Suggestions and Comments:

Signature of the Evaluator

 SCORE CARD FOR SENSORY EVALUATION OF CHOCOLATES

Name of the Evaluator: Date:

 Please evaluate the following samples using the 5-point hedonic scale.
 Write the preferred score in the columns as per evaluation.
 Rinse your mouth in between evaluating each sample.

Samples
Sensory Attributes
1 2 3 4 5
Colour & Appearance
Highly Appealing 5
Appealing 4
Moderately Appealing 3
Slightly Appealing 2
Not Appealing 1
Texture
Very Soft 5
Soft 4
Average 3
Hard 2
Very Hard 1
Taste
Very Good 5
Good 4
Fair 3
Poor 2
Very Poor 1
Flavour
Very Pleasant Flavour 5
Pleasant 4
Fair 3
Poor 2
Very Poor 1
Overall Acceptability
Highly Acceptable 5
Acceptable 4
Moderately Acceptable 3
Fairly Acceptable 2
Not Acceptable 1

Suggestions and Comments:

Signature of the Evaluator

 SCORE CARD FOR SENSORY EVALUATION OF CAKES

Name of the Evaluator: Date:

 Please evaluate the following samples using the 5-point hedonic scale.
 Write the preferred score in the columns as per evaluation.
 Rinse your mouth in between evaluating each sample.

Samples
Sensory Attributes
1 2 3 4
Colour & Appearance
Highly Appealing 5
Appealing 4
Moderately Appealing 3
Slightly Appealing 2
Not Appealing 1
Texture
Soft and Velvety 5
Moderately Soft and Velvety 4
Slightly Soft 3
Hard and Compact 2
Dry / Not Acceptable 1
Taste
Highly Acceptable 5
Acceptable 4
Moderately Acceptable 3
Fairly Acceptable 2
Not Acceptable 1
Flavour
Pleasant Fresh Flavour 5
Moderately Fresh Flavour 4
Mild Characteristic Flavour 3
Predominant Flavour of any ingredient 2
Stale and Undesirable 1
Overall Acceptability
Highly Acceptable 5
Acceptable 4
Moderately Acceptable 3
Fairly Acceptable 2
Not Acceptable 1

Suggestions and Comments:

Signature of the Evaluator