SEED COURSE

Unit 1: Composition of food and nutritional value

Chapter 1: Composition of food and nutritional value

Introduction

Food is any nutritious substance that people eat or drink in order to maintain life. The food one
eats provides energy for all body functions and to form new cells for the body. A variety of food
is eaten according to taste, body requirement and availability. All living ones whether plant or
animal need food for growth, repair and maintenance of body. Foods are derived from plants,
animals and single-cell organisms.

1.1.2 Functions of food

Food supply the human body with the necessary body building material, energy yielding
material and protective materials. Food also supplies elements and compounds indispensable
for metabolism.  
1.1.2 Functions of food
Food supply the human body with the necessary body building material, energy yielding
material and protective materials. Food also supplies elements and compounds indispensable
for metabolism.  
1.1.3.1 Water
The content of water in various foods ranges from a few percent in dried commodities (milk
powder) to about 90% in many fruits and vegetables.
Functions
The indigenous water that is immobilized in the plant and animal tissues by the structural
elements and various solutes contributes to the maintenance of the conformation of the
polymers, serves as a solvent for different constituents, and interacts in metabolic processes.
The water content of important food is given in table 1.1.
                                
                                  Table1.1 Water content of important food

Water %
Milk 86.8
Fresh Fruits 80-90
Fresh Vegetables 80-90
Fish 70-80
Lamb 71-73
Beef 70-73
Chicken 73-75
Pork 68-70
Cereals 12-14
Pulses, seeds 10-50
Nuts   3-5
1.1.3.2. Carbohydrates

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Carbohydrates are widely distributed in plant and animal tissues. The carbohydrates commonly
occurring in foods are starch, glucose, fructose, sucrose and lactose.

Functions:

About 50-70 % of energy value in the average diet is provided by carbohydrates. Glucose
derived by digestion of carbohydrates is stored as glycogen in liver and muscle tissues and
used as the main source of energy in the body. Hence food must always contain adequate
amounts of carbohydrates. They also serve as a source of carbon skeletons for synthesis of
various organic compounds.  Some carbohydrates are dissolved in tissue fluids or perform
different biological functions, such as in free nucleotides or as components of nucleic acids, or
in being bound to proteins and lipids.  Some plant polysaccharides are only partly utilized for
energy. However, as dietary fiber they affect, in different ways, various processes in the
gastrointestinal tract.
Important sources of carbohydrates
            The important sources of carbohydrates in the diets of children and adults are cereals,
millets, roots, tubers, pulses, sugar and jaggery, while milk and sugar are important sources
(Table 1.2) in the diets of infants.

                    Table 1.2 Carbohydrate content of some important foods

Name of food Carbohydrate%

Jaggery 94 – 95
   
Cane sugar 99
Sago 87 – 89
Arrow root flour 85 – 87
Honey 79 – 80
Dried fruits (Raisin, dates, etc.) 67 – 77
Cereals and millets (rice, jowar, etc.) 63 – 79
Pulses (Bengal gram, red gram, etc.) 56 – 60
Roots and tubers (Potato, tapioca, 22 – 39
sweet potato, etc.)
Nuts and oilseeds 10 - 25
Fresh fruits 10 - 25
Fresh vegetables 2 - 20
Milk (fluid) 4-5
1.1.3.3. Lipids
   The term lipid is applied to a group of naturally occurring substances characterized by
their insolubility in water and solubility in organic solvents. They occur in plant and animal
tissues.

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Functions
Oils and fats serve as the main source of energy. They also provide the essential fatty acids.
They are good source of fat soluble vitamins. Fat serve as an insulating material in the
subcutaneous tissues and around vital organs. It provides materials for the synthesis of
cholesterol and certain hormones. Lipoproteins and glycoproteins are essential for maintaining
cellular integrity. Fat is essential for maintaining good health.

The lipid content in foods is given in nutrition information labeling predominantly as total fat,
which is often triglycerides.  The lipids of numerous food fishes, such as mullet, codfish, rays
and shark as well as some crustaceans and mollusks also include wax esters. Some shark oils
are very rich in hydrocarbons, particularly in squalene.  The lipid fraction of food raw materials
harbors different sterols, vitamins and pigments that are crucial for metabolism.  Thus the
composition of the extracted crude fat depends on the kind of food and the polarity of the
solvent used for extraction. Fats from different sources have different types of fatty acids. Fat
content of different groups of food is presented in Table 1.3.

Table 1.3 Fat content of different groups of food

Food groups Fat content %

Butter 85
Peanut 50
Soybean 21
Sunflower seed 45
Egg yolk 32
Cotton seed 30
Beef Meat 6
Lamb 5.7
Pork 5
Chicken(white meat) 1
Milk (fresh) 3.5-4.0
Wheat 2
Rice 2
Corn 6.5
Yam 1
Cassava 1
Potato 0.4
Lean fish muscle <1
Fruits, vegetables <1
1.1.3.4. Protein

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Food proteins are of two types. Proteins of animal origin (meat, milk and eggs) are
almost completely utilized if taken alone whereas vegetable proteins (pea, bean, pulses) are not
completely digested and about 10 to 40% remain unabsorbed and excreted in the feces. The
protein in foods is present mainly as crude protein (i.e., as Nx6.25). The nitrogen-to-protein
conversion factor (N:P) of 6.25 has been recommended for most plant and animal food products
under the assumption that the N content in their proteins is 16% and they do not contain non-
protein N. The N content in the proteins in various food, however, is different because it
depends on the amino acid component of protein compounds, such as free peptides and amino
acids, nucleic acids and their the non-protein N may constitute up to 30% of total N. 

1.1.3.5. Minerals
Minerals are needed in small amounts but are important for the body’s basic growth and
structure. There are about 25 minerals that are essential to life and are present in the body.
There are certain major and minor minerals that are nutritionally important. Major minerals
include calcium, phosphorus, magnesium, sodium, potassium, and chloride. Trace elements
include iron, iodine, zinc, selenium, chromium and copper.

Functions

Some minerals perform important functions like formation of bones and teeth, hemoglobin of
blood cells, thyroid hormone, components of hair, nail and cell integrity etc. Egg, meat, milk,
nuts, vegetables, beans, fruits and salt are the chief sources of mineral elements in diet. Milk
products supply the majority of calcium and phosphorus in the diet. Mineral compositions of
certain selected food are present in table 1.5.

                  Table 1.5 Mineral compositions in some food (mg/g)

Crop/muscl Ca P Mg K Na Zn Fe Cl
e
Muscle                
Chicken 3.3 214 30.5 294 54.6 0.41 1.0 --
Beef 4.2 216 27 417 55.2 2.92 2.0  
Pork 5.47 176 21.3 283 82.8 5.5 3.0  
Pulses                
Soybean 2.1 4.9 2.1 14 0.06 0.01 0.07 0.06
Peanut 0.6 3.7 1.6 7.1 0.05 0.03 0.02 0.07
Vegetables                
Beet root 0.3 0.5 0.01 3.4 0.9 0.01 0.01 --
Tomato 0.1 0.3 0.2 3.0 0.06  -- 0.01 0.6
Green 0.5 0.4 0.3 2.6 -- -- -- 0.4
beans
Cereals                
Wheat 0.5 4.3 1.8 4.5 -- -- -- --
Milk mg/ml 121 65 12.5 144 60 -- -- 108
Mineral content of edible portion of fish

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Fish is a good source of minerals particularly small fish which are eaten with bones are good
source of calcium, phosphorus, iron and copper. The only rich source of iodine commonly
included in the human diet is fish. Dried fish is an important part of the village diet which
provides substantial amount of calcium and phosphorus. Most shellfish especially oysters are
rich in calcium, magnesium, copper, zip, and iodine. Mineral content of edible portion of fish is
given in Table.1.6.
Table 1.6 Mineral content of edible portion of
Mineral Average content (mg%) fish

Potassium 300
Chloride 200
Phosphorus 200
Sulphur 200
Sodium 63
Magnesium 25
Calcium 15
Iron 1.5
Manganese 1.0

Zinc 1.0

Fluorine 0.5
Arsenic 0.4
Copper 0.1
Iodine 0.1

1.1.3.6. Vitamins
The vitamins are organic micronutrients, required in only milligram or microgram
quantities per day. They are required for carrying out vital functions of the body and many of
them are involved in the utilization of major nutrients like proteins, fats and carbohydrates.
Although they are needed in small amounts, they are essential for the health and well being of
the body.

Classification; They are classified into water-soluble and fat-soluble. The fat soluble vitamins
A, D, E and K have diverse roles. The water soluble vitamins comprise vitamin C and eight B

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vitamins namely thiamin, riboflavin, niacin, pyridoxine, cobalamine, folate, pantothenic acid and
biotin.

Functions: Most of the B vitamins are essential components of specific coenzymes for

enzymes participating in energy metabolism and other specialized activities. Vitamins regulate
body processes such as energy production, blood clotting and calcium balance. Vitamins help to
maintain organs and tissues healthy.

Sources: Vitamins are found in fruits, vegetables, grains, legumes, meat, fish and dairy
products. Mineral compositions of certain selected food are present in table 1.7.Vitamins
present in edible portion of fish is presented in table 1.8.

Table 1.7 Vitamin compositions of certain selected food (100g)

Food Vitamin A (μg) Riboflavin Folic acid (μg) Vitamin B12 (μg
% RDA % RDA
(mg)% RDA )% RDA
Leafy 1072 0.2 50 Nil

vegetables 179 12 25 --

Other 155 0.08 5 Nil

26 5 2 --
vegetables
Pulses 15.5 0.2 112 Nil
3 12 56 --
Egg 190 0.4 78 2
32 23 39 180
Chicken 11 0.14 19 NR
2 8 9 --
Mutton 9 0.14 6 2.0
1 8 3 200
Beef 20 0.2 19 2.0
3 12 9 200
Fish 23 0.2 5.4 4
4 12 3 450
Liver (sheep, 6690 1.7 180 90
goat, lamb) 1115 100 90 9000
Buffalo’s  Milk 48 0.1 5.6 0.14
8 6 3 14
Cow’s 53 0.19 8.5 0.14
Milk 9 11 4 14

Fish as a sources of vitamins: Fishes are good sources of fat soluble vitamins A,D and E and
of water soluble vitamins of B group. Fish liver oils are known as the richest source of vitamin A
and D. Vitamin A content upto 30,000 IU/g and of vitamin D upto 250,000 IU/g of liver has been
found in some species. Fish liver oils are very often used for medicinal purposos because of

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these vitamins. Fish oils are also rich in vitamin E than any other animal fat. Fish flesh is an
excellent source of thiamin, riboflavin, niacin, pyridoxine, biotin, pantothenic acid and vitamin
B12. Table 1.8 gives an account of the various vitamins present in the edible fish flesh.

               Table 1.8 Vitamin content of edible flesh of fish

Vitamin Average content (µg %)

Vitamin A 25
Vitamin D 15
Vitamin E 12
Vitamin B  

 Thiamin 20
 Riboflavin 120
 Nicotinic acid 3000
 Vitamin B12 1
 Pantothenic acid 500
 Pyridoxine 500
 Boitin 5
 Folic acid 80
 Vitamin C 3000
1.1.4. Factors affecting food composition
i) Raw Materials: The content of different components in food raw materials depends on the
species and variety of the animal or plant crop; on the conditions of life, and age of the farm
harvesting of the plants; on the feeding, conditions of life, and age of the farm animals or the
fishing season for fish and marine invertebrates; and on post harvest changes that take place in
the crop during storage.

ii) Processed foods: The composition of processed foods depends on the recipe applied and
on changes taking place due to processing and storage.  These changes are mainly brought
about by endogenous and microbial enzymes, active forms of oxygen, heating, chemical
treatment, and processing at low or high pH.

iii) Changes occurring in food due to processing:  Leaching of soluble, desirable and

undesirable components, such as vitamins, minerals and toxins occurs during washing,
blanching and cooking. These are lost in dripping after thawing or due to cooking. Loss of
moisture and volatiles occur due to evaporation and sublimation.

During salting, pickling, seasoning, frying or smoking there is leaching of nutrients and
absorption of desirable or harmful compounds. Due to enzyme activity, desirable or harmful
compounds are formed. There is also development of typical flavor in cheese or decarboxylation
of amino acids in fish marinades. Desirable or objectionable products are generated due to

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interactions of reactive groups induced by heating or chemical treatment, such as flavors or
carcinogenic compounds in roasted meats, or trans-fatty acids in hydrogenated fats. Different
products of oxidation of food components, mainly of lipids, pigments, and vitamins are also
formed. There is a loss of nutrients and deterioration of dried fish due to attacks by flies, mites
and beetles.
Unit 2: Moisture in Foods

Chapter 1: Moisture in Foods

2.1. Water in Food

Water is the major component of many foods. Its quantity, location and orientation profoundly
influence the structure, appearance and taste of foods. Stability, wholesomeness, and shelf life
are significant features of foods that are, to a large degree, influenced by the water content.
Fresh foods contain large quantity of water and hence effective forms of preservation is needed
for long time storage.Water content of different groups of foods is presented in table
2.1.1 Removal of water by drying or converting it into ice crystals by freezing greatle alters the
native properties of foods. The physical properties, quantity, and quality of water within food
have a strong impact on food effectiveness, quality attributes, shelf life, textural properties and
processing.

Table 2.1.1   Water content of different groups of foods

Food Water content %

Fruits
Apple,grapes,oranges 90
Pears 80-85
Tomato, strawberries 90-92
Vegetables
Banana, peas 73-80
Beet root, potatoes,carrots 86-94
Cabbage, cauliflower, lettuce 90-95
Meat
Fish 65-84
Beef/Mutton 55-70
Chicken 65-75
Pork 55-60
2.1.2. Structure and Properties of water

 Water is a familiar material, but it has been described as the most anomalous of chemical
compounds. Although its chemical composition, HOH, or H 2O, is universally known, the
simplicity of its formula belies the complexity of its behavior. Its physical and chemical properties
are very different from compounds of similar complexity, such as HF and H 2S. Water has high
melting (0ºC) and boiling points (100ºC). It exhibits large values of surface tension, dielectric
constant, heat capacity and heats of phase transition (heats of fusion, vapourisation and
sublimation).It exhibit an unusual attribute to expand on solidification. The thermal conductivity
of water is large compared to other liquids. The thermal conductivity is moderately large
compared to other nonmetallic solvents.

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The Water molecule

 Although a water molecule is electrically neutral as a whole, it has a dipolar character. The high
polarity of water is caused by the direction of the H-O-H bond angle, which is 104.5 o, and by an
asymmetrical distribution of electrons within the molecule.

 O

 /104.5°\

 +H H+

In a single water molecule, each hydrogen atom shares an electron pair with the oxygen atom in
a stable covalent bond. However, the sharing of electrons between H and O is unequal because
the more electronegative oxygen atom tends to draw electrons away from the hydrogen
nuclei. The electrons are more often in the vicinity of the oxygen atom than in the vicinity of the
hydrogen atom. The result of this unequal electron sharing is the existence of two electric
dipoles in the molecule, one along each of the H-O bonds. The oxygen atom bears a partial
negative charge and each hydrogen atom a partial positive charge. Because the molecule is not
linear, H-O-H has a dipole moment. Due to this, water molecules can interact through
electrostatic attraction between the oxygen atom of one water molecule and the hydrogen of
another.

2.1.3.Types of Water in foods

Most natural foods contain water up to 70% of their weight. Water in foods is classified in to two
types: (a) bound water and (b) free water

a) Bound water: Bound water is the equilibrium water content of a sample at some temperature
and low relative humidity. It does not freeze at low temperature (-40Cº or lower). It is
unavailable as a solvent for additional solutes. it moves with a macromolecule in experiments
involving sedimentation rates, viscosity or diffusion. It exixts in the vicinity of solutes and other
nonaqueous substances and has properties differing significantly from those of bulkwater in the
same system.

b)Free water: Water that can be extracted easily from foods by squeezing or cutting or pressing
is called as free water

2.1.3.1.Bound Water

Water that is held so tightly by another molecule (usually a large molecule such as a protein)
that it no longer has the properties of free water; water that is not easily removed from the food
is called bound water. This water is not free to act as solvent for salts and sugars. It can be
frozen only at very low temperatures. It exhibits no vapour pressure. Its density is greater than
water.

The water molecules are bound to polar groups or ions on molecules such as starchs, pectins,
and proteins. This water is held firmly. The subsequent water layers are held less firmly.

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Constitutional: It forms an integral part of a non aqueous constituent forming <0.03%. It is
constituted by a monolayer of water molecules absorbed on the polar absorption site of the
molecule is almost immobilized and thus behaves, in many respects, like part of the solid or like
water in ice.

Vicinal: It is the bound water that strongly acts with specific hydrophilic sites of non-aqueous
constituents to form a monolayer coverage; water-ion and water-dipole bonds forming 0.1 to
0.9%.

Multilayer : Bound water that forms several additional layers around hydrophilic groups, water-
water and water-solute hydrogen bonds. It forms 1-5%.

2.1.3.2.Free or entrapped water

Water that can be extracted easily from foods by squeezing or cutting or pressing is called as
free water. Flow is unimpeded; properties close to dilute salt solutions. Free water is held within
matrix or gel, which impedes flow forming 5-96%. Entrapped water is immobilized in capillaries
or cells but if released during cutting or damage, it flows freely.

2.1.4. Water activity

Water activity or aw is a measurement of water content. It is defined as the vapour pressure of
a liquid divided by that of pure water at the same temperature; therefore, pure distilled water has
a water activity of exactly one.

aw = P / P0

Where, P is the vapor pressure of water in the substance, and P0 is the vapor pressure of pure
water at the same temperature.

As the temperature increases, aw typically increases, except in some product with crystalline salt
or sugar. Higher awsubstances tend to support more microorganisms. Bacteria usually
require aw of at least 0.91, and fungi at least 0.7. Water migrates from areas of high aw to areas
of low aw. For example, if honey (aw ≈ 0.6) is exposed to humid air (aw≈ 0.7) the honey will
absorb water from the air.

Many of the chemical and biological processes that cause deterioration of foods, and ultimately
spoilage, are water dependent. In general water activity aw represents the water which is made
available for the microbial action.

Microbial growth is directly linked to water activity. Essentially, water activity is the measure of
the degree to which water is bound within the food, and hence is unavailable for further
chemical or microbial activity

Relative humidity of moist air is defined in the same way except that by convention, relative
humidity is reported as a percentage whereas water activity is expressed as a fraction. Thus if a
sample of meat sausage is sealed within an airtight container, the humidity of the air in the head
space will rise and eventually equilibrate to a relative humidity of, say 83%, which means that
the water activity (aw) of the meat sausage is 0.83.

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2.1.4.1. Water activity and Shelf life of Foods

Water activity is an important consideration for food product design and food safety. Food
designers use water activity to formulate products that are shelf stable. If a product is kept
below a certain water activity, then mold growth is inhibited. This results in a longer shelf-life.

Water activity is used in many cases as a critical control point for Hazard Analysis and Critical
Control Points (HACCP) programs. Samples of the food product are periodically taken from the
production area and tested to ensure water activity values are within a specified range for food
quality and safety. Measurements can be made in as little as five minutes, and are made
regularly in most major food production facilities

2.1.4.2. Water activity of some foods

Many of the chemical and biological processes that cause deterioration of foods, and ultimately
spoilage, are water dependent. Microbial growth is directly linked to water activity. No microbes
can multiply at a water activity below 0.6. Dehydration is arguably the oldest form of food
preservation; the sun drying of meat and fish has been traces to the beginning of recorded
history. Drying relies on removing water, thus making it unavailable for microbial growth.

Water activity of some food

Substance aw
Distilled Water 1
Tap water 0.99
Raw meats 0.99
Milk 0.97
Juice 0.97
Cooked bacon < 0.85
Saturated NaCl solution 0.75
Point at which cereal loses crunch0.65
Dried fruits 0.60
Typical indoor air 0.5 - 0.7
Honey 0.5 - 0.7
Dried fruit 0.5 - 0.6

Many of the chemical and biological processes that cause deterioration of foods, and ultimately
spoilage, are water dependent. Microbial growth is directly linked to water activity. No microbes
can multiply at a water activity below 0.6. 

Dehydration: Dehydration is arguably the oldest form of food preservation; the sun drying of
meat and fish has been traced to the beginning of recorded history. Drying relies on removing
water, thus making it unavailable for microbial growth. Water is a facilitator of biochemical
deterioration of foods. Dry foods are much more stable than wet foods, because any water
remaining in them has low activity.

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Salting or curing has the same effect. A saturated solution of common salt has a water activity
of close to 0.75. Thus by adding sufficient salt to foods, the water activity can be lowered to a
level where most pathogenic bacteria are inactivated but the moisture content remains high.

Intermediate moisture content foods (IMF), such as pet food and continental sausages; rely on
fats and water-binding humectants such as glycerol to lower water activity. Fat, being essentially
hydrophobic, does not bind water, but acts as filler for IMF to increase the volume of the
product. The water activity of the salted food is 0.8.

None of the dangerous pathogenic bacteria associated with food, such

as Clostridium or Vibrio spp. which cause botulism and cholera, can multiply at water activity
values below about 0.9. Thus, drying or providing sufficient water-binding humectants is an
effective method of preventing the growth of food-poisoning bacteria.

Only osmophilic yeast and some molds can grow at water activities in the range 0.6 to 0.65.
Thus, by reducing the water activity below these values, foods are microbial stable. That is,
unless the packaging is such that the food absorbs moisture and spoilage can occur, for
example, when condensation occurs within a hermetically sealed package subject to rapid
cooling.

There are various chemical reactions that proceed, and may be accelerated, at low values of
water activity.

1. Maillard reactions leading to lysine loss and brown color development peaks at values
around 0.5 to 0.8.
2. Nonenzymatic lipid oxidation increases rapidly below aw = 0.4.
3. Enzymatic hydrolysis decreases with water activity down to aw = 0.3 and is then
negligible.

Water is facilitator of biochemical deterioration of foods. Dry foods are much more stable than
wet foods, because any water remaining to them has low activity, aw. Freezing removes water
from the food matrix by forming ice crystals. Although the ice crystals remain in the food, the
remaining water which is in contact with the food matrix becomes concentrated with solutes and
it’s aw becomes low. Freezing is therefore akin to drying and this is the rationale for preserving
food by freezing. Most micro-organisms cease functioning below the water activity of about 0.7.

Unit 3: Food lipids

Chapter 1 Food Lipids

3.1.1. Food Lipids

The lipids are heterogenous group of compounds related to the fatty acids. They are insoluble in
water and soluble in other solvents such as ether, chloroform and benzene. Chemically they are
esters of fatty acid and some alcohols. The lipids are widely distributed in plant and animal
kingdom. The lipids include fats, oils, waxes and related compounds. Oils are liquid at 20º C
and are solids at 20º C
3.1.2.Types of fat
                                          

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1. Predominantly saturated fats (solid at room temperature) include all animal fats (e.g. milk fat,
lard, tallow), as well as palm oil, coconut oil, cocoa fat and hydrogenated vegetable oil
(shortening).
2. All other vegetable fats, such as those coming from olive, peanut, maize (corn oil),
cottonseed, sunflower, safflower, and soybean, are predominantly unsaturated and remain
liquid at room temperature, hence known as oils.

However, both vegetable and animal fats contain saturated and unsaturated fats. Some oils
(such as olive oil) contain, in majority, monounsaturated fats, while others present quite a high
percentage of polyunsaturated fats (sunflower, rape).

3.1.3.Fatty acids
These are aliphatic monocarboxylic acid that are liberated by hydrolysis from naturally
occurring fats. The fatty acids present in natural fats are of two types. 1. Saturated and 2.
unsaturated.

1. Saturated fatty acids: These fatty acid are straight chain containing no doble bond. They are
named after hydrocarbons with the same number of carbon atoms but one terminal methyl
group (CH3) is replaced by a carboxyl group (COOH).

2. Unsaturated fatty acids

There are two main types of unsaturated fats (i) monounsaturated (containing one
double bond) and (ii) polyunsaturated (containing more than one double bond). Most
monounsaturated and polyunsaturated fats have good qualities, with one exception - trans-fatty
acids. Trans fatty acids are, technically speaking, an unsaturated fat but offer no health benefits.
1. Monounsaturated fatty acids
If there is one double bond is present, the fatty acid is known as a monounsaturated fatty acid.
This is found in significant amounts in most types of fats of plant origin, such as nuts, avocado,
pears, rapeseed oil and olive oil. Monounsaturated fat do not raise blood cholesterol and
evidence shows that they reduce blood cholesterol levels if they replace saturated fat in the
diet Oleic acid is the main monounsaturated fat in our diets and this is sometimes called omega-
9 (because the double bond is in position 9 of the fatty acid chain). It is found in significant
amounts in most types of nuts, avocado, pears, rapeseed oil and olive oil and spreads made
from these.
2. Polyunsaturated fatty acids
If there is more than one double bond, then the fatty acid is known as a polyunsaturated fatty
acid. These come mostly from vegetable sources, such as sunflower oil or seeds, but are also
found in nuts, green leafy vegetables and oily fish such as mackerel and sardines.
Polyunsaturated fatty acids can actively reduce blood cholesterol levels. The polyunsaturated
fatty acdsfound in oily fish specifically appear to have no effect on blood cholesterol levels, but
they do alter the consistency of blood.
Types of polyunsaturated fatty acids:
There are two 'types' of polyunsaturated fats that are found in our diet, which are also known as
essential fatty acids. They are omega 3 and omega 6. Essential fatty acids are so called
because the body cannot make them but they are essential to the body's normal functioning,
therefore, must be supplied through diet. Humans are unable to make these essential fatty
acids, because they do not have the particular destaurase enzymes that insert double bonds in
position 3 and 6 of the fatty acid chain.
3.1.4.Functions of lipid

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1. Source of energy: Lipids are important sources of metabolic energy (ATP). In fact, the lipids
are the most energy rich of all classes of nutrients: gross energy value of lipid is 9.5
Kcal/g, protein 5.6 Kcal/g, carbohydrate 4.1 Kcal/g and the net values were 9 K.cal /g,
4.0K.cal/g and 4.k.cal/g for fat, protein and carbohydrate respectively. The freefatty
acids derived from triglycerides (fats and oils) are the major aerobic fuel sources for energy
metabolism.
2. Forms part of membrane: Lipids are essential components of all cellular and subcellular
membranes (Lipid classes that are involved include phospholipids, and sterol esters).
3. Source of fat soluble vitamins: Lipids serve as biological carriers for the absorption of the
fat soluble vitamins A, D, E and K.
4. Source of essential fatty acids: Lipids are a source of essential fatty acids.  Linoleic and
linolenic acids are essential for the maintenance and integrity of cellular membranes, required
for optimal lipid transport (bound tophospholipids as emulsifying agents), and are
the  precursors of the prostaglandin hormones.
5. Mechanical cushion/support: Lipids play a role as mechanical cushion/support for the vital
body organs.
6. Source of essential steroids: Lipids are a source of essential steroids, which in turn perform
a wide range of important biological functions. The sterol cholesterol is involved in the
maintenance of membrane systems, for lipid transport, and as a precursor of vitamin D3, the
bile acids, and the steroid hormones – androgens, estrogens, adrenal hormones, and
corticosteroids.
7. Contribute food flavour, taste and texture: Fat play a role in food flavor / mouth feel,
palatability, texture and aroma.
3.1.5.Properties of fat

(a) The fats are insoluble in water, but readily soluble in ether, chloroform, benzene, carbon
tetra chloride

(b) They are readily soluble in hot alcohol but slightly soluble in cold.

(c) They are themselves good solvents for other fats, fatty acids etc.

(d) They are colourless, odourless, tasteless and neutral in reaction.

(e) Several neutral fats are readily crystallised, e.g. mutton, beef

(f) Their melting points are low.

(g)The specific gravity is about 0.86. Hence the fatreadily float in water.

They spread uniformly over the surface of wates and this spreading effect is to lower
surface tension.

3.1.6. Lipoproteins

Chylomicrons are droplets of nearly pure triglycerides, coated by a very thin layer of protein.
Chylomicrons carry triglycerides from the small intestine, where they are absorbed during
digestion, to the fat depots. The composition of lipoproteins is presented in Table. The nonpolar

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triglyceride and cholesterol are hidden inside an outer coat of hydrophilic polar heads. This
structure gives these lipid-rich struc tures water solubility, well adapted for the transport of lipids
via the blood from the small intestine to liver

Composition of Blood plasma Lipoprotein

---------------------------------------------------------------------------------------------------------
Type  Density Protein Triglycerides Phospholipids Cholesterols
 g/ml % % % %
Chylomicrons  0.92-0.96 1.7  96 0.8 1.7
Very low Density 
(VLDL) 0.95-1.00 10.0 60  18 15
Low density (LDL) 1.00-1.06 25.0 10 22 45
High density HDL 1.06-1.21 50.0 3 30 10
3.1.6.1. Functions

The various types of lipoproteins have different functions. 

Chylomicrons and VLDLs: Chylomicrons and VLDLs are the principal carriers of triglycerides

and Choleterols in blood.The concentrations are increased in atheroscorosis and coronary
thrombosis. 

LDLs:In LDLs the predominant lipid is cholesterol and phospholipids. Increased in

atherosclerosis and coronary thrombosis, etc.

HDLs: HDLs are predominant lipid is phospholipid and proteins.

LDLs and HDLs are involved in the cholesterol transport. LDLs carry about 80% of cholesterol
while the remaining is carried by HDLs. LDLs carry cholesterols to cells for their use where as
HDLs carry excess cholesterol away from the cells to the liver for processing and excretion from
the body. The levels of LDL correlate directly with heart disease, where as HDL levels correlates
inversely with heart disease risk. Thus HDL is some times referred to as “good” cholesterol and
LDL as “bad” cholesterol. 

Chapter 2:  Fish lipids

3.2.1. Fish lipids

Lipid in fish generally carries natural flavour components and provides and preserves other
generated during cooking, pickling or other processing. A certain amount of fat and fatty acid
assists in providing smoothness of texture during mastication of lean fish. In fatty fish the
influence of fat on texture is even more important. The proximate composition of fish varies
widely from species to species and even within the same species from one individual to another
depending on age, sex, environment and season. The principal components of fish may be
divided into five categories viz., water, protein, lipid, carbohydrate and ash.

Proximate composition of seafood

Type of fish Moisture % Protein % Fat % Ash Carbohydrate %

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%
Fatty fish 68.8 20.0 10.0 1.2 Negligible
Lean fish 81.8 16.4 0.5 1.3 < 0.5
Crustaceans 76.0 18.8 2.1 3.1 < 0.5
Mollusks 81.0 12.0 1.5 2.6 2.9

There is variation in the amount of lipid content of dark and white meat of some food fishes.

Variation of lipid content of dark and white meat of some fishes

Fish Kind of Moisture % Crude Crude fat

species meat protein % %
Tuna D 66.4 22.9 6.7

W 68.5 22.9 4.5

Sardine D 70.0 15.9 12.8

W 72.0 23.1 2.9

Mackerel D 54.2 14.9 29.7

W 65.5 21.2 13.1

Herring D 57.8 15.5 28.2

W 74.0 22.0 13.0

3.1.3. Distribution of Fat in Fish

Distribution of Fat in Fish

The term lipid will be used for total fat component in fish. However, term fat is used for selected
anatomical deposits, which are mostly triglyceride. In lean fish, the dark (red or lateral line)
muscle has about twice the lipid of white muscle. The percentage of cellular lipid in the white
muscle is normally altered by season. The lean muscle fish generally have more fat in livers
(e.g. cod) which show seasonal variation. In the fatty fish species, the muscle shows fluctuating
levels of seasonal variation in neutral fat

a. Neutral fat (Triglycerids)

Triglyceride or the neutral fat forms the major constituent of fish lipid. There are variations in the
amount of neutral fat in muscle. The belly flap is a high fat section of many fishes. (E.g. In
mackerel -29% lipid in belly flaps, 18.3% lipid in dark muscle and 7.6% in while muscle). In male
mackerel the skin fat forms 40% of the total fat in the whole fish. Triglyceride distributed through
fish muscle tends to have a homogeneous fatty acid composition Most species of marine
organisms try to obtain an optimal fat and fatty acid composition and their behavior and food
preferences lead towards this objective.

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In the triglycerides there is usually a polyenoic fatty acid such as 20.5ω3 or 22:6ω3 in
the 2 position, a saturated or monoenoic fatty acid in the 1 position and a monoenoic fatty
acid in the 3 position. However, none of these are hard and fast rules because of the super
abundance of fatty acid types and other factors. The triglycerides of marine mammals are
different, with the polyethylenic fatty acid concentrated at position 1 and 3.

Triglyceride

1 
CH2O – COR - Saturated or monoenoic
│
2CHO – COR - 20.5ω3 or 22:6ω3
│
3CH2O – COR - Monoenoic

b. Basic cellular lipids (Phospholipids)

Lean white fish muscle contain a minimum of about 0.7% of basic cellular lipid, of which 85-95%
is ‘polar’ lipids, mostly phosphatidyl ethanolamine and phosphatidyl choline. The balance of this
type of basic lipid includes sterol ester and free sterol, free fatty acid and triglyceride.

This basic mixture represent the structural lipid of cell walls, and that any excess of triglyceride
and/or certain other non-polar lipids such as wax esters or glyceryl ethers, provide the ‘fat’ of
fatty fish.

3.2.3.Fatty acids of fish lipid

The fatty acid s present in fish lipid is classified into three groups, saturated acids, monoenoic
acids and polyenoic acids.

1. Saturated Fatty Acid s

The saturated fatty acids present in fish lipids are myristic or tetradecaenoic (14:0), palmitic or
hexadecaenoic(16:0) and stearic or octadecaenoic(18:0). Palmitic (16:0) acid, is the principal
saturated fatty acid (10-30% of the total), myristic (14:0) acid (5-10% of the total) and stearic
(18:0) acid 1-3% of all fatty acid s. The 20:0, 22:0 and 24:0 fatty acid s are just detectable at
levels of 0.01-0.1%. These fatty acid s can all be biosynthesized by the organisms, but they are
also freely absorbed from dietary fats.

If a propionate molecule primes the two-carbon fatty acid chain extension process instead of an

acetate molecule odd-chain fatty acid s are also formed. Bacteria also contribute odd number
straight-chain fatty acid s. The fatty alcohols of copepod esters also have C15 and C17 methyl-
branched and odd carbon structures which are probably oxidized to corresponding acids and
then deposited in the fats of capelin, mackerel and herring.

2. Monoenoic Fatty Acids

The monoenoic acids present in fish lipids are, oleic C18:1 or 9-octadecenoic (1ω7), gadoleic or
11-eicosenoic (20:1 ω9) and cetoleic or 11-docosenoic (22:11 ω1). The monoenoic fatty acids,
palmitoleic acid (16:1) and oleic (18:1 ω9) of fish oils can be synthesized by fish and other

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marine organisms from acetate units. Palmitoleic acid can be carbon extended to cis-vanccenic
acid (18:1 ω7), which forms 10-30% of the total 18:1 isomers. The origin of the 20:1 ω9 is by
chain elongation of 18:1 ω9. The proportion of the minor isomers 20:1 ω11 and 20:1 ω7 relative
to the major isomer 20:1 ω9 are similar in several fish oils. In pacific herring oil 20:1 ω11 is
present in more than the usual proportions. The dominant 22:1 isomer in marine fish oil is 22:1
ω11. In herring only the 22:1 ω9 isomer is biosynthesized.

3. Polyenoic Fatty Acids

The important polyenoic acid present in fish lipids are Eicosapentaenoic (EPA) (C20:5 ω3) and
Docosahexaenoic (DHA) (C22:6 ω3). They give marine oils their most specific characteristics.
The polyenoic fatty acid s with five, (EPA) and six ethylenic bonds (DHA) originate in unicellular
phytoplankton or in some seaweed.

The average fatty acid composition of phytoplankton includes all the principal fatty acid found in

the oils and lipids of the higher organisms. The two common ‘plant’ C18 fatty acid s 18:2 ω6
(linoleic acid) and 18:3 ω3 (linolenic acid) do not accumulate in most fish oils to the extent of
more than 1% or 2% of fatty acid s. This is also true of marine invertebrates feeding directly on
plants with lipids rich in these acids. In many invertebrate lipids 20:5 ω3 is the dominant
polyunsaturated fatty acid . This is possibly due to dietary algae.

3.2.4. Wax Easters and Glyceryl Ethers

Wax esters of marine organisms fall into two main classes, those rich in 16:0 and those rich in
22:1 acids. The chain length total tends to be in the range C32 to C36. The copepod wax ester,
rich in 22:1 alcohol, has a high content of 14:0 acids. These species have a high proportion of
16:0 fatty alcohols in their wax esters as part of their body lipid. The ‘castor oil’ fish Ruvettus
pretiosus contains wax esters, which are responsible for the purgative effect.

Two related materials; I-glyceryl ethers (alkyldiacyglycerols) and II=plasmalogens(alk-1-

enyldiacylglcerols) may also be present in fish fat or oil.

I-glyceryl ethers (alkyldiacylglycerols) Plasmalogens (alk-1-enyldiacylglcerols)

CH2-O- R                                           CH2-O-CH=CH-R

│                                                        │

CH-O-CO-R                                       CH-O-CO-R

│                                                         │

CH2-O-CO-R                                     CH2-O-CO-R

Structurally related materials such as hydroxyalkyl glycerols or methoxy glyceryl ethers and
hydrocarbons such as squalene are also form minor components in fish lipids.

3.2.5. Role of Fish Lipids in Human Nutrition

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Role of Fish Lipids in Human Nutrition

To achieve a balanced diet there is a need to reduce total fat intake and it is also important to
make sure that the type of fat we eat is right. The fat that is not beneficial to human is the hard
"saturated" fat which comes mainly from the fat of land animals such as cows and sheep.

1. Food with low fat: Fish is a good food for a low fat diet. It is low in calories and many types
of fish do not contain any saturated fat. The nutritional value of fish will vary slightly according to
the location it is harvested, the cut of fish, and the age of the fish. The method used for cooking
will have an effect on it also.

2. Reduce the cholesterol level in the blood: Cholesterol is the type of fat which is naturally
produced by our bodies and is also found in the diet. It is essential for life but too much of it
circulating in the bloodstream is a problem, and it is usually deposited in the lining of the blood
vessels, causing them to narrow. The heart then has to work harder to pump blood around the
body. Blood clotting can result and the cholesterol deposits can be very hard. Tissues can
become deprived of oxygen when the blood vessels become blocked. Unsaturated fats can help
to reduce the cholesterol level in the blood, thus lowering the risk of heart disease. Oil-rich fish
such as mackerel, sardines, herring and sprats are rich in unsaturated fats containing Omega-3
fatty acids which are valuable for health.

3. Source Omega-3 fatty acids: Two fatty acids, eicosapentaenoic acid (EPA) and
docosahexaenoic (DHA), collectively known as omega-3, are essential fatty acids. Omega-3
fatty acids are not synthesised in the body. Oil-rich fish, such as salmon, trout, mackerel, herring
and sardines are excellent sources of Omega-3 fatty acids.

i.Schizophrenia symptoms can be eliminated or at least vastly diminished by oral

supplementation with EPA.

ii.DHA is the building block of human brain tissue and is particularly abundant in the grey matter
of the brain and the retina. Low levels of DHA have been associated with depression, memory
loss, dementia and visual problems. DHA is particularly important for fetuses and infants; the
DHA contents of the infant’s brain triples during the first three months of life. Optimal levels of
DHA are therefore crucial for pregnant and lactating mothers. They cannot be made in the body,
hence are essential in the diet.

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iii.The special Omega-3 oils from fish have been shown to have a lowering effect on blood fats.
This decreases the chance of the blood vessels clogging with cholesterol. Omega-3 can also
make blood less "sticky", and it therefore flows more easily around the body. This can reduce
the risk of a heart attack. They also help to reduce blood pressure a little and keep the heart
beat steady. Omega-3 oil in fish can reduce the risk of dying from heart attacks.

4. Prevent cancer: Fish oils can help to prevent cancer cells progressing to the tumor stage.
They may also reduce inflammation and provide relief for people suffering from rheumatoid
arthritis and even some skin disorders such as psoriasis.

5. Needed for the development of Brain: Omega-3 oils can play an important part in aiding
the development of brain. Expectant mothers are advised to eat a lot of oil-rich fish in the last
three months of pregnancy to assist the baby's brain growth. A good supply of Omega-3 oils
assists the development of nerves and eyesight.

Chapter 3. Oxidation of lipids

3.3.1.Intoduction
Lipid oxidation is one of the major causes of food spoilage. In edible oils and fat-
containing foods, it leads to the development of various off flavors and off odors, generally
known as oxidative rancidity, which renders the foods less acceptable. It may also be able to
decrease the nutritional value of food and in some cases may produce potentially toxic
products. It may be sometimes desirable as in the case of cheese.
3.3.2.Types of Oxidation
Oxidaion is caused by a biochemical reaction between fats and oxygen called as
autoxidation and it is the main reaction involved. The lipids of foods can be oxidized by both non
enzymic and enzymic mechanisms. Lipid oxidation generally occurs after a long induction
period. Once started it is generally a very rapid reaction.
3.3.2.1.Non enzymic oxidation
   (i)Non enzymic oxidation
Non enzymatic lipid oxidation (Autoxidation) proceeds by a free radical mechanism. It is
catalysed by light and free radical-producing substances and yields hydroperoxide (ROOH).
This primary product is relatively unstable. It enters into numerous reactions involving substrate
degradation and interaction, resulting in numerous compounds of various molecular weight ,
flavour generating and biological significance.
Free radical mechanism. 
A free radical is a compound with an odd number of unpaired electrons.

                                         H     H      H
                                    — C —C — C —
                                         H     H      H

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                                                ↓

                                         H     H      H
                                    — C —C — C —      +      * H
                                         H      *    H                    

  

When initiated two free radicals are formed. These radicals are very reactive and generally do
not have long life time.
3.3.2.2.Enzyme catalysed lipid oxidation
Enzyme catalysed lipid oxidation
Oxidation of lipids is also by enzymes that start with the action of lipolysis. Released
polyunsaturated fatty acids are then oxidized by either lipoxygenase or cyclooxygenase to form
hydroperoxides or endoperoxides, respectively. Then these compounds are hydrolysed to yield
a variety of breakdown products, which are responsible for the characteristic flavours of natural
products.
3.3.2.3.Effects of Lipid Oxidation in Foods
Effects of Lipid Oxidation in Foods

When lipids in food are oxidised, some of the product formed impart odour and flavours, usually
undesirable, to the food. The free radicals generated during the oxidation reaction and some of
the molecules formed when oxidized compound decombos (aldehydes, acids, alcohols, ketones
etc.) can interact with and alter other constituents including pigments, vitamins, proteins
and  amino acids.

3.3.2.4.Pro-Oxidants
Pro-Oxidants: Prooxidants are those compounds that promote oxidation. Transition metals,
those possessing two or more valency states and a suitable oxidation reduction potential
between them (e.g.copper, iron, manganese, cobalt and nickel), are effective pro-oxidants. The
proxoidants, if present even at very low concentrations (0.1ppm), can decrease the induction
period and increase the rate of oxidation.Trace amount of metals are naturally occurring in all
food tissues, extracted oils and all fluids of biological origin (eggs, milk and fruit juices). They
are present in both free and bound forms. Heme compounds are also important pro-oxidants in
food.
3.3.2.5.Antioxidant
Antioxidant: An antioxidant is a molecule that can delay onset, or slow the rate of oxidation of
oxidisable material. Oxidation reactions can produce free radical. In turn, these radicals can
start chain reactions. Antioxidants terminate these chain reactions by removing free radical
intermediates, and inhibit other oxidation reactions by acting as hydrogen donors or free radical
acceptors.
               ROO.  + AH → ROOH + A.
The phenolic antioxidants are more suitableas they are excellent hydrogen or electron donors
and their radical intermediates are relatively stable due to resonance.

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Antioxidants are found in varying amounts in foods such as vegetables, fruits, grain cereals,
eggs, meat, legumes and nuts. Natural antioxidants are ascorbic acid and tocopherols.
Synthetic antioxidants are widely used as preservatives in food and as ingredients in dietary
supplements to prevevt food deterioration. They include propyl gallate (PG), isoamyl gallate,
tertiary butylhydroquinone (TBHQ), butylated hydroxyl anisole (BHA), butylated hydroxyl toluene
(BHT) and nordihydroquaiaretic acid (NDGA) Antioxidants can be directly added to vegetable
oils or to melted animal fats after they are rendered. Food products can also be dipped in or
sprayed with solutions of antioxidants.
Unit 4: Metabolism of lipids

Chapter1: Digestion and absorption of lipids

4.1.1.Introduction
Foods are enzymatically digested to prepare them for absorption. During digestion in the
gastrointestinal tract of mammals, the three major nutrients (carbohydrates, lipids, and proteins)
undergo enzymatic hydrolysis into their building block components. This is necessary for their
absorption, since the cells lining the intestine are able to absorb them into the bloodstream only
as relatively small molecules. Lipids must be hydrolyzed into fatty acids and glycerol.
4.1.2.Digestion
The digestion of triglycerides begins in the small intestine. In this region the zymogen,
prolipase is secreted by the pancreas (Fig.1). There it is converted into lipase, which in the
presence of bile salts and a special protein called colipase, binds to droplets of triglycerides and
catalyzes the hydrolytic removal of one or both of the outer fatty acid residues. Monoglycerides
remain unhydrolyzed. The fatty acids and the uncleaved gycerides are emulsified into fine
droplet by peristalsis, the churning action of the intestine, aided by the detergent effect of the
bile salts and the monoglycerides, which are amphipathic molecules. Phospholipids are split by
phospholipases to the acyl chains, glycerol and choline. Cholesterol esters are converted to
cholesterol and free fatty acids.
4.1.3.Absorption
The fatty acids, glycerol and monogycerides, in these droplets are absorbed by intestinal
cells, where they are largely reassembled into triglycerides. The free fatty acids are activated by
thiokinase in the presence of coenzyme A and ATP for the resynthesis of triglyceride. Some free
glycerol passes directly to the lymp vessel. The others will be activated by glycerokinase in the
presence of ATP to form glycerol 3 phosphate and combine with acyl CoA to form triglycerides.
All the long chain fatty acids present are reincorporated into the triglycerides. The triglycerides
do not pass into the blood capillaries but into the small lymph vessels in the villi. The choline
from phospholipids may be absorbed and send to liver via lymph vessels. Cholesterol is
absorbed into the lymphatic vessels and converted into cholesterol esters and transported.
Chylomicrons: The lymph draining the small intestine, called chyle, has a milky appearance
after a fat-rich meal, due to the suspended chylomicrons, droplets of highly emulsified
triglycerides, about 1µm in diameter. Chylomicrons contain triglycerides, free and esterified
cholesterol; have a hydrophilic coat of phospholipids and a special protein, which function to
keep the chylomicrons suspended. The chylomicrons pass from the thoracic duct into the
subclavian vein and then to liver.
Emulsification: The emulsification and digestion of lipids in the small intestine is facilitated by
the bile salts. The major human bile salts are sodium glycocholate and sodium taurocholate,
derivative of cholic acid, the most abundant of four major human bile acids. The bile salts are
powerful emulsifying agents secreted by the liver into the bile, which empties into the upper
portion of the small intestine. After the fatty acids and monoglycerides of the emulsified fat

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droplets have been absorbed in the lower small intestine, the bile salts aiding this process are
also reabsorbed. They return to the liver, to be used over again.
4.1.4.Metabolism
Metabolism of Triglycerides
Triglycerides are first converted to fatty acids and glycerol mostly in adipose tissue. The
fatty acids are released into the plasma where they combine with serum albumin. Long chain
fatty acids are oxidized in liver, heart, kidney, muscle, lung, brain and adipose tissue. Glycerol is
utilized by liver, kidney, intestine and lactating mammary gland where the activating enzyme
glycerokinase is present.
b) Metabolism of fatty acids
The fatty acids components of the lipids entering the liver also have several different
pathways

1. Oxidation to CO2 with ATP production: Free fatty acids may be activated and oxidized to
yield acetyl-CoA and ATP. The acetyl-CoA is oxidized via the citric acid cycle to yield ATP by
oxidative phosphorylation. Fatty acids are the major oxidative fuel in the liver.

2. Synthesis of fatty acids: There are three types of fatty acid synthesis. (1) Elongation of
existing short chain fatty acid in the mitochondria (2) Microsomal system of chain elongation and
(3) The cytoplasmic synthesis of fatty acid from acetyl CoA.
3. Biosynthesis of cholesterol: Some of the acetyl-CoA derived from fatty acids (and from
glucose) will be used as the major precursor for the biosynthesis of cholesterol, which in turn is
the precursor of the bile acids and bile salts, which are essential for the digestion and
absorption of lipids.
4. Biosynthesis of lipids of plasma lipoproteins( Triglyceride and phospholipids): Fatty
acids are also used as precursors for the synthesis of the lipid portion (triglycerides and
phospholipids) of the plasma lipoproteins, which carry lipids to adipose or fat tissue for storage
as triglycerides.
5. Fomation of ketone bodies: Excess acetyl-CoA released on oxidation of fatty acids and not
required by the liver is converted into the ketone bodies, acetoactate and D-β-hydroxy butyrate,
which are circulated via the blood to peripheral tissues, to be used as fuel for the citric acid
cycle. The ketone bodies may be regarded as a transport form of acetyl groups. They can
supply significant fraction of the energy to some peripheral tissues, up to one-third in the case of
the heart

Chapter 2: Metabolism of Fat

4.2.1.Introduction
The lipids of metabolic significance include synthesis and degradation of triglycerides,
phospholipids, steroids together with long chain fatty acids, glycerol and ketone bodies.
Oxidation of triglycerides takes place in the adipose tissue. The complete metabolism of fat in
the body leads to the oxidation to CO2 and water, and the liberation of energy equivalent to
9kcal /g of fat.
4.2.2.Oxidation of fatty acids to CO2 with ATP production
Fatty acids are oxidized by β, α, and ω oxidation. β- Oxidation is the most important pathway for
the production of energy. The term oxidation means the oxidation takes place in the β -carbon in
the fattyacid with the removal of 2 carbon atoms at a time from the carboxyl end of the
molecule. The saturated fatty acids containing even number and odd number of carbon atoms
and the unsaturated fatty acids are oxidized by β -oxidation.

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4.2.2.1.β-Oxidation of saturated fatty acids
Saturated fatty acids are oxidized to acetyl-CoA by β oxidation. It takes place in mitochondria.
Five steps are involved and each step involves acy1-CoA derivatives catalyzed by separate
enzymes, utilizes NAD+ and FAD as coenzymes, and generates ATP. Fatty acid oxidation is an
aerobic process, requiring the presence of oxygen.
Step 1. Activation of Fatty Acids
Long chain fatty acids are first converted to an ‘active fatty acid’ or acyl CoA in the
cytosol in the presence of ATP, coenzyme A and Mg2+ catalyzed by the enzyme acyl-CoA
synthase (thiokinase). But activation of lower fatty acids occurs within the mitochondria.
Thiokinase is found both inside and outside the mitochondria.

Thiokinase
Fatty acid + ATP + coenzyme A + Mg2+ → Acyl CoA + AMP

The presence of inorganic pyrophosphatase ensures that activation goes to completion

by facilitating the loss of the additional high-energy phosphate associated with pyrophosphate.
Thus, in effect, two high energy phosphates are expended during the activation of each fatty
acid molecule.
Acyl-CoA synthetases are found in the endoplasmic reticulum and inside and on the
outer membrane of mitochondria. Several acyl-CoA synthetases have been described, each
specific for acids of different chain length.
Transport into mitochondria
(i)Transport of smaller fatty acids
Small fatty acids are able to penetrate the inner membrane of mitochondria and become
oxidized within the mitochondria.
(ii) Transport of Long-Chain Fatty Acids
Long-chain fatty acids penetrate the inner mitochondrial membrane only
as carnitine derivatives.An enzyme,carnitine acyl transferase I, present in outer mitochondrial
membrane, converts long-chain acyl CoA to acylcarnitine, which is able to penetrate the inner
membrane of mitochondria and gain access to the oxidation system of enzyme.
Carnitine-acyl carnitine translocase present in mitochondria, catalyses the transfer the
acyl carnitine into inner membrane. Carnitine acyl transferase II, present in the inner
mitochondrial membrane, converts acylcarnitine to long-chain acyl CoA and carnitine. Acyl CoA
then undergoes further reactions of β-oxidation.
Step 2. Dehydrogenation of Aceyl CoA
Acyl CoA is dehydrogenated by Aceyl CoA dehydrogenase to form α-β unsaturated acyl CoA.
NAD+ is the coenzyme. It is converted to NADH + H + which is reoxidised via electron transport
chain.
Aceyl CoA dehydrogenase 
Acyl CoA + NAD+ ↔ α-β unsaturated acyl CoA + NADH +H+
Step 3. Conversion of α-β unsaturated acyl CoA to β hydroxyl acyl CoA
α-β unsaturated acyl CoA is converted to β hydroxyl acyl CoA by the addition of a water
molecule catalysed by the enzyme enoyl-CoA hydratase.
Enoyl-CoA hydratase.
α-β unsaturated acyl CoA + H2O↔ β hydroxyl acyl CoA
Step 4. Dehydrogenation at the β-carbon of β-hydroxyacyl CoA
The β-hydroxy derivative undergoes further dehydrogenation on the β-carbon by β-hydroxyacyl-
CoA dehydrogenase to form the corresponding β-ketoacyl-CoA compound. In this case, NAD+ is
the coenzyme involved in the dehydrogenation. The NADH+H+ formed is reoxidised via electron
transport chain.

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β-hydroxyacyl-CoA dehydrogenase
β hydroxyl acyl CoA + NAD+↔ β-ketoacyl-CoA + NADH+H+
Step 5. Cleavage by thiolase
Finally, β-ketoacyl-CoA is spilt at the 2, 3- position by thiolase (β-ketoacyl-CoA-thiolase), which
catalyzes a thiolytic cleavage involving another molecule of CoA. The products of this reaction
are acetyl-CoA and an acyl-CoA derivative containing two carbons less than the original acyl-
CoA molecule that underwent this oxidation.
Thiolase 
β-ketoacyl-CoA ↔ Acetyl-CoA + acyl-CoA
The acyl-CoA formed in the cleavage reaction renters the oxidative pathway at reaction `1.
In this way, a long-chain fatty acid may be degraded completely to acetyl-CoA (C2 units). In the
case of palmitic acid the reactions are repeated 7 times and 8 molecules of acetyl CoA are
formed.
Since acetyl-CoA can be oxidized to CO 2 and water via the citric acid cycle, the complete
oxidation of fatty acids is achieved.
Production of ATP
Transport of electrons from FADH2 and NADH (formed during β-oxidation) in the respiratory
chain will lead to the synthesis of five high energy phosphates (ATP) for each of the first seven
acetyl-CoA molecules formed by β-oxidation of palmitate. At the end, 8 molecules of acetyl CoA
are formed.
No.of ATP derived from β-Oxidation                                 : 7x5   = 35  
No. of ATP formed on oxidation of 8 acetyl-CoA molecules
                                                         (via citric acid cycle) : 8x12 = 96
                                                                                                       ------
                                                                   Total               :           131
 ATP utilized for initial activation of the fatty acid              :              -2  
                                                                                                        ------
                                                         Net total yield  :            129        
Calorific value per mole of palmitic acid:          
The calorific value is 129x7.6=980 K.cal/mole. The calorific value per mole of combustion of
palmitic acid is 2340 K.cal/mole. The process captures as high-energy phosphate in the order of
41% of the total energy of combustion of the fatty acid.
4.2.3. Oxidation of a fatty acid with an odd number of carbon atoms
Fatty acids with an odd number of carbon atoms are oxidized by the pathway of β - oxidation,
producing acetyl-CoA until a three- carbon (propionyl-CoA) residue remains. This compound is
then converted to succinyl-CoA, a constituent of the citric acid cycle and metabolized.
Propionyl-CoA carboxylase
Propionyl CoA + CO2 + H2O → D-methylmalonyl-CoA
D-Methylmalonyl-CoA is converted to its steroisomer, L- methylmalonyl-CoA, by methylmalonyl-
coA racemase before its final isomerization to succinyl-CoA by the enzyme methylmalonyl-CoA
isomerase.
Methylmalonyl-CoA racemase
D-Methylmalonyl-CoA ↔ L- methylmalonyl-CoA
Methylmalonyl-CoA isomerase
L- methylmalonyl-CoA ↔ Succinyl-CoA
Thus the propionyl residue from an odd-chain fatty acid is the only part of a fatty acid that is
glucogenic.
4.2.4. Oxidation of unsaturted fatty acids
Oxidation of unsaturated fatty acids occurs by a modified beta- oxidation pathway.
1. Initial reaction 

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The CoA ester of these acids are degraded by the enzymes normally responsible for β -
oxidation until either aΔ3-cis- acyl-CoA compound or Δ4-Cis-acyl-CoA compound is formed,
depending upon the position of the double bonds.

2. Reaction of Isomerase

The former compound is isomerized (Δ3 cis-Δ2 1 CoA isomerase) to the corresponding D2 -
trans-CoA stage of β - oxidation for subsequent hydration and oxidation.

3. One cycle of Beta Oxidation

Any Δ4 -cis-acy1-CoA either remaining, as in the case of linoleic acid, it is converted to Δ2 -
trans enoy1-CoA by an NADP dependent enzyme, Δ2 - trans - Δ3-cis dienoy1-CoA
reductase.

4. Action of Acyl-CoA dehydrogenase

Δ-cis (or trans) Δ2 enoy1-CoA isomerase will attack the trans double bond to

produce Δ2 - trans enoy1-CoA, an intermediate in beta oxidation.

This compound is further metobolised via β - oxidation

5.  Action of  2, 4-Dienoyl-CoA reductase

Action of Enoyl-CoA Isomerase

Enoyl Co A isomerase then coverts Δ3 isomer to Δ2 isomer

∆3-cis (or trans)   ∆2 - trans enoy1-CoA isomerase will attack the trans  double bond to

produce ∆2- trans enoy1-CoA, an intermediate in β- oxidation.
This compound is further metabolized via β-oxidation.

Chapter 3: Biosynthesis of lipids

The process of synthesis of fatty acid from acetyl CoA and triglycerides is called lipogenesis.
Other compounds like, phospholipids, cholesterol and ketone bodies are also synthesized.

(i) Synthesis of fatty acid

There are three types of fatty acid synthesis. (a) Elongation of existing short chain fatty
acid in the mitochondria (b) Microsomal system of chain elongation and (c) The cytoplasmic
synthesis of fatty acid from acetyl CoA.
a. Elongation of smaller fatty acid mitochondrial System
Mitochondria catalyses the incorporation of acetyl CoA into smaller chain fatty acids into longer
chain fatty acids under anaerobic conditions.
           The enzymes are mostly the same as those involved in β-oxidation except the α - β
unsaturated acyl CoA reductase which converts α-β unsaturated acyl CoA to a saturated
compound requiring NADPH+H+. Thiolase is not used in this pathway. Pyidoxal phosphate is
required for the enzyme condensing acetyl CoA with acyl CoA.
b. Microsomal System of elongation of smaller fatty acid

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This is the main pathway for the elongation of existing fatty acid molecules. Elongation of fatty
acid chains occurs in the endoplasmic reticulum. This pathway the (“microsomal system”)
converts fatty acyl-CoA to higher fatty acids using malonyl CoA as acetyl donor and
NADPH+H+as reducing agent catalyzed by the microsomal fatty acid elongase system of
enzymes.
The acyl groups that may act as a primer molecule include the saturated series from C 10-
C16 upward, as well as unsaturated C18fatty acids.
Elongation of stearyl-CoA in brain increases rapidly during myelination in order to provide C22
and C24 fatty acids that are needed for the synthesis of sphingolipids.
4.3.3. Synthesis of fatty acids in cytoplasm
 The main pathway for the synthesis of fatty acids from acetyl CoA occurs in the cytoplasm. This
system is present in many tissues, including liver, kidney, brain, lactating mammary gland and
adipose tissues. Its cofactor requirements include NADPH, ATP, Mn2+, biotin, and HCO3 (as a
source of CO2). Acetyl-CoA is the immediate substrate, and free palmitate is the end product.
The reactions are as follows
1.  Formation of malonyl-CoA.
The first reaction is the formation of malonyl-CoA by the carboxylation of acetyl-CoA in the
presence of ATP, biotin and acetyl-CoA carboxylase. Bicarbonate is the source of CO 2. The
reaction takes place in two steps:
(1) carboxylation of biotin (involving ATP) and
(2) transfer of the carboxy group to acetyl-CoA to form malonyl-CoA
Acetyl-CoA carboxylase.
Acetyl-CoA + ATP+ biotin + HCO3- → Malonyl-CoA + ADP + Pi
Fatty acid synthase
Two types of fatty acid synthase are found to be involved in the synthesis of fatty acids. In
bacteria, plants, and lower forms, the individual enzymes of the system are separate, and the
acyl carrier protein (ACP). However, in yeast, fish, higher animals and birds, the synthase
system is a multienzyme complex and ACP is part of this complex. ACP of both synthase
systems has 4’- phosphopantetheine and cysteine -SH group.
2. Reaction of acetyl CoA and malonylCoA with Enzyme
An acetyl-CoA molecule combines with the cysteine -SH group of ACP catalyzed by acetyl
transacylase. Then a malonyl-CoA combines with the adjacent -SH on the 4’-
phosphopantethenine of enzyme catalyzed by malony1 transacylase, to form acetyl (acyl)-
malonyl enzyme.
3. Action of 3-ketoacyl synthase
The acetyl group attacks the methylene group of the malonyl residue, catalyzed by 3-ketoacyl
synthase, and liberates CO2, forming 3-ketoacyl enzyme (acetoacetyl enzyme). This frees the
cysteine-SH group, hitherto occupied by the acetyl group. Decarboxylation allows the reaction to
go to completion.
4. Reduction of 3-ketoacyl group of Acyl ACP
The 3-ketoacyl-S-enzyme is reduced to form a 3-hydroxyl acyl-S-enzyme. NADPH and H + are
needed for the reaction.
5. Dehydration of 3-hydroxyl acyl-S-enzyme by hydratase
A water molecule is removed from the 3-hydroxyl acyl-S-enzyme to form an unsaturated acyl-S-
enzyme. This reaction is catalysed by hydratase.
6. Reduction of unsaturated acyl-S-enzyme by enoyl reductase
Unsaturated acyl-S-enzyme is reduced by enoyl reductase to form a saturated acyl-S-enzyme.
Repeat of the sequence of reactions
A new malonyl-CoA molecule combines with the -SH group of the enzyme. The sequence, of
the reactions (No 3-6) is repeated 6 more times, a new malonyl residue being incorporated
during each sequence, until a saturated 16 carbon acyl radical (palmityl) has been assembled.

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It is then liberated from the enzyme complex by the activity of a seventh enzyme in the complex,
thioesterase (deacylase).
The free palmitate must be activated to acy-CoA before it can proceed via any other metabolic
pathway. Its usual fate is esterification into triglycerides.
The equation for the overall synthesis of palmitate from acety-CoA and is shown below.
   Acetyl CoA                 Malonyl CoA                                                         Palmitate
CH3COSCoA + 7HOOC.CH2CO.S.CoA+ 14NADPH + 14H+ →CH3(CH2)14COOH +7CO2 +
6H2O+8CoA.SH + 14NADP+
Sources of acetyl CoA-Carbohydrates and glucose
The pathway involves glycolysis followed by the oxidative decarboxylation of pyruvate to
acetyl-CoA within the mitochondria and subsequent condensation with oxaloacetate to form
citrate, as part of the citric acid cycle.
This is followed by the translocation of citrate into the extra mitochondrial compartment via the
tricarboxylate transporter, where in the presence of CoA and ATP, it undergoes cleavage to
acetyl-CoA and oxaloacetate catalyzed by ATP-citrate lyase.
The acetyl-CoA is then available for malonyl-CoA formation and synthesis to palmitate.
4.3.4.Synthesis of triglyceridess
.  (ii) Synthesis of triglycerides
Triglycerides are synthesized from glycerol and fatty acids. Glycerol is a product of the
metabolism of adipose tissue, and only tissues that possess the activating enzyme, glycerol
kinase, can utilize it. This enzyme, which requires ATP, is found in liver and kidney, among
other tissues.
1. Synthesis of glycerol 3-phosphate
Glycerol kinase catalyzes the conversion of glycerol to glycerol 3-phosphate.
Glycerol kinase
Glycerol + ATP  → Glycerol 3-phosphate + ADP
This pathway connects with the stages of the glycolysis pathway triose phosphate, because
glycerol 3-phosphate by NAD + in the presence of glycerol-3-phosphate dehydrogenase.
Liver and kidney are able to convert glycerol to blood glucose by making use of the above
enzymes, some enzymes of glycolysis, and the specific enzymes of the gluconeogenic
pathway, fructose-1,6-bisphosphatase and glucose 6- phosphatase.
2. Activation of fatty acid to acyl CoA
 Fatty acids are activated to acyl-CoA by the enzyme acyl-CoA synthetase, utilizing ATP and
CoA.
Acyl-CoA synthetase
Fatty acids +ATP + CoA. → Acyl-CoA + ADP
3. Formation of phosphadidate
Two molecule of acyl-CoA combine with glycerol 3-phosphate to form phosphatidate (1,2-
diglyceride phosphate). This takes place in two stages via lysophosphatidate, catalyzed first by
glycerol-3-phosphate acyltransferase and then by 1-acylglycerol-3-phosphate acyltransferase.
Glycerol-3-phosphate acyltransferase

2 Acyl-CoA + Glycerol 3-phosphate →1-Acylglycerol-3-phosphate

1-Acylglycerol-3-phosphate acyltransferase
1-Aacylglycerol-3-phosphate→Phosphatidate (1,2- Diglyceride phosphate)
4. Formation of diglyceride
Phosphatidate (1, 2- diglyceride phosphate) is converted by phosphatidate phosphohydrolase to
a 1,2 diglyceride.
Phosphatidate phosphohydrolase 
Phosphatidate (1,2- diglyceride phosphate) → 1,2 diglyceride.

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5. Fomation of triglyceride
A further molecule of acyl-Co.A is esterified with the diglyceride to form a triglyceride, catalyzed
by diglyceride acyltransferase.
Diglyceride acyltransferase
Diglyceride + acyl-Co.A→ Triglyceride + CoA
In intestinal mucosa, a monoacyl-glycerol pathway exists whereby monoglyceride is converted
to 1,2- diglyceride as a result of the presence of monoglyceride acyltransferase.
Most of the activity of these enzymes resides in the endoplasmic reticulum of the cell, but some
is found in mitochondria, e.g, glycerol-3-phosphate acyltransferase
4.3.5. Synthesis of phospholipids

The phospholipids are synthesized either from phosphatidate, e.g. phosphatidylinositol, or from
1,2-diacyl glycerol, eg, phosphatidylcholine and phosphatidylethanolamine.

1. Formation of phosphatidate

It is formed by the reaction between 1,2 diacylglycerol and ATP in the presence of a kinase.

                                      Kinase                                      

1,2 diacylglycerol + ATP   → 1,2 diacylglycerolphosphate + ADP

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2. Fomation of phosphatidyl choline (lecithin)

Cholie+ ATP → Chloine monophosphate

Chloine monophosphate + CTP → CDP-Chloine + PPi
CDP-Chloine + 1,2 diacylglycerol → Phosphatidyl choline + CMP
3. Fomation of phosphatidyl ethanolamine (Cephalin)

Cephalin is formed from Ethanolamine and CTP

Ethanolamine+ ATP → Ethanolamine monophosphate

Ethanolamine monophosphate + CTP → CDP-Ethanolamine + PPi
CDP-Ethanolamine + 1,2 diacylglycerol → Phosphatidyl Ethanolamine + CMP 

4. Formation of Phosphatidyl inosital

In the synthesis of phosphatidyl inosital, cytidine triphosphate (CTP), a high energy phosphate
formed from ATP, reacts with phosphatidate to form a cytidine- diphosphate- diacylglycerol
(CDP-diacyglycerol). Finally, this compound reacts with inositol, catalyzed by the enzyme CD-
diacylglycerol inositol transferase, to form phosphophatidylinositol.

Cytidine triphosphate (CTP) + phosphatidate ↔ CDP-diacylglycerol

CD-diacylglycerol inositol transferase

CDP-diacylglycerol + Inositol → Phosphophatidylinositol

4.3.6. Synthesis of Cholesterol

Cholesterol is made from acetyl CoA. Three molecule of acetyl CoA combine to yield
mevalonate, which becomes phosphorylated to 3- phosphor-5- pyrophospho-mevalonate.

Thiolase

Acetyl CoA + acetyl CoA → Acetoacetyl CoA + CoA

Synthase

Acetoacetyl CoA + acetyl CoA + H2O→ 3 hydroxy 3methylglutaryl CoA + CoA + H+

Hydroxmethyl glutaryl CoA reductase

3 hydroxy 3methylglutaryl CoA + 2NADPH + 2H+ → mevalonate + CoA +2NADP+

On the loss of CO2 and phosphate, 3-isopentenyl pyrophosphate is formed. Stepwise assembly
of six molecules of the latter ultimately yields the linear hydrocarbon squalene. It then cyclizes to
form lanosterol, which is then converted to cholesterol. Cholesterol synthesis is inhibited by
dietary cholesterol.

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Formation of ketone bodies
Excess acetyl-CoA released on oxidation of fatty acids and not required by the liver is converted
into the ketone bodies, acetoactate and D-β-hydroxy butyrate, which are circulated via the blood to
peripheral tissues, to be used as fuel for the citric acid cycle. The ketone bodies may be regarded as a
transport form of acetyl groups. They can supply significant fraction of the energy to some peripheral
tissues, up to one-third in the case of the heart
        Ketone bodies are formed in the liver but utilized in the extrahepatic tissue. Enzymes responsible are
associated with mitochondria. Two units of acetyl CoA formed in β-oxidation of fatty acids condence to
form acetoacetyl CoA by a reversal of thiolase reaction. Two pathways exist for the formation of
acetoacetone.

1. Action of acetoacetyl CoA deacylase: There is simple deacylation catalysed by the enzyme

acetoacetyl CoA deacylase.

2. Action of HMG-CoA: The acetoacetyl CoA condense with another molecule of acetyl CoA to form β-
hydroxy β-methyl glutaryl CoA by HMG CoA synthase.

3. Action of HMG CoA lyase: β-hydroxy β-methyl glutaryl CoA is splitted into acetoacetic acid and
acetyl CoA by HMG CoA lyase present in mitochondria. From acetoacetic acid, acetone and βhydroxyl
butyrate are formed.

These compounds are carried from liver to extrahepatic tissue mainly kidney and muscle where
they are oxidized for energy production afte conversion to acetyl Co A. Ketone body formation occurs if
the concentration of circulating free fatty acids increases by the lipolysis of triglycerides in adipose tissue.
Unit 5: Food Carbohydrates

Chapter 1: Naturally Occurring Carbohydrates in Food

5.1.1.Introduction
Nature commonly utilizes carbohydrates as source of energy, structure-forming material, water-
maintaining hydrocolloids and even sex attractants. Plants use carbon dioxide from the air,
water from soil and energy from sun to produce carbohydrates and oxygen through a process
called photosynthesis.
Carbohydrates are organic compounds containing carbon, hydrogen and oxygen with the
general formula Cn(H2O)n. They may be simple or complex molecules. Important food
carbohydrates include simple sugars, dextrins, starches,celluloses, hemicelluloses, pectin, and
gums. They are an important source of energy or fiber in the diet and they are important
constituents of food because of their functional properties. They are used as sweeteners,
thickeners, stabilizers, gelling agents, and fat replacers.
The simplest carbohydrates are called monosaccharides or sugars and they have the general
formulae CnH2nOn.The most common ones contain six carbon atoms. Disaccharide contains two

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sugar units, trisaccharides contain three,oligosaccharides contain several units,
and polysaccharides are complex polymers containing as many as several thousand units
of monosaccharides linked by means of glycosidic bonds.
5.1.2 Monosaccharides

Monosaccharides are simple carbohydrates containing between three and eight carbon atoms,
but only those with five and six carbon atoms are common. Most important ones are glucose,
galactose and fructose with general formula C6H12O6.

Glucose is the most abundant simple carbohydrate unit in nature. Glucose plays a key role in
both foods and the body. Glucose imparts a mildly sweet flavor to food. It is usually joined to
other sugars to form disaccharides, starch, or dietary fiber. Glucose makes up at least one of
the two sugar molecules in every disaccharide. In the body glucose supplies energy to cells.
The body regulates blood sugar levels to assure constant fuel source for vital functions of the
body. Glucose is the only fuel source for brain.

Fructose is the sweetest of all sugars. it occurs in fruits and vegetables. Honey contains
fructose and glucose in equal proportion and it is responsible for the sweet taste of honey. High-
fructose corn syrup is added to sweeten many foods like soft drinks, fruit beverages, desserts,
candies, jellies and jams.

Galactose rarely occurs as a monosaccharide in food. It is bonded to glucose to form lactose

"the milk sugar".

Monosaccharide and their natural derivatives

Pentoses

L-Arabinose Plant gums, hemicellulose, Alcoholic fermentation, furan-2, aldehyde

saponins, protopectin production

Accompanies L-arabinose

Reduction to xylitol; sucrose substitute;

D-xylose alcoholic fermentation; production of furan-
2 aldehyde
Hexoses

D-Glucose Plants and animals, honey, Alcoholic fermentation;

inverted sugar, saponins
D-Fructose
Fruit, traces in plant honey,
D-Galactose  sweetener ; energy pharmacopeial
material; nutrient; food preservative

Constituent of milk, dairy

L-Fucose products
Preparation of dairy products
(Milk sugar)

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algae, plant mucus and gums

Oligo-and polysaccharides,
plant mucus and gums,
D-Mannose saponins, glycosides

Algae, plant mucus. Oranges

L-Rhamnose Plant mucus and gums,

pectins saponins, glycosides

Preparation of mannitol, which is used as

an alternative sweetener in food products
Hexuloses

D-Fructose Fruits, honey, inverted sugar, Noncavity-causing sweetener; sweetener

maple syrup for diabetics; food humidifier and
preservative
Chitin, Chitosan
D-Glucosylamine Pharmaceutical aid; antiarthritic drugs; ion
exchanger

Rowan berries
L-Sorbose
Synthesis of ascorbic acid
5.1.3. Disaccharides
Disaccharide contains of two monosaccharides linked together by glycosidic bond.
(A gycosidic bond is a type of covalent bond that joins a carbohydrate (sugar) molecule to
another group, which may or may not be another carbohydrate).

Sucrose: Sucrose or the table sugar is the most common disaccharide. It contains glucose and
fructose linked by α-1, 2-glycosidic bond. Sucrose provide natural sweetness of honey, maple
syrup, fruits, and vegetables. It is manufactured from juices of sugar cane and beet root. White
sugar and powdered sugar are highly purified nearly100 percent sucrose. It is the one that is
listed as sugar in the label of food products.

Lactose: Lactose, known as milk sugar, contains one glucose and one galactose molecules.
Lactose gives milk and other dairy products a slightly sweet taste. Human milk has a higher
concentration of lactose ( approx. 7g/100ml) than cow's milk (4.5g/100ml).

Maltose: Maltose contains two glucose units linked by α1-4 glycosidic bond. It is the building
block of starch. Maltose is formed by the breaking of starch. Human digestive enzymes in the
mouth and intestine break down starch into maltose. The sweet taste formed in the mouth when
raw rice is chewed is due to the breaking of starch to maltose. Starch breaks down to maltose in
the germinating seeds.

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5.1.4 Oligosaccharides
Oligosaccharide contains 3-10 monosaccharide units linked together by glycosidic bonds.
Common ones include raffinose and stachyose.
Raffinose is a trisaccharide composed of three monosaccharide units.-one galactose,
one glucose and one fructose.
Stachyose is a tetrasaccharide, composed of one molecule each of glucose and fructose and
two molecules of galactose.
They are present in cabbage, beans, broccoli, asparagus, other vegetables and grains. They
can be hydrolysed to D galactose units and sucrose by α-glycosidase, an enzyme not found in
the human digestive system but is readily broken by intestinal bacteria and is responsible for the
familiar gaseous effect of such foods.
5.1.5. Polysaccharides
A polysaccharide contains more than 10 monosaccharide units linked together by glycosidic
bond. The most important polysaccharides are starches, cellulose, pectins and gums. All are
complex polymers with different properties, which depend on the mono saccharides that make
up the structure the linkage by which they are linked and the degree of branching of the
molecules.
Occurrence
All organism cells, including those of animals, contain components of carbohydrates in their
membranes. Frequently, carbohydrates exist in naturally derivatives forms, including aminated
forms, as in chitin and chitosan; esterified; alkylated as in glycosides; oxidized; reduced; or
linked to proteins, lipids, and other structures such as glycoproteins.
i. Starch: It is the important energy giving polysaccharide present in food. It is present in (1)
cereals such as rice, wheat, corn, millet and barley, (2) legumes such as peas, beans, and
lentils and (3) tubers such as potatoes, yam, and cassava. Starch imparts a moist gelatinous
texture to food. The starch in flour absorbs moisture and thickens gravy. Other types of
polysaccharides present in food are given in the following table.
ii. Cellulose: It is a polysaccharide with the formula (C 6H10O5)n consisting of a linear chain of
more than 100 to 1000 D-glucose units linked by β(1-4) glycosidic bond. Cellulose is the
structural component of the primary cell wall of plants and different types of algae. It is secreted
as biofilm by some bacteria. It is the most important organic compound present in earth. It forms
about 30-355 of all plants. Higher values are found in wood (40-45%) and cotton
(90%).  Humans cannot digest cellulose but it acts as a hydrophilic bulking agent for feces.It
forms insoluble dietary fiber.
iii. Pectin: Pectin is a structural polymsaccharide present in the primary cell eallsof terrestrial
plants like. It is present in the peels of apples, citrus and sugar beet.  It extracted and used as
gelling, thickening and stabilizing agent in confectionary (sweets), dairy (milk drinks) and fruit
preparations such as jams and jellies. It forms a natural part of diet but does not contribute
significantly to nutrition. It is a source of dietary fiber.
iv. Natural Gums: Natural gums are polysaccharides of natural origin, capable of increasing the
viscosity of solutions even at very low concentrations. They are found in the sead coatings and
in the woody elements. Natural gums obtained from plant sources are gum arabic, gum ghatti,
gum tragacanth etc. while that obtained from marine sources are agar, alginic acid, sodium
alginates and carageenan.They are used as thickening agents, gelling agent, emulsifying
agents and stabilizers.
Other types of polysaccharides present in food are presented in Table 5.1.5.1.
Table 5.1.5.1.Other types of polysaccharides present in food
Types of Source Uses
polysaccharides

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Agar Red algae (Rhodophyceae Microbiological nutrient; gelforming
Alginates sp.) agent; Emulsifier; bread staling
Carrageenans Brown retardant; meat texturizer; meat
Dextran algae(Pheophyceae) substitute 
Inulin Red seaweed Thickener; gel-forming agent; food
(Rhodophyceae) and beer foam stabilizer
  Frozen sugar beet (continued)
Endive, Jerusalem Gel-forming agent; stabilizer; protein
  artichoke fiber texturizer; milk fat
anticoagulant; milk clarifying agent
    Blood substitute
Prebiotic
   
 
   
 
   
 
   
 
 
 
 

 
5.1.4.1. Occurrence

All organism cells, including those of animals, contain components of carbohydrates in their
membranes. Frequently, carbohydrates exist in naturally derivatives forms, including aminated
forms, as in chitin and chitosan; esterified; alkylated as in glycosides; oxidized; reduced; or
linked to proteins, lipids, and other structures such as glycoproteins.

Starch: Plants store energy as starch for use during growth and reproduction. Good sources of
starch include (1) grains such as rice , wheat, corn, millet and barley, (2)legumes such as peas,
beans, and lentils and (3) tubers such as potatoes, yam, and cassava. Starch imparts a moist
gelatinous texture to food. The starch in flour absorbs moisture and thickens gravy.

Chapter 2:  Role of Fiber in Food

5.2.1.Introduction

1.                   Dietary fiber consists of non-starch polysaccharides such ascellulose, arabinoxylons,

inulins, pectin, gum, mucilage, cellulose, chitins, hemicellulose, beta glucans and lignin.
Dietary fiber comes from the portion of plants that is not digested by enzymes in the
intestinal tract. Part of it, however, may be metabolized by bacteria in the lower gut.

There are two main types

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i.                    i. Water-soluble fibers: Pectin and gum are water-soluble fibers found inside plant cells.
Beans, oat bran, fruits and vegetables contain soluble fiber. They slow the passage of food
through the intestines but do nothing to increase fecal bulk.
ii. Water insoluble fiber: These include cellulose, hemicelluloses and lignin. They are
metabolically inert and absorb water as they move through the digestive system.  Such fibers
increase fecal bulk and speed up the passage of food through the digestive tract. Wheat bran
and whole grains contain the most insoluble fiber, but skins of vegetables and beans also are
good sources.
5.2.2.Benefits of Fiber

i) Prevent constipation: Insoluble fiber binds water, making stools softer and bulkier.

Therefore, fiber especially that found in whole grain products is helpful in the treatment and
prevention of constipation, hemorrhoids and diverticulosis. Diverticula are pouches of the
intestinal wall that can become inflamed and painful. In the past, a low-fiber diet was prescribed
for this condition. A high-fiber diet gives better results once the inflammation has subsided.

ii) Lower Carbohydrate digerstion and absorption

Attract water and form viscous gel during digestion, slowing and emptying of the stomach and
intestinal tract and thus protect carbohydrates from digestive enzymes, and delaying absorption
of glucose.

iii) Lower cholesterol levels: Low blood cholesterol levels (below 200 mg/dl.) have been
associated with a reduced risk of coronary heart disease. The body eliminates cholesterol
through the excretion of bile acids. Water-soluble fiber binds bile acids, and hence a high-fiber
diet may result in an increased excretion of cholesterol. Some types of fiber appear to have a
greater effect than others. The fiber found in rolled oats is more effective in lowering blood
cholesterol levels than the fiber found in wheat. Pectin has a similar effect in that it, too, can
lower the amount of cholesterol in the blood.

iv) Reduce the risk of some cancers: Dietary fiber may help reduce the risk of some cancers,
especially colon cancer. This idea is based on information that insoluble fiber increases the rate
at which wastes are removed from the body. This means the body may have less exposure to
toxic substances produced during digestion. A diet high in animal fat and protein also may play
a role in the development of colon cancer.

v) Reducing weight: High-fiber diets may be useful for people who wish to lose weight. Fiber
itself has no calories, yet provides a "full" feeling because of its water-absorbing ability. For
example, an apple is more filling than a half cup of apple juice that contains about the same
calories. Foods high in fiber often require more chewing, so a person is unable to eat a large
number of calories in a short amount of time.

5.2.3. Sources of Fiber

          Dietary fiber is found only in plant foods: fruits, vegetables, nuts and grains. Meat, milk
and eggs do not contain fiber. The form of food may or may not affect its fiber content. Canned
and frozen fruits and vegetables contain just as much fiber as raw ones. Other types of
processing, though, may reduce fiber content. Drying and crushing, for example, destroy the
water-holding qualities of fiber.

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The removal of seeds, peels or hulls also reduces fiber content. Whole tomatoes have
more fiber than peeled tomatoes, which have more than tomato juice. Likewise, whole wheat
bread contains more fiber than white bread.

Table 5.2.1 Different dietary fractions in Indian food

Food Group   Food Item Fibre Content  g /100g edible Soluble (%
portion    TDF*)
Crude TDF* Soluble
Fibre           
Cereals Rice 0.2 4.11 0.92 22.4
  Wheat 0.3 12.48 2.84 22.7    
  Bajra 1.2 11.33 2.19 19.3
  Maize 2.7 11.54 1.65 14.2
  Jowar 1.6   9.67 1.64 17.0
  Ragi 3.6 11.85 0.89 7.50
Pulses, dhals Lentil 0.7 10.31 2.04 19.8
  Chick pea 1.2 15.30 2.56 16.7
  Pigeon Pea 0.9 9.14 2.33 25.4
  Green gram 0.8 8.23 1.69 20.5
Vegetables Cluster beans 3.2 5.7 1.6 28.0
  Brinjal 1.3 6.3 1.7 27.0
  Cabbage   2.8 0.8 28.6
  Cauliflower 1.2 3.7 1.1 30.3
  Bhendi 1.2 3.6 1.0 26.9
Roots and Potato 0.4 1.7 0. 6 33.5
Tubers
  Carrot 1.2 4.4 1.4 30.6
  Onion 0.6 2.5 0.8 32.0
Green leafy Spinach 0.6 2.5 0.7 28.0
Vegetables
  Amaranth 1.0 4.0 0.9 22.5
Fruits Orange 0.3 1.1 0.5 45.5
  Banana 0.4 1.8 0.7 38.9
  Apple 1.0 3.2 0.9 28.1
  Tomato 0.8 1.7 0.5 28.5    
5.2.4. Adverse effect

Although fiber is important, it is just one part of a balanced diet. Too much fiber inhibit
the absorption of calcium, iron, zinc, copper and magnesium that is absorbed from foods.
Deficiencies of these nutrients could result if the amount of fiber in the diet is excessive,
especially in young children.

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Fiber supplements are sold in a variety of forms from bran tablets to purified cellulose.
Many laxatives sold as stool softeners actually are fiber supplements. Fiber's role in the diet is
still being investigated. It appears that the various types of fiber have different roles in the body.
For these reasons fiber supplements should be avoided. Eating a variety of fiber-rich foods is
the best way to receive the maximum benefits from each type of fiber present in foods, and
obtain necessary nutrients.

Chapter  3. Browning Reactions

5.3.1. Introduction

Browning is a common colour change seen in food during pre-preparation, processing or

storage of food. It occurs in varying degrees in some foods. The colour produced range from
cream or pale yellow to dark brown or black. 

Browning reactions observed in food may be classified as enzymatic browning or non-enzymatic

browning.

5.3.2.Enzymatic Browning

Fruits such as apples, pears, peaches, apricots, and bananas, and vegetables such as potatoes
quickly turn brown when their tissue is exposed to oxygen. Such oxygen exposure occurs when
the food is sliced or bitten into or when it has sustained bruises, cuts or other injury to the peel.

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This “browning reaction” is related to the work of an enzyme called phenolase (or
polyphenoloxidase), a conjugated enzyme in which copper is present.

Phenolase is classified as an oxidoreductase. The substrates for phenolase are phenolic

compounds present in the tissues of the fruits and vegetables. Phenolase hydroxylates
monophenols to o-diphenol and oxidizes o-diphenols to o-quinones. The o-quinones then enter
into a number of other reactions, which produce the “undesirable” brown discolorations.
Quinone formation is enzyme and oxygen-dependent. Once the quinones have formed, the
subsequent reactions occur spontaneously and no longer depend on the presence of phenolase
or oxygen.

Prevention

Enzymatic browning can be prevented or slowed in several ways. Immersing the “injured” food
(for example, apple slices) in cold water slows the browning process. The optimum temperature
for enzymes to act is 43ºC(109ºF).The lower temperature decreases enzyme activity, and the
water limits the enzyme’s access to oxygen. Refrigeration slows enzyme activity even more,
and boiling temperatures destroy (denature) the enzyme. A long-used method for preventing
browning involves lowering of pH to 2.5-2.7 by the addition of acids such as ascorbic acid, malic
or citric acid. Phenolase works very slowly in the acidic environment created by the added
acids. In addition, the vitamin C (ascorbic acid) present in lemon juice functions as an
antioxidant. It is more easily oxidized than the phenolic-derived compounds, and its oxidation
products are colorless.

5.3.3. Non enzymatic reaction or Maillard reaction

The non enzymatic browning or Maillard reaction is a chemical reaction between an amino acid
and a reducing sugar, usually requiring heat. When aldoses and ketoses are heated with

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amines, a variety of reactions ensue, producing numerous compounds some of which
are flavours, aromas and dark coloured polymeric material. They may be produced slowly
during storage and much more rapidly at the high temperature encountered during frying
roasting or backing.
The reducing sugar reacts with the amine to form a Schiff base (an imines) which may
cyclate to form glucosamine. In the case of glucose the Schiff base undergo a reaction called
Amadori rearrangement to give 1-amino-1-deoxy-D-fructose or Amadori compound. The
Amadori compounds are early intermediates in the browning reaction sequence. Amadori
compounds undergo transformation via different pathways starting with four different
intermediates formed from them. The result is a complex mixture of intermediates and
products. 
The Maillard reaction occurs in three main steps:

1. Initial step- formation N glycoside: The carbonyl group of the sugar reacts with the amino
group of the amino acid, producing N-substituted glycosylamine and water. 

2. After formation of N glycoside the immonium ion is formed and then isomerizes, this reaction
is called Amadori rearrangement and forms a compound called ketosamine:

3.The ketosamine products then either dehydrates into reductones and dehydro reductones,
which are caramel, or products -short chain hydrolytic fission products such as diacetyl, acetol
or pyruvaldehyde which then undergo the Strecker degradation and produce short-chain
hydrolytic fission products and brown nitrogenous polymers and melanoidins.

Important intermediates are formed by rearrangements and eliminations are 1-, 3- and 4-
deoxydicarbonyl compounds called 1-, 3-, and 4-deoxyosones. hey finally form 5- hydroxy
methyl furfural In the process, hundreds of different flavor compounds are created. These
compounds in turn break down to form yet more new flavor compounds, and so on. Each type

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of food has a very distinctive set of flavor compounds that are formed during the Maillard
reaction. It is these same compounds have been used over the years to create artificial flavors.

5.3.4. Food products with Maillard reactions

The Maillard reaction is responsible for many colors and flavors in foods such as bread, biscuit,
malted barley as in malt whiskey or beer, roasted meat, dried or condensed milk, roasted coffee
etc 6-Acetyl-2,3,4,5-tetrahydropyridine i s responsible for the biscuit or cracker-like flavor
present in baked goods like bread and popcorn. The structurally related compound 2-acetyl-1
pyrrpoline has a similar smell and occurs also naturally without heating and gives varieties of
cooked rice their typical smell.

Maillard reaction may result in a reduction in nutritional properties and the formation of
potentially toxic and mutagenic compounds. In a food system, the reactants are mostly amino
acids (free forms or peptide-bound) and reducing sugars. Since up to 50% of the food groups
have been processed before consumption, some of the amino acids and reducing sugars is lost
during processing. Maillard reactions affect protein bioavailability by derivatizing protein-bound,
dietary limiting amino acids such as lysine, arginine, and histidine. Maillard reaction products
also exhibit antinutritive effects by mechanism involving complex formation with micronutrients,
destruction of vitamins, and by acting as inhibitors of digestive enzymes.

High temperature, low moisture levels and alkaline conditions promote the Maillard reaction.
The rate of Maillard reactions increases as the water activity increases, reaching a maximum at
water activities in the range of 0.6 to 0.7. However, as the Maillard reaction produces water,
further increases in water activity may inhibit Maillard reactions. Pentose sugars react more than
hexoses, which react more than disaccharide. Different amino acids produce different amounts
of browning.

5.3.5. Browning reactions which occur in meat

The browning reactions which occur when meat is roasted or seared have often been referred
to as Maillard reaction browning. However, lean meat contains very few, if any, reducing sugars.
Furthermore, red meat undergoes more extensive browning than does white meat. The
browning reactions in lean meat are most likely due to the breakdown of the tetrapyrrole rings of
the muscle protein, myoglobin. Thus, the browning of meat is technically not a Maillard browning
since it does not involve the reaction with a reducing sugar.

5.3.6. Caramelization

Caramelization is a browning reaction formed by heating carbohydrates like sucrose or reducing

sugars. Reactions are facilitated by small quantity of acids, base and certain salts.

Caramelization is an entirely different process from Maillard browning, though the results of the
two processes are sometimes similar to the naked eye (and taste buds). The final product
caramel contains a complex mixture of polymeric compound, formed from unsaturated cyclic
compounds. Flavour and aroma compounds are also formed. Heating causes the dehydration of
sugar molecule with introduction of double bonds or formation of anhydro rings. Intermediates
such as 3-deoxy osones and furans are formed. The unsaturated rings may condense to form
useful, conjugated double-bond containing, brown coloured polymers. Catalysts increase the

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reaction rate and are used to direct the reaction to specify types of caramel colour, solubility and
acidities.

To make caramel a carbohydrate is heated alone or in the presence of acid, a base or salt. The
carbohydrates most often used are sucrose, but fructose, glucose, invert sugar, malt syrups and
molasses may also be used. Acid used are food grade sulfuric, sulfurous, phosphoric, acetic
and citric acids. Bases that may be used are ammonium, sodium, potassium and calcium
hydroxides. Salts that may be used are ammonium, sodium, potassium carbonates,
bicarbonates, phosphates, sulphates or bisulphates.

There are four classes of caramel.

Class I caramel :Prepared by heating a plain carbohydrate

Class II caramel :Prepared by heating a carbohydrate in the presence of a sulphite

Class III caramel :Prepared by heating a carbohydrate in the presence of a source of

ammonium ion.

Class IV caramel :Prepared by heating a carbohydrate in the presence of a both sulphite and

ammonium ions

Caramelization may sometimes cause browning in the same foods in which the Maillard
reaction occurs, but the two processes are distinct. They both are promoted by heating, but the
Maillard reaction involves amino acids, as discussed above, while caramelization is simply the
pyrolysis of certain sugars.

Unit 6: Metabolism of Carbohydrates

Chapter 1: Digestion and absorption of carbohydrates

6.1.1. Introduction
The most abundant carbohydrates ingested by human beings are the polysaccharides, starch
and cellulose, furnished by plant foods and glycogen, provided by foods of animal origin.
Starch and glycogen are completely hydrolyzed by enzyme action in the gastrointestinal tract to
yield free D-glucose.  
6.1.2.Digestion
This process begins in the mouth during chewing, through the action of amylase secreted by
the salivary glands. Salivary amylase hydrolyzes many of the α(14) glycosidic linkages of
starch and glycogen to yield a mixture of maltose, glucose and oligosaccharides. The digestion
of digestible polysaccharides to yield D-glucose is continued and completed in the small
intestine, largely by the action of pancreatic amylase, made by the pancreas and secreted via
the pancreatic duct into the upper protein of the small intestine. This segment of the small
intestine in which most of its digestive activity occurs, is called the duodenum.
         Disaccharides are hydrolyzed by enzymes located in the outer border of the epithelial
cells lining the small intestine. Sucrose is hydrolyzed to D-glucose and D-fructose by sucrose,
also called invertase: lactose is hydrolyzed to D-glucose and D-galactose by lactose β-
galactosidase is hydrolyzed by maltose, yielding two molecules of D-glucose. The larva stores

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the glucose as glycogen and releases glucose as and when needed to maintain blood glucose
level.
         Cellulose cannot be enzymatically digested and used by most mammals for lack of
enzymes capable of hydrolyzing the β (14) linkages between successive D-glucose residues
of cellulose. Nevertheless undigested cellulose residues of plant foods provide bulk or fiber
(also called “roughage”) in the diet and are desirable for proper motility of the intestine.
Cellulose can be digested by ruminant animals, but only indirectly. The rumen bacteria
hydrolyze cellulose to yield D-glucose, which they ferment to yield lactate, acetate, and
propionate, absorbed into the blood. Lactate and propionate are converted by the liver into
blood sugar in ruminants.
6.1.3.Absorption
   In the epithelial cells lining the small intestine, D-fructose, D-galactose and D-mannose are
converted into D-glucose. The resulting mixture of simple hexoses is absorbed into the epithelial
cells lining the small intestine and brought via the blood to the liver.
Fig.6.1.1 Digestion and absorption of carbohydrates
6.1.4. Metabolisms involving glucose
1.      Metabolisms involving glucose
The absorbed free glucose is phosphorylated to glucose 6 phosphate by hexokinase
using ATP. The fructose, galactose and mannose absorbed are also converted into glucose
6 phosphate. This compound is metabolized by five major metabolic path ways.
i.        Conversion into blood glucose: Glucose 6-phosphate is dephosphorylated by
glucose 6-phosphate to yield free-D-glucose, which passes into the systemic blood to be
transported to other tissues.
Glucose 6 phosphatase
Glucose 6-phosphate   → Glucose  + Pi
ii.      Conversion into glycogen: Glucose 6-phosphate not immediately needed to form
blood glucose is converted into liver glycogen by the sequential action of
phosphoglucomutase and glycogen synthase.
iii.    Conversion into fatty acids and cholesterol: Excess glucose 6-phosphate not used
to make blood glucose or liver glycogen is degraded via glycolysis and pyruvate
dehydrogenase into acetyl-CoA, which is converted into malonyl CoA, and thence into
fatty acids. These are used to form triglycerides and phospholipids, which are in part
exported to other tissues by plasma lipoproteins. Some acetyl-CoA is also used by the
liver to make cholesterol.
iv.    Oxidative degradation to CO2: Acetyl-CoA, yielded from glucose 6-phosphate via
glycolysis and decarboxylation of pyruvate, may be oxidized via the citric acid cycle. The
ensuing electron transport and oxidative phosphorylation yield energy in the form of
ATP. Normally, however, fatty acids are the major oxidative fuel for the citric acid cycle
in the liver.
v.      Degradation via the Pentose Phosphate Pathway: Glucose 6-phosphate is the
substrate for the pentose phosphate pathway, yielding (1) reducing power in the form of
NADPHI, needed in the reducing steps in the biosynthesis of fatty acids and cholesterol
and (2) D-ribose 5-phosphate, a precursor in nucleotide biosynthesis. Through the action
of various regulatory enzymes and through hormonal regulation, the liver directs the flow
of glucose residues into these different pathways according to the prevailing supply and
demand economy of the organism.

Chapter 2: Glycogenesis and Glycogenolysis

6.2.1.Introduction

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The monosaccharides formed from carbohydrate digestion the liver. They are used for energy
or stored, depending upon the body’s need. Galactose and fructose are converted into glucose
in the liver.
Metabolic pathways of glucose are as follows
1. Coversion into Glycogen and degradation 
 i) Glycogenesis in which the excess glucose is converted into glycogen as a cellular 
storage compound.
 ii) Glycogenolysis involves the breakdown of glycogen into glucose, which provides a glucose
supply for glucose-dependent tissues.
2. Oxidative degradation to CO2
i) Glycolysis is the pathway in which the oxidation metabolism of glucose molecules forms ATP
and pyruvate.
ii) Citric acid cycle in which pyruvate from glycolysis enters the citric acid cycle or Krebs cycle
in aerobic organism or to lactate or alcohol in anaerobic condition and in  anaerobic organisms.
3. Gluconeogenesis which is involved in the synthesis of glucose molecules from simple other
compounds such as fatty acid or amino acid.
6.2.2.Coversion into Glycogen and degradation

      The absorbed free glucose is phosphorylated to glucose 6   phosphate by hexokinase using ATP.
The fructose, galactose  and mannose absorbed are also converted into glucose 6 phosphate.
This compound is then metabolized by major metabolic path ways. Glucose 6-phosphate is
dephosphorylated by glucose 6-phosphate to yield free-D-glucose, which passes into the systemic
blood to be transported to other tissues.

                          i). Glycogenesis

Glycogenesis is the process of biosynthesis of glycogen from glucose and occurs mainly in
muscle and liver. The following steps are involved

6.2.2.1. Formation of glucose 6-phosphate Glycogen

i). Glycogenesis

Glycogenesis is the process of biosynthesis of glycogen from glucose and occurs mainly in
muscle and liver. The following steps are involved

Formation of glucose 6-phosphate

Glucose is phosphorylated to glucose 6-phosphate, a reaction that is common to the first

reaction in the pathway of glycolysis from glucose. This reaction is catalyzed by hexokinase in
muscle and glucokinase in liver.

Hexokinase

Glucose + ATP → Glucose 6-phosphate + ADP

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6.2.2.2. Conversion of Glucose 6- phosphate to glucose 1- phosphate

Glucose 6-phosphate is converted to glucose 1-phosphate in a reaction catalyzed by the

enzyme phosphoglucomutase.

                                Phosphoglucomutase

           Glucose 6- phosphate ↔ glucose 1- phosphate

6.2.2.3. Formation of UDPG

Next, glucose 1- phosphate reacts with uridine triphosphate (UTP) to form the active nucleotide
diphosphate glucose (UDPGIc).

The reaction between glucose 1- phosphate and uridine triphoaphate is catalyzed by the
enzyme UDPGlc pyrophosphorylase.

                           UDPGlc pyrophosphorylase

          UTP + Glucose 1- phosphate ↔ UDPGlc + PPi

The subsequent hydrolysis of inorganic pyrophosphatase pulls the reaction to the right of the
equation.

6.2.2.4. Action of glycogen synthase

By the action of the enzyme glycogen synthase, the C1 of the activated glucose of UDPGlc
forms a glycosidic bond with the C4 of a terminal glucose residue of glycogen, liberating uridine
diphosphate (UDP).

Glycogen synthase

UDPGlc + Glycogen primer( n ) → Glycogen(n+1)

A preexisting glycogen molecule, or “glycogen primer”, must be present to initiate this reaction.
Further glucose residues are attached in the 1-4 position to make a short chain that is acted
upon by glycogen synthase.

The addition of a glucose residue to a preexisting glycogen chain, or “primer,” occurs at the
nonreducing, outer end of the molecule so that the “branches” of the glycogen “tree” become
elongated as successive α1-4 linkages are formed. 

6.2.2.5. Action of Branching enzyme

When the chain has been lengthened to a minimum of 11 glucose residues, a second enzyme,
the branching enzyme (amylo[α 1-4 ] [α 1-6] -transglucosidase), transfers a part of the α 1-4
chain (minimum length 6 glucose residues) to a neighboring chain to form a α 1-6 linkage, thus
establishing a branch point in the molecule.

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The branches grow by further additions of a 1-4 glucosyl units and further branching speeding
up glycogenesis.

6.2.3.Glycogenolysis

Glycogenolysis is the breakdown of glycogen into glucose. It is not the reverse of glycogenesis
but is a separate pathway. Degradation of glycogen involves the follwing steps.

6.2.3.1 Step 1. Action of phosphorylase

First step is catalyzed by phosphorylase. This enzyme is specific for the phosphorlytic
breaking (phosphorolysis; of hydrolysis) or the 1-4 linkages of glycogen to yield glucose 1-
phosphate.

                                     Phosphorylase

            Glycogen(C 6 )n + P i ↔ Glycogen (C 6) n-1 + Glucose 1- phosphate

The terminal glucosyl residues from the outermost chains of the glycogen molecule are removed
sequentially until approximately four glucose residues remain on either side of a 1-6 branch.
6.2.3.2. Step 2. Action of glucan transferase

Another enzyme ( α -[1-4 ] α -[1-4 ] glucan transferase ) transfers a trisaccharide unit from one
branch to the other, exposing the 1-6 branch point. The 1-6 linked glucose is hydrolysed by
amylo 1-6 glucosidase. Then the action ofphosphorylase can proceed. The combined action of
phosphorylase and these other enzymes leads to the complete breakdown of glycogen.

THe glucose 6-phophate can be formed from glucose 1- phosphate by the action of
phosphoglucomutase. In liver and kidney, glucose-6- phosphatase, coverts glucose 6-
phosphate, to glucose enabling glucose to diffuse from the cell into the blood. This is the final
step in hepatic glycogenolysis, which is reflected by an increase in the blood glucose.

6.2.3.3. Regulatory Action of cyclic AMP

Regulation of glycogenesis and glycogenolysis by hormones
1. Epinephrine, norepinephrine glucagons:
Cyclic AMP (cAMP) integrates the regulation of glycogenolysis and glycogenesis. The principal
enzymes controlling glycogen metabolism glycogen phosphorylase and glycogen
synthase are regulated by a complex series of reactions.
cAMP is the intracellular intermediate compound or second messenger through which
many hormones act. It if formed from ATP by an enzyme, adenylyl cyclase, occurring in the
inner surface of cell membranes. Adenylyl cyclase in activated by hormones such
as epinephrine and norepinephrine, acting through β - adrenergic receptors on the cell
membrane and additionally in liver by glucagons, acting through an independent glucagon
receptor.
2. Insulin cAMP is destroyed by a phosphodiesterase, and it is the activity of this enzyme that
maintains the normally low level of cAMP. Insulin increases its activity in liver, thereby lowering
the concentration of cAMP.

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           Phosphorylase differs between liver and muscle. In liver the enzyme exists in both an
active and an inactive form. Active phosphorylase (phosphorylase a) has one of its serine
hydroxy1 groups phosphorylated in an ester linkage. By the action of a specific
phosphatase, protein phosphatase-1, the enzyme is in activated tophosphorylase b in a
reaction that involves hydrolytic removal of the phosphate from the serine
residue.Phosphorylase kinase reactivates it by rephosphorylation with ATP. Pathway of
glycogenesis and glycogenolysis in the liver is presented in Fig.6.2.4.

Chapter 3: Oxidative degradation of glucose to CO2

6.3.1. Introduction
Oxidative degradation of glucose to CO2 and energy production takes place via to important
pathways. Glycolysisor Embden- Meyerhoff pathway is the major pathway for the utilization
of glucose for the production of energy and is found in the cytosol of all cells. Glycolysis can
function under aerobic and anaerobic conditions. Two molecules of pyruvate are produced.
Pyruvate is then converted to Acetyl CoA. In the second pathway, Citric acid cycle, acetyl CoA
is further oxidized to CO2 and H2O in mitochondria.
6.3.1.1. Glycolysis

Glucose is converted into two molecules of pyruvate, chemical energy in the form of ATP is
produced, and NADH- reduced coenzymes are produced. This metabolic pathway takes place
in almost all cells. All of the enzymes of the glycolysis pathway are found in the
extramitochondrial soluble fraction of the cell, the cytosol. They catalyze the reactions under
aerobic and anaerobic conditions.

The over all reactions of glycolytic reactions are presented in below.

Glycolysis

Anaerobic condition

Glucose → Pyruvate → Lactic acid

Aerobic condition

Pyruvate → CO2 + H2O

Glycolysis is a ten step process in which every step is enzyme catalyzed. Details of
individual steps with in the glycolytic pathway are now considered.

Step1: Phosphorylation of Glucose

Glucose enters into the glycolytic pathway by phosphorylation to glucose 6-

phosphate, accomplished by the enzyme hexokinase. However, in liver parenchyma cells and

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in pancreatic islet cells, this function is carried out by glucokinase. ATP is required as phosphate
donor, and it reacts as the Mg-ATP complex.

The terminal high-energy phosphate of ATP is utilized, and ADP is produced. The
reaction is accompanied by considerable loss of free energy as heat and therefore, under
physiologic conditions, may be regarded as irreversible.

Hexokinase is inhibited in an allosteric manner by the product, glucose 6- phosphate.

The function of glucokinase is to remove glucose from the blood following digestion
and absorption. It is specific for glucose.

Step 2: Converstion of Glucose 6 Phosphate to Fructose 6-Phosphate

Glucose 6 phosphate is converted to fructose 6-phosphate by phosphoglucose

isomerase, which involves an aldose-ketose isomerization.

Step 3: Phosphorlyation of Fructose 6-Phosphate to Fructose 1, 6-Bisphosphate

This reaction is catalyzed by the enzyme phosphofructokinase to produce fructose 1,

6-bisphosphate fromFructose 6 phosphate. The phosphofructokinase reaction is irreversible
under physiologic conditions.

Step 4: Cleavage of Fructose 1,6-Bisphosphate

Fructose 1, 6-bisphosphate is split by aldolase (fructose1, 6-bisphosphate aldolase)

into two triose phosphates, glyceraldehyde -3-phosphate and dihydroxyacetone phosphate.

Step 5: Inter conversion of triose phosphates

Glyceraldehyde-3-phosphate and dihydroxy acetone phosphate are interconverted

by the enzymephosphotriose isomerase.

At this stage 2 molecules glyceraldehyde 3-phosphate are formed. Dihydroxyacetone

phosphate is also formed from glycerol of fat, which is phosphorylated to glycerol 3 phosphate
and then to dihydroxy acetone phosphate. 

Step 6: Oxidation of Glyceraldehyde 3-Phosphate to 1, 3 Bisphosphoglycerate

Glyceraldehyde-3-phosphate is converted to 1,3 bisphosphglycerate by
glyceraldehyde 3-phosphate dehydrogenase using NAD+ as the coenzyme. Finally, by
phosphorolysis, inorganic phosphate (pi) is added, forming1, 3 bisphosphoglycerate,
and the free enzyme.

Energy released during the oxidation is conserved by the formation of a high-energy

sulfur group that becomes, after phosphorolysis, a high- energy phosphate group in
position 1 of 1, 3 bisphosphoglycerate.

Step 7: Transfer of phosphate group from 1, 3 bisphosphoglycerate

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1, 3-bisphosphoglycerate is oxidized to 3- phosphoglycerate by phosphoglycerate
kinase. This high- energy phosphate is captured as ATP in a further reaction with ADP.

Since two molecules of triose phosphate are formed per molecule of glucose
undergoing glycolysis, two molecules of ATP are generated at this stage per molecule of
glucose.

Step 8: Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate

In the next reaction 3-phosphoglycerate is converted to 2-phosphoglycerate by

the enzyme phosphoglycerate mutase.

Step 9: Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate

The subsequent step is catalyzed by enolase that promotes the reversible removal
of a water molecule from 2-Phosphoglycerate to form phosphoenolpyruvate.

Step 10: Conversion of Phosphoenol pyruvate to pyruvate

The high-energy phosphate of phosphoenol pyruvate is transferred to ADP by the

enzyme pyruvate kinase to generate, at this stage, two molecules of ATP per molecule of
glucose oxidized and enolpyruvate is formed. Enolpyruvate formed is converted
spontaneously to the keto form pyruvate. This is an irreversible step.

6.3.1.2. Energy production under anaerobic and aerobic condition

a. Aerobic condition
Under aerobic condition, pyruvate is taken up into mitochondria, and after conversion to
acety-CoA is oxidized to CO2 by the citric acid cycle. The reducing equivalents from the
NADH+H+ formed in glycolysis are taken up into mitochondria for oxidation.
Two triosephosphates are produced from each molecule of hexose metabolized.
Dihydroxyacetone phosphate can be isomerized by isomerase to glyceraldehyde 3 phosphate
which is then converted to 1, 3 diphosphoglyceric acid by glyceraldehyde 3 phosphate
dehydrogenase system requiring NAD+ and inorganic phosphate.
ATP Production:                                                                    ATP
Conversion of 1, 3 diphosphoglycerateto 3 phosphoglycerate : 2
Conversion of phosphoenol pyruvate to enol pyruvate             : 2
Action of glyceraldehyde 3 phosphatedehydrogenase
and ETC          (2x3)                                                                   : 6
                                                                                                --------
                              Total                                                             :10
                                                                                                --------
  ATP molecules  used  up in the initial reactions                     :  2
                                                                                                ---------
Net ATP production                                                    :  8
                                                                                    ---------
b. Anaerobic condition
If anaerobic conditions prevail, the reoxidation of NADH by transfer of reducing
equivalents through the respiratory chain to oxygen is prevented. Then, pyruvate is reduced by
the NADH+H+ to lactate, the reaction being catalyzed by lactate dehydrogenase. The
reoxidation of NADH via lactate formation allows glycolysis to proceed in the absence of oxygen

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by regenerating sufficient NAD+ for another cycle of the reaction catalyzed by glyceraldehyde-3
phosphate dehydrogenase.

Lactate dehydrogenase
Pyruvate + NADH + H+ ↔ NAD+ + Lactate
During fermentation, pyruvate is reduced by NADH to ethyl alcohol being catalysed by alcohol
dehydrogenase.
  Alcohol dehydrogenase
Pyruvate + NADH + H+ ↔ NAD+ + Ethyl alcohol
ATP production:                                                                      ATP
     
  Conversion of two triose phosphates to lactic acid (or ethanol): 4.
  ATP molecules used up in the initial reactions                         :  2
                                                                                                 ----------               
Net ATP production                                                      : 2
                                                                                     ----------

6.3.1.3. Oxidation of Pyruvate to Acetyl CoA

Under aerobic condition pruvate derived from glucose by glycolysis, is dehydrogenated to yield
acetyl-CoA and CO2by pyruvate dehydrogenase, located in the mitochondria.

The reaction is as follows:

                                  Pyruvate Acetyl CoA

CH3COCOOH+ NAD+ + CoA-SH → CH 3 S ~ CoA+ NADH + CO2

Thiamine pyrophosphate, coenzyme A, lipoic acid and NAD+ are coenzymes needed for the
reaction. The acetyl CoA is then fed into citric acid cycle and oxidized to CO2 in citric acid cycle
and  energy is producd.

6.3.2. Citric acid cycle

The citric acid cycle (Kreb’s cycle, Tricarboxylic acid cycle) is a series of reactions in
mitochondria that bring about the catabolism of acetyl residues, liberating hydrogen equivalents,
which, upon oxidation, lead to the release of most of the free energy which is captured as ATP
of most of the available energy of tissue fuels. The acetyl residues are in the form of acety1-
CoA (CH3-CO-S-CoA, active acetate), an ester of coenzyme A. Coenzyme A contains the
vitamin pantothenic acid.
             The major function of the citric acid is to act as the final common pathway for the
oxidation of carbohydrate, lipids, and protein. This is because glucose, fatty acids, and many
amino acids are all metabolized to acetyl-CoA or intermediates of the cycle. It also plays a major
role in gluconeogenesis, transamination, deamination, and lipogenesis. Several of these
processes are carried out in many tissues but the liver is the only tissue in which all occur to a
significant extent. Reactions of the citric acid cycle liberate reducing equivalents and CO2. The
reactions of citric acid cycle the following 9 steps:
1. Condensation of acety1- CoA with oxaloacetate to form citrate

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            The initial condensation of acety1- CoA with oxaloacetate to form citrate is catalyzed by
condensing enzyme, citrate synthase, which effects synthesis of a carbon to carbon bond
between the methyl carbon of acety1-CoA and the carbony1 carbon of oxaloacetate.
2. Conversion of citrate to isocirtrate via cis-aconitate
            Citrate is converted to isocitrate by the enzyme aconitase (aconitate hydratase), which
contains iron in the Fe2+ state in the form of an iron- sulfur protein (Fe:S) This conversion takes
place in two steps: dehydration to cis-aconitate, some of which remains bound to the enzyme,
and rehydration to iocitrate. 
3. Dehydrogenation of isocitrate to oxalosuccinate
Isocitrate undergoes dehydrogenation in the presence of isocitrate dehydrogenase to
form oxalosuccinate. The linked oxidation of isocitrate proceeds almost completely through the
NAD+ dependent enzyme isocitrate dehydrogenase. 
4. Decarboxylation   α -ketoglutarateof oxalosuccinate to
       There follows decarboxylation of oxalosuccinate to α -ketoglutarate, also catalyzed by
isocitrate dehydrogenase. A CO2 molecule is liberated.  Mn2+(or Mg2+) is an important
component of the decarboxylation .
5. Decarboxylation of α-ketoglutarate to succiny1-CoA
            Next, α -ketoglutarate undergoes oxidative decarboxylation. The reaction is catalyzed by
an α -ketoglutarate dehydrogenase complex, which requires cofactors thiamin pyrophosphote,
lipoate, NAD+, FAD and CoA results in the formation of succiny1-CoA, a high- energy thioester
and NADH. Arsentic inhabits the reaction, causing the substrate,α -ketoglutarate to accumulate.
 6.  Conversion of succinyl-CoA to succinate
            Succinyl-CoA is converted to succinate by the enzyme succinate
thiokinase   (succiny1CoA synthetase). High-energy phosphate (ADP+Pi →ATP) is synthesized
at the substrate level because the release of free energy from the oxidative decarboxylation
of α -ketoglutarate is sufficient to generate a high- energy phosphate..
7. Dehydrogenation of succinate to fumarate        
Succinate is metabolized further by undergoing a dehydrogenation catalyzed by
succinate dehydrogenase to form fumerate. It is the only dehydrogenation in the citric acid cycle
that involves the direct transfer of hydrogen from the substrate to a flavorprotein without the
participation of NAD+.
8. Addition of water to furmarate to give malate.
Furmarase (furmarate hydratase) catalyzes the addition of water to furmarate to give
malate. In addition to being specific for the L-isomer of malate, furmarase catalyzes the addition
of the elements of water to the double bond of furmarate in the tans configuration. 
9. Dehydrogenation of malate to form oxaloacetate
Malate is converted to oxaloacetate by dehydrogenation catalysed by the  enzyme
malate dehydrogenase, a reaction requiring NAD+.
Thus the citric acid cycle is completed. An acetyl group, containing two carbon atoms, is
fed into the cycle by combining it with oxaloacetate. At the end of the cycle a molecule of
oxaloacetate is generated.
            The enzymes of the citric acid cycle, except for the α-ketoglutarate and succinate
dehydrogenase, are also found outside the mitochondria. As a result of oxidations catalyzed by
dehydrogenase enzymes of the citric acid cycle, three molecules of NADH and one molecule of
FADH2 are produced for each molecule of acety1-CoA catabolized in one revolution of the
cycle. These reducing equivalents are transferred to the respiratory chain in the inner
mitochondrial membrane for reoxidation.
6.3.2.2. Respiratory chain in the inner mitochondrial membrane and ATP
production

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Pyruvate           ADP+Pi→ATP ADP+Pi→ATP                            ADP+Pi→ATP

Isocitrate                         ↑            ↑                                                            ↑

-ketoglutarate → NAD → FMN → Co Q→Cytb→CytC1→ CytC→ Cyta→ Cyta3

Malate

During passage along the chain, reducing equivalents from each NADH generate three high-
energy phosphate bonds by the esterification of ADP to ATP in the process of oxidative
phosphorylation. However, FADH2 produces only two high- energy phosphate bonds because it
transfers its reducing power to Co Q, by passing the first site for oxidative phosphorylation in the
respiratory chain. A further high-energy phosphate is generated at the level of the cycle itself
(i.e., at substrate level) during the conversion of succinyl -CoA to succinate. Thus, 12 ATP
molecules are generated for each turn of the cycle. 

 ATP production in mitochondria

Name of enzyme Reaction Catalyzed ATP molecules

formed
 Isocitrate dehydrogenase  Respiratory chain oxidation of  3
NADH +H+
 -Ketoglutrate  Respiratory chain  3
dehydrogenase
oxidation of NADH +H+
 Succinate thiokinase  Phosphorylation at substrate level  1
 Succinate dehydrogenase  Resiphorylation chain oxidation of  2
FADH 2
 Malate dehydrogenase  Respiratory chain oxidation of  3
NADH +H+
 Net  12
Net ATP production from glucose
1. Glycolysis
One mole. of glucose → 2 moles of pyruvate
ATP produced                              =  8

2. Pyruvate → Acetyl CoA

ATP produced                              =  2x3=6

3. Citric acid cycle

Acetyl CoA →CO2 + H2O
ATP produced                              =  12 x2=24

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Net production                           =30+8=38

Citric acid cycle intermediates are used for other metabolic purposes. It is
an amphibolic pathway it functions not only in the oxidative degradation of carbohydrates, fatty acids
and amino acids but also as a source of precursors for other metabolic pathways. 

Chapter 4: Gluconeogenesis

6.4.1. Gluconeogenesis

Gluconeogenesis is the synthesis of glucose from noncarbohydrate and then conversion to

glycogen. The major substrates for gluconeogenesis are the glucogenic amino acids, lactate,
glycerol, and propionate. Liver and kidney are the major tissues involved, since they contain a
full complement of the necessary enzymes.

Gluconeogenesis meets the needs of the body for glucose when carbohydrate is not available in
sufficient amounts from the diet. A continual supply of glucose is necessary as a source of
energy, especially for the nervous system and the erythrocytes. Failure of gluconeogensis is
usually fatal. Below a critical blood glucose concentration, there is brain dysfunction, which can
lead to coma and death. Glucose is also required in adipose tissue as a source of glyceride-
glycerol, and it probably plays a role in maintaining the level of intermediates of the citric acid
cycle in many tissues. Even under conditions where fat may be supplying most of the caloric
requirement of the organism, there is always a certain basal requirement for glucose.

Glucose is the only fuel that will supply energy to skeletal muscle under anaerobic conditions. In
addition, gluconeogenic mechanisms are used to clear the products of the metabolism of other
tissues from the blood, e.g. lactate, produced by muscle and erythrocytes, and glycerols, which
is continuously produced by adipose tissue.

Gluconeogenesis involves glycolysis, the citric acid cycle, plus some special reactions

The energy barriers obstruct a simple reversal of glycolysis

i)between pyruvate and phosphoenolpyruvate,

ii)between furctose 1,6 bisphosphate and furctose 6-phosphate,

iii)between glucose 6-phosphate and glucose, and

iv)between glucose 1-phosphate and glycogen.

These reactions are all nonequilibrium, releasing much free energy as heat and therefore
physiologically irreversible. These reactions are circumvented by the following special reactions.

1. the conversion of pyruvate into phosphoenolpyruvate 

2.Conversion of fructose 1,6-bisphosphate into fructose 6- phosphate  

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6.4.1.1. Conversion of Pyruvate into Phosphoenolpyruvate

Pyruvate carboxylase, present in mitochondria, converts pyruvate to oxaloacetate in the

presence of ATP, the B vitamin biotin, and CO2 . The function of the biotin is to bind CO2 from
bicarbonate onto the enzyme prior to the addition of the CO2 to pyruvate.

A second enzyme, phosphoenolpyruate carboxykinase, catalyzes the conversion of

oxaloacetate to phosphoenolpyruvate. High energy phosphate in the form of GTP or ITP is
required in this reaction, and CO2 is liberated.

Thus, with the help of these two enzymes catalyzing endergonic transformations and lactate
dehydrogenase, lactate can be converted to phosphoenolpyruvate, overcoming the energy
barrier between pyruvate and phosphoenolpyruvate.

6.4.1.2. Conversion of Fructose 1,6-Bisphosphate into Fructose 6- phosphate

The conversion of fructose1,6 bisphosphate to fructose 6-phosphate, necessary to achieve a

reversal of glycolysis, is catalyzed by a specific enzyme, fructose-1,6 bisphosphatase. 

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This enzyme is present in liver and kidney and in striated muscle. It is absent in heart muscle
and smooth muscle.

6.4.1.3. Conversion of glucose 6-phosphate into Glucose

The conversion of glucose 6-phosphate to glucose is catalyzed by another specific

phosphatase, glucose-6-phosphatase. It’s presence allows a tissue to add glucose to the blood. 

6.4.1.4. Conversion of glucose 6-phosphate into Glycogen

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The break down of glycogen to glucose 1-phosphate is carried out by phosphorylase. The
synthesis of glycogen involves an entirely different pathway through the formation of uridine
diphosphate glucose and the activity of glycogen synthase.

Phosphoglucomutase 
Glucose 6-phosphate ↔glucose 1-phosphate

Glucose 1-phosphate uridylyltransferase 

Glucose 1-phosphate + UTP →UDP-glucose + PPi

Glycogen synthase 
UDP-glucose+ Glycogen Primer (Glucose)n → Glycogen (glucose)n+1

These key enzymes allow reversal of glycolysis to play a major role in gluconeogenesis, the
relationships between gluconeogenesis and the glycolytic pathway.

After transamination or deamination, glucogenic amino acids form

either pyruvate or members of the citric acid cycle. The reactions described above can
account for the conversion of both glucogenic amino acids and lactate to
glucose or glycogen. Thus, lactate forms pyruvate and enter the mitochondria before
conversion to oxaloacetate and ultimate conversion to glucose.

The source of pyruvate and oxaloacetate for gluconeogenesis is mainly amino acid catabolism.
Some amino acids are catabolized to pyruvate, oxaloacetate, or precursors of these. Muscle
proteins may break down to supply amino acids. These are transported to liver where they are
deaminated and converted to gluconeogenesis inputs.

6.4.1.5. Conversion of propionate into succinylCoA

Propionate resulting from fatty acid matabolism enters the main gluconeogenic pathway via the
citric acid cycle after conversion to succinyl-CoA. Propionate is first activated with ATP and CoA
by an appropriate acyl-CoA synthetase.

Propionyl-CoA synthetase

Propionate + ATP + CoA → Propionyl CoA

Propionyl CoA formed undergoes a CO2 fixation reaction to form D-methylmalonyl-CoA,

catalyzed by propionyl-CoA carboxylase. This reaction forms a malonyl derivative and requires
the vitamin biotin as a coenzyme.

Propionyl-CoA carboxylase

Propionyl CoA + CO 2 + H2O → D-methylmalonyl-CoA

D-Methylmalonyl-CoA must be converted to its steroisomer, L- methylmalonyl-CoA, by

methylmalonyl-coA racemase before its final isomerization to succinyl-CoA by the enzyme
methlmalonyl-CoA isomerase.

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Methylmalonyl-CoA racemase

D-Methylmalonyl-CoA ↔ L- methylmalonyl-CoA

M ethylmalonyl-CoA isomerase

L- methylmalonyl-CoA ↔ Succinyl-CoA

It is converted into malate which is then converted into phosphoenol pyruvate and finally to
glucose.

Succinyl-CoA → Malate → Phosphoenol pyruvate → Glucose

Thus glucose can be formed from fatty acids

Chapter 5: Biological oxidation

6.5.1.Introduction   
Oxidation is chemically defined as the removal of electrons and Reduction is the gain of
electrons. The free oxidation takes place with the production of flames and much heat , while in
the living organism oxidation takes place near room temperatures in aqueous solution or at
lipoprotein surfaces, and the free energy of combustion is efficiently recovered and utilised.
Substances like glucose or citric acid are stable indefinitely in the air are smoothly oxidised by
living cells. The biological combustion is under the control of enzymic catalysis. An iron
containing enzyme system( the cytochrome system) present in the tissues enables molecular
oxygen to serve as an oxidixing agent.
The enzymes involved in biological oxidation–reduction are (1) dehydrogenases with
carriers of reducing equivalents, and (2) oxidases. Many of these enzymes are organised in
multienzyme systems which function as the terminal respiratory chain. The first process in the
terminal respiratory chain consists in the activation of certain specific hydrogen atoms by
specific dehydrogenases. The second process involves the transfer of the hydrogen atoms
(reducing equivalents) from metabolites to carriers of reducing equivalents. The final stage in
terminal oxidation consists in the combination of the reducing equivalents from the carrier with
oxygen.
6.5.2.Dehydrogenases and carriers of reducing equivalents
a) Aerobic dehydrogenases
They catalyze the removal of hydrogen from a substrate and use either oxygen or
artificial substances such a methylene blue as hydrogen acceptor. H 2O2 is formed as a product.
They are flavoprotein enzymes having FMN (flavin mononucleotide) or FAD (Flavin adenine
dinucleotide) as prosthetic groups. Many of the flavoprotein enzymes contain a metal for which
they are known as metal for which they are known as metalloflavoprotein enzymes.
(i) D-amino acid dehydrogenase (D-amino acid oxidase):  It is an FAD-linked enzyme..It is
found particularly in liver and kidney.  It catalyzes the oxidative deamination of the unnatural
(D-) forms of amino acids.
(ii) L-amino acid dehydrogenase (L-amino acid oxidase):  It is an FMN-linked enzyme. It is
found in kidney. It catalyzes the oxidative deamination of naturally occurring L-amino acids.
(iii) Xanthine dehydrogenase (Xanthine oxidase): It occurs in milk and liver. In the liver, it
converts purine bases to uric acid.  It contains FAD as the prosthetic group.  It is highly
significant in the liver and kidneys of birds which excrete uric acid as the end product of purine

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metabolism and also of protein and amino acid catabolism. It is a metalloflavoprotein containing
nonheme iron and molybdenum.  It also oxidizes all aldehydes.
6.5.3. Anaerobic dehydrogenases

(a) They catalyze the removal of hydrogen from a substrate but not able to use oxygen as
hydrogen acceptor.

(b) They transfer hydrogen from one substrate to another by oxidation-reduction reaction not
involving a respiratory chain.

(c) They perform oxidation of metabolite utilizing several components of a respiratory chain

(i) Dehydrogenase dependent on Nicotinamide Coenzymes:

(a) They are linked as coenzymes either to NAD (Nicotinamide adenine dinucleotide) or to
NADP (Nicotinamide adenine dinucleotide phosphate).

(b) The coenzymes are reduced by the particular substrate of the dehydrogenase and
reoxidized by a suitable electron acceptor and synthesized from the vitamin niacin (nicotinic acid
and nicotinamide).

(c) NAD-linked dehydrogenases catalyze oxidoreduction reactions in glycolysis, the citric acid
cycle and in the respiratory chain of mitochondria.

(d) NADP-linked dehydrogenases are found in fatty acid and steroid synthesis in the
extramitochondria. They are also found in hexose monophosphate shunt.

(e) Some nicotinamide coenzyme-dependent dehydrogenases contain zinc, particularly alcohol

dehydrogenases from liver and glyceraldehydes-3-phosphate dehydrogenase from skeletal
muscle. The zinc ions do not take part in the oxidation and reduction.

(ii) Dehydrogenases dependent on Riboflavin Prosthetic Groups:

(a) Most of the riboflavin-linked anaerobic dehydrogenases are concerned with electron
transport in the respiratory chain.

(b) Succinate dehydrogenase, acyl-CoA dehydrogenase and mitochondrial glycerol-3-

phosphate dehydrogenase transfer electrons directly from the substrate to the respiratory chain.

(c) In the dehydrogenation of reduced lipoate, and intermediate in the oxidative decarboxylation
of pyruvate and a -Ketoglutarate, the flavoprotein(FAD) due to the low redox potential acts as a
carrier of electrons from reduced lipoate of NAD+. The electron transferring flavoprotein in an
intermediary carrier of electrons between acyl-CoA dehydrogenase and the respiratory chain.

(iii) The Cytochromes:

(a) The cytochromes excepting cytochrome oxidase are anaerobic dehydrogenases. They are
involved as carriers of electrons from flavoproteins to cytochrome oxidase in the respiratory
chain.

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(b) They are iron-containing hemoproteins in which iron becomes Fe+++ and Fe++ oxidation
and reduction. The cytochromes in the respiratory chain are b,c1, c, a and a3.

(c) Cytochromes are also found in the endoplasmic reticulum (cytochromes P-450 and b5) plant
cells, bacteria and yeast.

6.4.2.1. Oxidases and carriers of reducing equivalents

Oxidases are enzymes that catalyze the removal of hydrogen from a substrate but use only
oxygen as a hydrogen acceptor to form water as a reaction product (with the exception of
uricase and monoamine oxidase which form H2O2).  They are conjugated proteins containing
copper as prosthetic groups.
(i) Cytochrome oxidase: Cytochrome oxidase is a hemoprotein widely distributed in plants and
animal tissues. It is the terminal component of respiratory chain found in
mitochondria.  Cytochromes a and a3 are combined with the same protein and the complex is
known as cytochrome aa3. Cytochrome aa3 contains 2 molecules of heme A, each having one
Fe atom.. 2 atoms of Cu are also present which are associated with the cytochrome oxidase
activity. The terminal cytochrome aa3 is responsible for the final combinant of reducing
equivalents with molecular oxygen. It has a high affinity for oxygen. It is the only one in the
chain which signifies the irreversible reaction.. It gives direction to the movement of reducing
equivalents in the respiratory chain and to the production of ATP, to which it is coupled.
(ii) Phenolase (tyrosinase, polyphenol oxidase, catechol oxidase):   It is a copper
containing enzyme. It converts monophenol to O-quinones.
3. Hydroperoxidases
They utilize hydrogen peroxide as a substrate. Two enzymes fall into this category (i)
Peroxidase, (ii) Catalase.
(i) Peroxidase: It is found in milk and leukocytes and the prosthetic group is protoheme.  It
catalyzes the reduction of hydrogen peroxide by the help of ascorbic acid, quinines and
cytochrome C which act as electron acceptors.
(ii) Catalase: It is hemoprotein and found in blood and liver. It uses one molecule of H 2O2 as a
substrate electron donor and another molecule as electron acceptor.  Its function is to destroy
H2O2 formed by the action of aerobic dehydrogenases.
6.5.5. Hydroperoxidases
They utilize hydrogen peroxide as a substrate. Two enzymes fall into this category (i)
Peroxidase, (ii) Catalase.
(i) Peroxidase: It is found in milk and leukocytes and the prosthetic group is protoheme.  It
catalyzes the reduction of hydrogen peroxide by the help of ascorbic acid, quinines and
cytochrome C which act as electron acceptors.
(ii) Catalase: It is hemoprotein and found in blood and liver. It uses one molecule of H 2O2 as a
substrate electron donor and another molecule as electron acceptor.  Its function is to destroy
H2O2 formed by the action of aerobic dehydrogenases.
Unit 7: Food proteins

Chapter 1: Native proteins and denatured proteins

7.1.1.Introduction
Proteins are important in foods, both nutritionally and as functional ingredients. They play an
important role in determining the texture of a food. Proteins are made up of sequence of amino
acids. There are 20 amino acids present in food proteins.
1. Essential amino acids and Nonessential amino acids

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       The body is able to synthesis about 10 amino acids and these amino acids are called non-
essential or dispensable amino acids as they can be synthesized in animals from other
compounds. They are glycine, cysteine, alanine, serine, proline, tyrosine, aspartic acid,
asparagine, glutamic acid and glutamine. The remaining amino acids are called essential or
non-dispensable as they cannot be synthesized in animals. They aremethionine, tryptophan,
threonine, valine, isoleucine, leucine, phenylalanine, lysine, arginine and histidine.
Arginine and histidine are essential for infants and children. 
      
2. Limiting amino acid
Proteins of different foods have different proportions of essential amino acids. Some of
them may contain required  mounts of essential amino acid and few of them may not have
adequate amounts of one or more of essential amino acids. An essential amino acid of a protein
which is present much below requirement is called as limiting amino acid. Most of the plant
proteins contain limiting amino acid.
Examples
(a) Tryptophan is the limiting amino acid in maize, bengal gram and red gram proteins.
(b) Lysine is the limiting amino acid in wheat protein.
(c) Methionine is the limiting amino acid in peanut protein.
Due to limiting amino acid quality of protein decreases.
Effect of limiting amino acid on protein utilization
When tissue proteins are synthesized all the essential amino acids must be present in proper
proportions in tissues. If one essential amino acid is absent in tissues due to lack of dietary
supply protein synthesis decreases and nitrogen balance is not maintained. More over biological
value of a protein with limiting amino acid is low.
The supplementary value of protein
One way of improving quality of dietary protein with limiting amino acid is by adding another
protein containing the missing amino acid. Some proteins are poor quality proteins as such due
to limiting amino acids. But they are complementary in limiting essential amino acid composition
i.e., a limiting essential amino acid in one protein is present in excess amounts in another
protein and vice versa. So they supplement each other and make good quality protein in diet.
This is known as supplementary value of proteins. For example wheat proteins and red gram
proteins are complementary proteins and as such both are low quality proteins due to limiting
amino acid.Wheat protein is limiting in lysine but good source of tryptophan whereas red gram is
limiting in tryptophan but a good source of lysine. When they are mixed they make up good
quality protein in diet by supplementing one another i.e., wheat protein effectively supplement
pulse protein and vice versa. Therefore chapati and dal combination improves quality of protein
in diet.
7.1.3.Structure of amino acids
(1)Structure of amino acids
The 20 amino acids that make up food proteins are linked by peptide bonds and have distinct
structures and properties. These amino acids consist of an α-carbon atom covalently attached
to a hydrogen atom, an amino group, a carboxyl group, and a side- chain R  group. These
amino acids differ only in the chemical nature of the side chain R group. 
The solubility, chemical reaction, net charge and hydrogen bond formation of the amno acids
are due to the chemical nature of the R group. Based on the degree of interaction of the R
groups with water, amino acids are classified into: hydrophilic (Polar) or  hydrophobic(non-
polar).
a. R- Group -Hydrophilic(Polar)
Hydrophilic or polar amino acids are soluble in water. They are charged or uncharged.

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i. Charged:  Arg, Asp, Glu, His and Lys are charged amino acids. The side chains of Arg
and Lys contain quanido and amino groups, respectively and hence are positively charged
(basic) at neutral pH. The imidazole group of His is also basic in nature and slightly positive at
neutral pH. Asp and Glu acids contain a carboxyl group and carry a net negative charge at
neutral pH. The charged amino acids are strongly hydrophilic. The overall charge of a protein
depends on the number of positive and negative amino acids present.
ii. Uncharged: The uncharged amino acids have polar groups that form hydrogen bond with
water. Ser and Thr are polar due the presence of a hydroxyl group. Tyr contains an ionisable
phenolic group that ionizes at alkaline pH and hence considered as polar. But at neutral pH it is
a nonpolar aminoi acid. The amide group of Asn and Gln can interact with water and hence
polar in nature. Cys is a polar amino acid. Majority of Cys molecules exist as Cystine, a dimer
formed by the oxidation of thiol groups to form a disulphide bond. Proline is an imino acid. 
b. Hydrophobic(non-polar): Amino acids with aliphatic and aromatic side chains are
hydrophobic and hence are insoluble in water. Ala, Ile, Leu, Met, pro and Val are aliphatic amino
acids. Phe, Try and Tyr are aromatic amino acids.
7.1.4.Structure of Protein
Structure of Protein
There are four levels in a protein structure: primary, secondary,
tertiary and quaternary.
i.Primary Structure: The primary structure of a protein refers to the linear seqence formed by
the amino acids linked by peptide bond formed by the condensation of the α-carboxyl group of
one amino acid with the α- amino group of the next amino
acid.                                                                                                      
  Peptide bond
ii. Secondary Structure: Secondary structure refers to the spatial arrangement of amino acid
residues of the polypeptide chain. Two forms of secondary structures are found in proteins: α-
helix and β-pleated structure. α-helix is the major form found in proteins. The axial length
occupied per rotation is 5.4Ǻ and each helical rotation involves 3.6 amino acid residues, each
residue extending the axial length by 1.5 Ǻ. The α-helix is stabilized by hydrogen bond. The β-
pleated structure is formed by hydrogen bonds between two strands of the same molecule
forming a sheat like structure known as β-pleated sheet.
                          α-helix β-pleated structure 
         
iii. Tertiary structure: Tertiary structure refers to the spatial arrangement formed when a linear
protein chain with secondary segments folds further into a compact three dimensional form. The
tertiary structure of a myoglobin molecule is shown in the diagram.
Tertiary structure of a myoglobin
iv. Quaternary structure
Quaternary structure refers to the spatial arrangement of a protein when it contains more than
one polypeptide chain.  Many biologically active proteins exist as dimmers, trimers, tetramers
etc. These structures are formed by specific protein-protein interactions. Eg. Haemoglobin-: The
four separate chains of haemoglobin assembled into a tetrameric protein subunit.
Quaternary structure of haemoglobin
7.1.5.Forces involved in the stability of protein structure

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The forces that contribute to protein folding into unique three-dimensional structure may be
grouped into two types.
(i)Intramolecular interactions due to force intrinsic to the protein molecule- van der Walls  and
steric interactions and (ii) intermolecular interactions affected by the surrounding solvent.
(i)Intramolecular interactions
a. Van der Walls  Interactions: These are dipole induced dipole and induced dipole-induces
dipole interactions between neutral atoms in protein molecules. When two such atoms approach
each other, each atom induces a dipole in the other via polarization of the electron cloud. These
interactions may be attractive or repulsive. The magnitude of these forces is dependent on the
interatomic distance. In proteins, numerous pairs are involved in van der Walls interactions, and
hence the sum of its contribution to protein folding and stability is very significant.
b. Steric strains: There is steric hindrance from side chains atoms and hence segments of a
polypeptide chain can assume only a limited number of configurations. Distortions in the
arrangement of the peptide chain or stretching and bending of the peptide bond will increase the
free energy of the molecule and hence folding of the polypeptide can occur only in such a way
that deformation of bond angles and bond lengths are avoided.
(ii))Intermolecular Interactions
1. Hydrogen bond
2. Electrostatic bond
3. Hydrophobic bond
4. Disulphide bonds
1. Hydrogen bond: The hydrogen bond involves the interaction of a hydrogen atom that is
covalently attached to an electronegative atom (N, O, or S) with another electronegative atom.
Proteins contain several groups capable of forming hydrogen bonds. The greatest number of
hydrogen bonds are formed between the N-H and C=O groups of the peptide bonds in α-helix
and β-sheet. The hydrogen bonds are ionic bonds. They are stable as long as they are
protected from water.
2. Electrostatic bond: Proteins contain several amino acid residues with ionisable
groups. At neutral pH, Asp Glu residues are negatively charged, and Lys, Arg and His are
positively charged. At alkaline pH, Cys and Tyr residues assume a negative charge.
Depending upon the relative number of negatively and positively charged residues, proteins
assume either a net negative or net positive charge at neutral pH. The pH at which net charge is
zero is called the  isoelectric pH(IEP).
Type of proteins lEP at pH
Casein 4.6
Ovalbumin 4.6
Gelatin 4.9
Myosin 5.4
Gluten 7.0
3. Hydrophobic interactions: The hydrophobic interaction occurs between water and nonpolar
groups of protein in aqueous solutions. Hence nonpolar groups tend to sggregate, so that the
area of direct contact with water is minimized. This water structure –induced interaction between
nonpolar groups in aqueous solutions is called hydrophobic interations. In proteins, hydrophobic

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interactions among nonpolar group are the major force driving protein folding into tertiary
structure in which the nonpolar groups are protected from the aqueous environment.
 4. Disulphide bonds: Disulphide bonds are covalent side chain cross links found naturally in
proteins.They can occur both intramolecularly or intermolecularly  When two Cys residues are
brought into proximity with proper orientation oxidation of the sulfhydryl groups by molecular
oxygen results in disulphide bonds. This bond stabilizes the folded structure of protein.
7.1.6.Nature of proteins 
Proteins play an important role in biological systems. Proteins are synthesized in ribosomes.
After synthesis some amino acids are modified by cytoplasmic enzymes. Proteins that are not
modified thus are called homoproteins and that are modifiedor complexed with nonprotein parts
are called heteroproteins or conjugated proteins.
Conjugated proteins
-
Type Examples
Nucleoprotein Ribosomes, Histones
Glycoprotein Ovalbumin, k-casein
Phosphoproteins α, and β caseins, phosphorylases
Lipoproteins Proteins of egg yolk, plasma proteins
Metalloproteins Hemoglobin, myoglobin and enzymes
7.1.7.Native proteins
        Proteins found in living tissues (within the cell and in the fluids) of animals and plants are
called native proteins. The formation of a unique three dimensional protein structure is the net
result of various repulsive and attractive covalent and noncovalent interactions. They are stabilized
by the following.
1. All colloidal particles have similar charge on the molecules. Like charges repel each other and the
particles remain dispersed. Protein macromolecules are hydrophilic colloids and are stabilized by a
layer of water. This acts as insulation and prevents bonding. Some amino acids with a ring structure
and carbon chains (-C-C-C-C-C-) are hydrophobic while the acidic group (COOH group) is
hydrophilic.
2. The free amino and carboxyl groups in a protein molecule have an electric charge depending
on the pH of the medium in which the protein is found. Proteins are said to be amphoteric, i.e., they
can react with acids and bases. Hence, they act as buffers in food preparations. In the presence of an
acid, (the amino group neutralizes the acid) the protein molecules are drawn towards the cathode
as a result of the net positive charge on them. In the presence of an alkali, (the carboxyl group
neutralizes the alkali) the protein molecule migrates towards the anode because of the net negative
charge on them. This migration of proteins in an electric field at a definite pH is called
electrophoresis. It depends on: charge on the molecule, size of the molecule, and shape of the
molecule.
3. At the isoelectric point (IEP) the charge on the protein molecule is neutralized and they show
no movement in an electric field. Proteins at the IEP are called zwitterions. At this point
hydrophobic colloids like casein flocculate and hydrophilic colloids show minimum hydration, e.g.,
gluten
4. When a fluid food containing protein is brought to its IEP, curdling is likely to occur (addition
of acid while curdling milk) as there is no charge which keeps protein dispersed.
7.1.8.Denaturation of Protein

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           Denaturation is defined as any non-proteolytic modification in the original structure of the
native proteins, giving rise to definite changes in physical, chemical, and biological
properties. The native structure of protein is the net result of various attractive and repulsive
forces intramolecular as well as interaction with surrounding water medium. This structure is
thermodynamically stable with lowest feasible free energy at physiological conditions. Any
change in the environment, such as temperature, pH, ionic strength, solvent composition etc.
will force the molecule to assume a new stable structure if the change does not drastically alter
the molecular. architecture.
            But major changes in the secondary, tertiary and quaternary structures without cleavage
of peptide bonds are called as denaturation. It may be a major change in which there is an
increase or decrease in the α-helix  and  β-sheet. But in many cases, denaturation involves a
loss of ordered structure. In a denatured protein the globular proteins become a random coil.

(i) Changes in protein during denaturation

Denaturation is brought about by the following: (1) Denaturing agents, such as acids, alkalis,
salts, (2) Increase in temperature and (3) Extensive beating.
(ii) Stages of denaturation
1st stage: Unfolding of helix of the protein molecules as the cross-links holding the helix is
disrupted. R groups are exposed. Rebonding takes place between adjacent R groups of protein
molecules leading to aggregation of the molecules, bringing about increased viscosity. This is
the first change in denaturation which involves structural alteration.
2nd stage: When sufficient proteins have united, the protein molecules are no longer dispersed as
a 'sol'. At this stage the protein is said to have coagulated, i.e., water is held in the capillary spaces
formed by united protein molecules and the coagulated proteins form a 'gel'.
3rd stage: If the liquid separates from the coagulated protein, the protein is said to
be, 'precipitated' or 'flocculated', i.e., 'curdling' takes place. These stages can be observed while
cooking scrambled eggs. If it is overcooked, liquid separates out.
(iii) Effects of denaturation
Properties of denatured proteins are completely different from their native form. Denaturation of
food is irreversible unless it occurs under very mild condition.
Biologically active proteins lose their biological activity. During heating enzymes are destroyed,
e.g., browning does not take place in boiled potato.
Food proteins lose their functional property. In some cases it is desirable. Denatured proteins
are easily attacked by proteolytic enzymes, e.g., cooked meats are more easily digested than
raw meats.
Denaturation of trypsin inhibitors in legumes improves the digestibility and biological value of
proteins present.
Heat denaturation results in improved flavour and texture, e.g., cooking improves flavour in
meat, and eggs give structure and improve texture cakes.
Denaturatin decrease solubility of protein. For example, cooked egg white is not solution in water.
Denatured proteins lose their ability to crystallize.
There is an increase in viscosity of food.

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Denaturing Agents
1. pH: Proteins are more stable against denaturation at their isoelectric pH. At neutral pH, most
proteins are negatively charged and a few are positively charged. The net electrostatic repulsive
energy is small and hence, most proteins are stable at neutral pH. But at extreme pH values the strong
intramolecular repulsions due to high net charge results in swelling and unfolding of the protein
molecule.
e.g. Caseinogen is dispersed as a sol in milk. When milk turns sour (pH 4.6 ; IEP it curdles (3rd stage
in denaturation). Egg albumin is colloidally dispersed as a sol. It has a pH of 7.2-7.8. But when the
pH is reduced to 4.6 (its IEP) by adding vinegar to water used for poaching eggs, the egg coagulates
faster.
2. Temperature: When a protein solution is gradually heated above a critical temperature, it
undergoes a sharp transition from the native to the denatured state.
 e.g. When egg white is heated at 60°C the protein ovalbumin gets denatured.  A  temperature
increases, coagulation takes place and egg white separates out as solid.  If the temperature is below
100°C, e.g., poaching, then coagulation is slow and coagulated protein is soft and easily digested.  If
temperature is above 100°C, e.g., boiling and roasting, coagulation is fast and coagulated protein is
hard and more difficult to digest.
3. Organic solvents: Organic solvents affect the solubility of protein hydrophobic and electrostatic
interactions and hydrogen bonding. Some nonpolar side chains are more soluble in organic solvelts than
in water, hydrophobic interactions are weakened by organic solvents.At low concentrations, some organic
solvents can stabilize proteins. At high concentrations, however all organic solvents cause denaturation of
proteins because of their solubilising effect on nonpolar side chain.
4. Surface denaturation:  This is brought about by mechanical means, e.g., beating egg white or
milk to foam. Surface denaturation of the protein takes place leading to pellicle or skin formation.
This stabilizes the foam (pellicle is seen when foam has subsided).  If such a foam is heated, as in egg
white foam, it becomes firm due to the coagulation of ovalbumin. If pH of ovalbumin is at its IEP,
then coagulation is faster. Hence, acidic sub stances (cream of tartar, lime juice, etc.) are added when
whipping egg white.
5.Salts: At low concentrations, ions interact with proteins via nonspecific electrostatic
interactions. When present in a high concentration salt precipitates proteins out of solution and
disperses them.  e.g., cured ham baked in white sauce. The high salt concentra tion of the ham can
cause the milk in white sauce to curdle.

6. Detergents:  Detergent such as sodium dodecyl sulphate are powerful protein denaturing agents.  It is
due to the interaction of the detergent to the  R-group disrupting the interactions involved.

Chapter 2: Food Proteins Sources

7.2.1.Introduction

Proteins are widely distributed in nature but only a few foods provide sufficiently higher quantity of
proteins. Eggs, fish, meats, nuts and poultry have protein ranging from 12-29%. Cereal contributes 1-2%
of proteins. Fruits and vegetables are low sources of protein foods. Syrups, oils and fat contain no protein.
Animal foods have high protein values and also have good quality. Soya bean and some nuts contain
protein of high quality.

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7.2.2. Muscle proteins
Meat is made of high biological value or complete proteins (15-25%) containing all of the essential amino
acids in amounts and proportions that can be used in synthesizing body proteins. The three primary types
of proteins are myofibril proteins, stroma proteins and sarcoplasmic proteins. Muscle bundles are groups
of myofibrils composed of several proteins including actin, myosine which may overlap to form
actomyosin. The connective tissue contains collagen and elastin. The sarcoplasmic proteins include
albumins, globulins, enzymes and pigment hemoglobin and myoglbin.

The meat of animals, fish, crustaceans and mollusks is used for the preparation of different
dishes, a variety of canned, smoked, marinated, salted and dried products and for the production of value
added products like sausages. In these products the functional properties of the animal proteins are
responsible for the desirable sensory attributes. An increasing quantity of the raw material is used for
manufacturing protein concentrates, preparations and hydrolysates which can be incorporated in various
food commodities as functional ingredients.

Table 2.1 Protein content of different types of meat

Type of meat Protein %

Chicken 25.9
Mutton 18.5
Beef 21.4
Pork 18.7

Seafood Protein: Nutritive value of fish and shellfish is due to their content of high quality protein.
Protein content of seafood is given in table2.2. It is comparable in biological value and in essential amino
acid composition with those of milk, meat and eggs. Fish is a rich source of all amino acids especially of
methionine, threonine, lysine and isoleucine, which are considered to be limited in plants and cereal
foods. Hence fish, when taken along with pulses and cereals can improve the protein content of the diet
and thereby improve the nutritional status. The shellfish protein is also of good biological value. The
amino acid composition of some fish protein is given in table:2.3.

Table 2.2 Protein content of seafood

Type of fish Protein %

Fatty fish 20.0
Lean fish 16.4
Crustaceans 17.8
Mollusks 13.0

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Table 2.3Amino acid composition of fish protein

Amino acid Mackeral Pomfret Sardine Oil Hammer Silver Sol
sardine Head belly
shark
Leucine 11.0 18.4 12.4 13.4 9.1 5.5 6.7
Phenylalanine 4.2 6.2 4.8 6.1 5.4 5.4 3.8
Valine 6.0 6.4 8.8 5.9 5.6 7.3 6.9
Methionine 4.3 4.7 2.9 3.8 3.1 2.1 1.9
Tryptophan 1.1 1.8 Traces Traces 2.1 -- --
Threonine 5.5 6.4 4.9 4.7 4.7 5.8 5.6
Arginine 6.8 8.2 6.2 7.0 6.3 13.5 19.2
Lysine 6.5 3.7 6.1 8.0 5.1 6.4 7.4
Histidine 1.9 0.7 0.8 1.7 3.1 3.9 2.4
7.2.3. Milk proteins
Protein represents 3-4% of the composition of milk. Casein is the primary protein of milk, comprising
approximately 80% of the milk protein. Milk proteins are of two types: casein (24-28g/L) and whey
proteins(5-7g/L). Milk proteins have found various applications in formulated foods and as meat
extenders, initially only casein was used. Whey protein concentrates are also used. Milk proteins have
high nutritional value due to amino acid composition and have higher content of phophoryl serine and
praline. Several milk proteins have antibacterial properties. The whey protein lactoferrin, beta
lactoglobulin, lactalbumin, α -casein, β-casein κ and –casein are biologically active peptides.
7.2.4. Egg proteins
Egg proteins
         Eggs provide nutritive value and culinary variety to food. (Table 2.4). The egg white is made up of
albumin. Protein in the egg yolk is primarily vitellin. Egg albumen is used various food formulations
because of its foaming properties and heat gelling ability, while egg yolk serves as an emulsifying agent.
                                               Table2.4 Protein content of egg

  Item %
Whole egg 11.8
egg white 11
egg yolk 17.5
7.1.1.4. Legume/Pulse Proteins
Legume /Pulse Proteins
i. Pulses: Pulses are important food crops due to their high protein and essential amino acid content

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Pulses are 20 to 25% protein by weight, which is double the protein content of wheat and three times that
of rice. While pulses are generally high in protein, and the digestibility of that protein is also high, they
often are relatively poor in the essential amino acid methionine, although Indian cuisine includes sesame
seeds, which contain high levels of methionine. Grains (which are themselves deficient in lysine) are
commonly consumed along with pulses to form a complete diet of protein.
ii. Green pea
Peas are high in fiber, protein, vitamins, minerals, and lutein. On dry weight basis peas contain
14.8% protein, 7.2% carbohydrates, and dietary fiber 30%.

iii. Dry Beans

           Beans are high in protein (20-30%), complex carbohydrates (8%), folate and iron. They also have
significant amounts of fiber and soluble fiber.

iv. Soyproteins
               Soybean contains about 43 percent high quality protein and 20 percent oil. It is rich in valuable
amino acid lysine (5%) in which is deficient in most of the cereals. Soyproteins are used in a variety of
traditional products such as soymilk or fermented items, defatted flour, grits, concentrates or isolates. The
traditional products are prepared by water extraction, cooking, coagulation of the proteinaceous curd,
roasting and fermentation often with other products. Different forms of concentrated or isolated protein
products are prepared by milling, toasting, extraction of fat and saccharides, and isolation of protein
fractions. The anti nutritional factors present in soybean are usually removed in processing. The ‘beany’
or ‘painty’ off-flavour in soymilk due to volatiles generated in lipoxygenase-catalysed reactions is
prevented by thermal denaturation of the enzyme before or during grinding with water.
. 
v. Peanuts: Peanuts contain about 20% protein. They are a good source of niacin, folate, fiber,
magnesium, vitaminE, manganese and phosphorus. They are free of trans-fats and sodium.

The flours, grits, and isolates of legumes, mainly peanuts, beans, broad beans and peas are used
.for food applications.
Table 2.5 Protein content of legumes

Item %
Bengal gram whole 17
Bengal gram, dhal 21
Bengal gram, roasted 22
Black gram, dhal 24
Cow pea 24
Field bean, dry 25
Green gram, whole 24
Green gram dhal 24
Horse gram, whole 22

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Khesari, dhal 28
Lentil 25
Peas green   7
Peas dry 20
Peas roasted 23
Red gram, dhal 22
Redgram tender 10
Soyabean 43

Source: Gopalan et. al., 2004,

7.1.1.5.Cereal proteins
         Cereals are important source of proteins because they contain 7.5 to 14% proteins depending upon
the type. They are used in foods mainly as flour used for the preparation of breads, noodles, cakes, and
other commodities by baking. The biological value of cereal proteins is significantly lower because of the
deficiency of lysine.
Texturised Proteins:

The main function of extrusion in protein processing is to denature and to texturize. As a result of the
high temperature and shear forces in the extruder, the intramolecular bonds in the protein molecule are
broken and the protein denatures. Subsequently, these bonds can recombine with their counter parts from
other molecules thus forming an intermolecular cross-linked network in the form of strands. The shear
forces in the extruder will align these strands to give the product the desired texture. For good alignment
and structure, the flow conditions in the die are very important. Examples are pasta products, restructed
muscle foods, instant soup and gravy bases, protein modification, meat analogs, frozen produce and
instant noodles

Chapter 3: Functional properties of food proteins

7.3.1. Introduction
Proteins have many useful functional properties in food. A functional property is a characteristic
of the protein that enables it to perform a specific role or function in a food.
1. Types of interactions important to protein structure and functionality
1. The ion-dipole interactions: Proteins contain a number of amino acids that have side groups that
contain electrical charges at certain pH values. The ion-dipole interactions between water and these
charged groups are fairly strong with an energy of about 5 Kcal/mole. With model peptides, it has been
shown that from four to seven water molecules can be associated with each residue of charged amino
acid.
2. Dipole-dipole interactions: Proteins also contain amino acids that have polar side chains, but that do
not have a charge. These polar molecules are dipoles and thus water can interact with them through
dipole-dipole interactions. Because the molecules involved all contain hydrogen as part of the dipoles, the
special class of dipole-dipole interactions known as hydrogen bonds can occur. These are typically
stronger than other dipole-dipole interactions and can have energies of from 2 to 6 Kcal/mole. The
nonionized polar amino acids in proteins typically have two water molecules strongly associated with
them.

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 3. Interaction of hydrophobic groups: Nonpolar amino acids are not soluble in water and thus, the
interaction of water with these molecules is minimal. Sometimes, how-ever, nonpolar groups are forced
into water as a part of a specific protein structure. The intrusion of hydrophobic groups into the aqueous
environment causes an ordering of the water molecules in their vicinity and thus a decrease in entropy. It
has been estimated that the removal of a hydrophobic group from contact with water yields a reduction in
the free energy of the protein of about 4 Kcal/mole. This is a strong driving force for the removal of these
groups from the aqueous environment. Any water associated with these groups is highly ordered and has
been termed hydrophobic hydration water.
2. Important functional properties of proteins

Functional properties of proteins are:

1. Proteins and hydration
2.Water-Holding Capacity (WHC)
3. Foam formation and foam stabilization by proteins
4. Viscosity
5. Gelation
6. Emulsification
7.1.2.. Role of proteins in hydration

The interactions of water with proteins are very important both to the structure of the proteins
and to their behavior in food systems. Water molecules tend to associate with themselves
through a network of hydrogen bonds. When solute molecules are placed in water, these
molecules will be soluble if water solute interactions have a lower free energy than do the
separate solute-solute and water-water interactions. The nature of these interactions are very
complex.

Factors affecting water binding by food proteins

            (1) Amino acid composition
            (ii) Protein conformation
            (iii) Ionic concentration and  Ion species
            (iv) pH
            (v). Temperature
i. Amino acid composition: Charged amino acids bind more water than do others and hence, proteins
that contain large amounts of these charged amino acids will tend to bind large amounts of water. The
water binding of proteins is based on amino acid composition.
ii. Protein conformation The amino acids are arranged in the final protein structure. If groups that are
capable of forming hydrogen bonds are removed from contact with the water and allowed to form
hydrogen bonds with themselves, the amount of water bound to the protein will be decreased. On the
other hand, if the protein molecule is unfolded, these groups may make better contact with the aqueous
phase and water binding may actually increase with denaturation.
iii. Ionic concentration and Ion species: Some proteins require the presence of trace amounts of
electrolytes to be soluble. These ions interact with the charged groups on the protein surface and also with
water present. This tends to increase the amount of water bound to the protein. At very high salt
concentrations, the salt and protein may compete for the water present and protein water binding and
solubility may be decreased. The extent of these effects depends to a large extent on the nature of the ions
present. The size of the hydrated radius of the ion seems to be of prime importance for determining the
dehydration effect of concentrated salts.

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iv. pH: The effect of pH on the water binding of proteins can be manifested in many ways. As the pH of a
protein solution changes, so does the number of charged groups on the molecule. As previously noted, the
number of charged groups is strongly related to the amount of water associated with a protein. Changes in
pH can also alter the conformation of the protein which may either expose or bury potential water binding
sites.
Types of bound water associated within proteins
 Types of water associated with proteins are of 5 types- structural, monolayer, hydrophobic, hydration,
unfreezable and capillary.
1. Structural: In some proteins, a molecule of water may be intimately associated with the final structure
of the molecule. In these cases, the water serves as a bridge to hydrogen bond charged groups within the
molecule. These molecules of structural water do not behave like bulk phase water and are unavailable for
chemical reactions.
2. Monolayer: The water molecules that are bound through ion-dipole or dipole-dipole interactions are
described as monolayer water, in which the molecules form a layer of tightly bound water around the
protein molecule.
3. Hydrophobic Hydration: Hydrophobic groups that are exposed to the aqueous phase tend to cause an
increase in the order of the water molecules near them. The nature of the water bound near these
hydrophobic groups is not well defined, but it
does                                                                                                                       not behave as normal bulk
water.
4. Unfreezable: All of the above types of water interact strongly with the protein molecule and exhibit
properties that are different from those of free water. One of the most notable characteristics of bound
water is that it does not freeze at normal temperatures and is thus termed unfreezable water.   This is also
the type of water that can be predicted from a knowledge of the amino acid composition of proteins as
previously mentioned.
5. Capillary water: Capillary water refers to water that is associated with proteins, but that freezes at
normal temperatures and is free to act as a solvent for small molecules. This water behaves very much
like bulk phase water, but is very difficult to remove from the protein mass. The amount of this type of
water associated with a protein is dependent upon the nature of the measurement being utilized. Some of
this water is entrained in a three-dimensional network of protein molecules. The incorporation of large
amounts of water into a protein structure can lead to the formation of a gel.
7.3.3.Water-Holding Capacity (WHC)

The ability of many foods to retain water is affected by the involvement of proteins in different
structures. In meat and fish tissues, the state of water depends on interactions of water
structures with proteins and other solutes. Furthermore, because of the fibrous nature and
compartmentalization of the muscle, water is also held in the meat by physical entrapment.
Alterations in the spatial arrangement of the proteins and in the integrity of tissue structures
caused by biochemical and processing factors are responsible for shrinking or swelling of the
material, and thus for retention or exudation of water.

WHC has a large impact on the texture and juiciness of meat and fish products. A decrease in
WHC brings about excessive cooking loss and thawing drip. Changes in WHC may also be
used for evaluating the effect of processing on the structure of proteins and on the quality of
muscle foods.

7.3.4. Foam formation and foam stabilization by proteins

Proteins function as foam forming and foam stabilizing components, for example in baked
goods, sweets, desert and beer. Foams are dispersions of gases in liquids. Proteins stabilize by

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forming feasible, cohesive foam around the gas bubbles. During impact, the protein is adsorbed
at the surface via hydrophobic areas. There is partial infolding (surface denaturation). The
reduction of surface tension caused by protein adsorption facilitates the formation of new
interfaces and further gas bubbles. The partially unfolded proteins associate while forming
stabilizing film.

The ideal foam forming and foam stabilizing protein is characterized by a low molecular weight,
high surface hydrophobicity good solubility, a small net charge is terms of the pH of the food,
and easy denaturability. Foams are destroyed by liquid and organic solvents such as higher
alcohols which due to their hydrophobicity displace proteins from the gas bubbles surface. A
very low concentration of egg yolk prevents the bursting of egg white. This is attributed to the
disturbance of protein associates by the lecithin.

The foam forming and foam stabilizing characteristics of protein can be improved by chemical
and physical modifications. A partial enzymatic hydrolysis leads to smaller, more quickly
diffusing molecules, better solubility and the release of hydrophobic groups.

7.3.5. Viscosity

Viscosity in food is often considered with flow properties. Ideal solutions maintain a constant
viscosity coefficient independent of shear stress or shear rate. Viscosity can be related to the
volume occupied by solute compared to the volume occupied by solvent. As a protein occupies
more volume in solution it has a greater chance of interacting with other molecules and for there
to be a resistance to flow.

When most protein solutions are exposed to increasing shear the viscosity coefficient
decreases. This behavior is called shear thinning and solutions that exhibit this behavior are
called pseudoplastic. As shear is applied interactions between protein molecules can be
weakened. If this results in the breaking of a network, shear thinning will occur and the apparent
viscosity will decrease with increased shear. This leads to an alignment of molecules in the
direction of shear which decreases resistance to flow. When the shear is removed it is possible
for interactions to occur again. If these interactions lead to an increase in apparent viscosity, the
solution is said to be thixotropic. A number of protein solutions exhibit pseudoplastic and
thixotropic behaviors.

Factors that can cause proteins to unfold can increase their viscosity. Factors having an
important effect on viscosity include: pH, temperature, concentration, ionic strength and gelation
on protein denaturation, extremes of pH can cause proteins to unfold and to increase their water
binding. As the compact protein unravels its effective diameter increases and it occupies a
larger percentage of the available volume.

Interactions are more probable and viscosity increases. Near the isoelectric point proteins can
form aggregates more easily than when they are highly charged. If these aggregates are poorly
hydrated or precipitate, the volume of the protein in solution will decrease and viscosity will be
low.

 If the aggregates are highly hydrated and remain is suspension the viscosity may increase.
Heating proteins can cause them to unfold. If this unfolding leads to increased interactions the
viscosity will increase.

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7.3.6. Gel formation

A gel is a continuous network of macroscopic dimensions immersed in a liquid medium

exhibiting no steady-state flow. Protein gels can be formed by the addition of salts, the action of
enzymes, changes in pH or by the application of heat.

Process of gelation : The process of gelation was a two-stage one involving an initial
denaturation or unfolding of a protein molecule followed by subsequent aggregation. The steps
involved in thermal gelation are protein unfolding, water binding, protein-protein interactions and
water immobilization when a protein is heated, the bonds that maintain its secondary and
tertiary structures are weakened and, at some temperature, broken. This breaking of non-
covalent bonds with its resulting alteration of protein structure is termed denaturation. In the
early stages of thermal denaturation, most protein molecules begin to unfold. This unfolding
often leads to a slight increase in the amount of water tightly bound to the protein. If protein-
protein interactions lead to the formation of a three-dimensional network capable of entraining
water molecules, a gel can form.

The application of heat will weaken these bonds and allow water to interact with the charged
groups. At some temperature, the attractive forces will have been weakened enough to allow for
extensive water-ion interactions. This causes an unfolding of the molecule and an increase in
water binding.

If a network can be imagined to extend into three-dimensions, it could be envisaged the

existence of "pockets" of bulk phase water that would be associated with the network. This
water would freeze at normal temperatures and would be freely available to participate in
chemical reactions.

The interactions could involve calcium or other ionic bridges, hydrophobic interactions, disulfide
bonds or others. As long as there are enough of them to form a network and there are enough
repulsive forces to prevent the network's collapse, a gel can be formed.

7.3.6.1. Factors that have an effect on the formation and properties

of protein gels

Many factors have been reported to have an effect on the formation and properties of protein
gels. Some of the more important of these are temperature, protein concentration, pH, salt
concentration, calcium concentration and free sulfhydryl concentration.

i)Temperature: Temperature can affect the formation of protein gels in a variety of way. Heat
can affect both the rate of denaturation and the rate of protein-protein interaction. A temperature
must be selected for gel formation that balances the rate of protein unfolding with that of
aggregation. The majority of the protein-protein interactions occur as the system is cooled.

ii)Protein concentration: The protein concentration determines both the likelihood of gel

formation and also the characteristics of the gel that forms. Below a certain level that varies
according to the protein utilized, gelation will not occur. When the level of protein is too low, a
protein net-work is difficult to establish. Protein-protein interactions then tend to occur within
molecules rather than between molecules and a gel framework cannot be established. As the
protein content in-creases, the likelihood of intermolecular crosslinks increases and at some

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concentration, gelation can occur. Further increases in protein content change the strength and
texture of the gel resulting in firmer gels as more water is tightly bond to protein molecules.

iii)pH: pH can have a marked affect on the structure of proteins and the amount of water that
can be bound to proteins. pH can also affect the extent of protein-protein interaction if they are
of an ionic nature. In other cases, maintenance of proper pH can prevent the collapse of a gel
network due to charge repulsion. The denaturation of proteins is highly pH dependent and so
the rate of protein unfolding can be influenced by the pH of heating. Proper pH adjustment may
be necessary to achieve the proper balance between the rate of denaturation and aggregation
as well as the forces of attraction and repulsion between adjacent protein chains that are
necessary to achieve a protein gel.

iv) Salts: Salts in general can affect the structure of protein molecules as well as the nature of
protein-water interactions. These affects markedly influence both the solubility of protein and
their rate of thermal denaturation. As with heating and pH there is generally an optimal level of
salt that favors gel formation.

v) Sulfhydryl groups: There is an optimal sulfhydryl concentration for gel strength. At low pH

values, the gels are rather soft and opaque and can be described as coagula. At higher pH
values the gels become more elastic, transparent and have greater gel strengths.

7.3.7. Emulsification

Emulsions are thermodynamically unstable mixtures of immiscible liquids. If energy is applied

the systems may be dispersed, but increased surface energy causes the phases to coalesce
unless an energy barrier to coalescence is established.

Emulsified droplets can be stabilized by the addition of molecules that are partially soluble in
both phases. In foods a number of small emulsifier molecules can serve this function.

Proteins capable of unfolding at the interface may also serve this function. Protein coats the lipid
droplet and provides an energy barrier to particle association and phase separation. In native
proteins most of the nonpolar amino acid side chains are located in the interior of the molecules.

Proteins have charged groups at the surface of the molecule and in contact with water
molecules. The favorable interaction of water with surface charge lowers the total energy of the
protein molecule. In some respects the protein may be envisioned as resembling the micelles of
phospholipids in the previous example. The hydrophobic groups are removed from contact with
the aqueous phase while charged groups maximize solvent contacts.

Once a protein molecule reaches the surface it must be able to unfold enough to expose
hydrophobic groups if it is to function as an emulsifier. 

 Molecule that contain crosslink such as disulfide bonds are more rigid and less able to unfold.
Such molecules are less effective in emulsion formation

Unit 8: Metabolism of Protein

Chapter 1: Digestion and Absorption of proteins

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 Metabolism of proteins

Proteins are enzymatically digested to prepare them for absorption. During digestion in the
gastrointestinal tract of mammals, proteins undergo enzymatic hydrolysis into their building
block components viz amino acids. This is necessary for their absorption, since the cells lining
the intestine are able to absorb them into the bloodstream only as relatively small
molecules.The amino acids are then metabolised.

8.1 Digestion and absorption of proteins

Ingested proteins are enzymatically hydrolyzed into amino acids in the gastrointestinal tract.
When protein enters the stomach, it stimulates the secretion of hydrochloric acid by the parietal
cells of the gastric glands and pepsinogen by the chief cells.

Action of HCl

Due to the secreted HCl the gastric juice has a pH between 1.5 and 2.5. The acidity of gastric
juice acts as an antiseptic and kills most bacteria and other cells. In addition, it causes globular
proteins to undergo denaturation or unfolding at this low pH, rendering their internal peptide
bonds more accessible to enzymatic hydrolysis.

Action of pepsin 

Pepsinogen, an interactive precursor or zymogen, is converted into active pepsin in the gastric
juice by the enzymatic action of pepsin itself. In this process, 42 amino acid residues as a
mixture of small peptides is cutoff. The rest of the pepsinogen molecule, which remains intact, is
the enzymatically active pepsin.

In the stomach, pepsin hydrolyzes the peptide bonds of ingested proteins involving the aromatic
amino acids tyrosine, phenylalanine, and tryptophan, thus cleaving long polypeptide chain into a
mixture of smaller peptides.

Action of Hormones

Secretin: As the acid stomach contents pass into the small intestine, the low pH triggers the
secretion of the hormone, secretin into the blood. Secretin stimulates the pancreas to secrete
bicarbonate into the small intestine to neutralize the gastric HCl. The pH then rises abruptly from
between pH 1.5 to 2.5 to about pH 7. In the small intestine the digestion of proteins continues.

Cholecystokinin: The entry of amino acids into the duodenum releases the hormone
cholecystokinin, which stimulates secretion of several pancreatic enzymes, whose optimum pHs
are near 7. Three of these, trypsin, chymotrypsin, and carboxypeptidase, are made by the
exocrine cells of the pancreas as their respective enzymatically inactive zymogens, trypsinogen,
chymotrypsinogen and procarboxy peptidase, synthesis of these enzymes as interactive pre
cursors protects the exocrine cells from destructive proteolytic attack.

Action of trypsin:

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After trypsinogen enters the small intestine, it is converted into its active form trypsin by
enterokinase, a specialized proteolytic enzyme secreted by intestinal cells. Once some free
trypsin has been formed, it also can catalyze the conservation of trypsinogen into trypsin. The
formation of free trypsin is brought about by removal of a hexa peptide from the amino-terminal
end of the trypsinogen chain. Trypsin hydrolyzes those peptide bonds whose carbonyl groups
are contributed by lysine and arginine residue.

Action of chymotrypsin

Chymotrypsinogen has a single polypeptide chain with a number of intra chain disulfide bonds.
When it reached the small intestine, it is converted into chymotrypsin by trypsin, which cleaves
the single long polypeptide chain of chymotrypsinogen at two points by excision of dipeptides.
The three segments formed from the original chymotrypsinogen chain are still held together,
however, by disulfide cross-linkages. Chymotrypsin hydrolyzed those peptide bonds involving
phenylalanine, tyrosine, and tryptophan residues. Trypsin and chymotrypsin thus hydrolyze the
polypeptides resulting from the action of pepsin in the stomach into smaller peptides.

Action of peptidases

Degradation of the short peptides in the small intestine is now completed by other peptidases.
The first is carboxypeptidase, a zinc containing enzyme, which the pancreas make as its
inactive zymogen, procarboxypeptides.

Carboxypeptidase removes successive carboxyl-terminal residues from peptides.

The small intestine also secrets an amino peptidase, which can hydrolyze off successive amino-
terminal residues of peptides. ingested proteins are ultimately hydrolyzed to yield a mixture of
free amino acids, which are then transported across the epithelial cells lining the small intestine.
The free amino acids enter the blood capillaries in the villi and are transported to the liver.

Enzymes involved in protein digestion and their peptide bond specificity

Table 8.1.1

Pepsin Tyr, Phe, Trp; Leu, Glu, Gln

Trypsin Lys , Arg
Chymotrypsin Tyr, phe, Trp
Carboxypeptidas Successive carboxy terminal
e residues
Aminopeptidase Successive amino terminal
resides(except proline)

Absorption

Ingested proteins are ultimately hydrolyzed to yield a mixture of free amino acids, which are
then transported across the epithelial cells lining the small intestine. The free amino acids enter
the blood capillaries in the villi and are transported to the liver.

Metabolism

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            The amino acids entering the liver, following absorption from the intestinal tract, also have several
important metabolic routes.

i). Transport to other tissues: Amino acids may pass into the systemic blood and thus to other organs, to
be used as building block in the biosynthesis of tissue proteins.

ii) Biosynthesis of liver proteins and plasma proteins: The liver constantly renews its own intrinsic
proteins, which have a very high turnover rate. The liver is also the site of biosynthesis of most of the
plasma proteins of the blood.

iii) Deamination and degradation: Amino acids which not needed for protein biosynthesis in the liver or
other tissues are deaminated and degraded to yield acetyl CoA and citric  acid  cycle intermediates. Citric
acid cycle inter mediates thus  formed may be converted into blood glucose  and glycogen  via the
glyconeogenesis pathway.

iv) Participation in the glucose – Alanine cycle: The liver also  participates in the metabolism of amino
acids arriving from other tissues. These amino acids are  deaminated and the resulting keto acids are
converted into blood glucose via  gluconeogenesis. The NH3 is converted into urea for excretion.

v) Conversion into Nucleotides and other Products:  Amino acids are precursors in the biosynthesis of
the purine and pyrimidine bases of the nucleotides and in the synthesis of specialized products such as
porphyrins, hormones and other nitrogenous compounds.
Dynamic state of protein or Constant turnover of proteins

            Proteins are in a constant state of degradation and resynthesis. All body proteins continuously
undergo degradation and synthesis. More than half of the protein in liver and intestinal mucosa are broken
down and resynthesised in 10days. Active resynthesis occurs even in starvation and active breakdown in
nitrogen equilibrium. Degradation of protein in one tissue is accompanied by systhesis in others. When
amino acids are oxidized, their nitrogen atoms are fed into the urea cycle in the liver and excreted as urea
in the urine, sweat and feces. Other nitrogenous excretory products include uric acid, creatinine and
ammonia.

The continuous turnover of protein in the adult is probably necessary for maintaining an amino acid pool,
and the capability to meet the demand for amino acids by various cells and tissues when they are
stimulated to make necessary proteins. The most active tissues for protein turnover are the
plasma proteins, intestinal mucosa, pancreas, liver, and kidney whereas the muscle, skin, and brain are
much less active

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Amino acid pool

There is no large reserve of free amino acids in the body, and any amount above that needed for systhesis
of tissue protein and the various nonproteinnitrogen-containing compounds is metabolized.  However, in
the cellular proteins themselves a metabolic pool of amino acids exists in a state of dynamic equilibrium
that may be called on at any given time to meet an appropriate need.  

Nitrogen balance

Since protein is the main source of nitrogen in body the dietary protein must make up nitrogen
lost from body to maintain nitrogen balance. Nitrogen balance is the difference in the amount of nitrogen
consumed and the amount of nitrogen excreted in the urine, sweat and feces. If an individual’s total
nitrogen content of the urine and feces equals the amount of dietary nitrogen then the individual is said to
be in nitrogen balance or equilibrium
          Fecal nitrogen (N) + Urinary nitrogen (N) = Dietary nitrogen (N)
          N output = N intake
Healthy adults are normally in zero nitrogen balance

Positive nitrogen balance

If the ratio of N intake to N output is greater than one then it is called as positive nitrogen balance or if the
N output is less than N intake then the individual is in positive nitrogen balance. In the positive nitrogen
balance most of dietary nitrogen is retained in the body and  less is eliminated from body. More over in
positive nitrogen balance the tissue protein content increases due to increased protein synthesis. Usually it
occurs during growth, pregnancy, lactation and post operative recovery.

Negative nitrogen balance

If the nitrogen output is more than the N intake then the individual is in negative nitrogen balance
or if the ratio of N intake to N output is less than one then the individual is in negative nitrogen balance.
In the negative nitrogen balance nitrogen lost is not replaced by dietary nitrogen. It occurs in malnutrition
and other wasting diseases where there is tissue breakdown like starvation, uncontrolled diabetes mellitus
and cancer. Menstruating women may have transient negative nitrogen balance if proper replacement for
nitrogen lost is not possible. Severe physical exercise may alsolead to transient negative nitrogen balance
because of atrophy of muscle. The nitrogen balance of the body depends on the following factors such as
the physiological state of the individual, adequacy of energy intake from carbohydrates and fats and the
capacity to adjust to levels of intake.
Protein Requirement
It is the minimum amount of dietary protein required. The minimum nitrogen intake that will allow the
state of nitrogen equilibrium is considered minimum nitrogen (protein) requirement. Average food
proteins have 16% nitrogen. To estimate the protein content of foods, the nitrogen content of food will be
first estimated and the nitrogen values will be multiplied by 6.25, the protein conversion factor. However
protein requirement also depends on the (a) Protein quality (b) Carbohydrate and fat contents (c) Physical
activity. 
8. Quality of protein

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Essential amino acid content determines quality of a protein. An ideal or a good quality protein is the one
which has amino acid composition of body protein synthesized at any given time. Further an ideal protein
must meet essential amino acid requirement. Unfortunately ideal proteins or good quality proteins are
limited.

Chapter 2: Amino acid catabolism and Urea Synthesis

Amino acids can undergo degradation in three different circumstances.

1. During the normal dynamic turn over of body proteins the amino acids released, if not needed for
synthesis of new body proteins, may undergo oxidative degradation.
2. When amino acids are ingested in excess of the body’s needs for protein synthesis, the surplus
may be catabolised, since amino acids cannot be stored.
3. During fasting or in diabetes mellitus, when cafbohydrates are either unavailable or not properly
utilized, body proteins are used as fuel. During this amino acids undergo oxidation to carbon
dioxide and water, in part via citric acid cycle.
8.2.2.Metabolic fate of Amino acid

Intracellular proteases hydrolyze internal peptide bonds of protein, releasing peptides, which are
then degraded to free amino acids by peptidases. Endopeptidases cleave internal bonds,
forming shorter peptides. Aminopeptidases and carboxypeptidases remove amino acids
sequentially from the amino acids. Extra cellular, membrane-associated, and long-lived
intracellular proteins are degraded in cellular organelles termed lysosomes by ATP-
independent processes. By contrast, degradation of abnormal and other occurs in the cystol.

Animals excrete nitrogen from amino acids and other sources as one of three end
products: ammonia, uric acid, or urea. Teleostean fish, which are ammonotelic, excrete nitrogen
as ammonia. Land animals convert nitrogen either to uric acid (uricotelic organisms) or to urea
(ureotelic organisms) and excreted in urine.

Free amino acids released from dietary or intracellular proteins are metabolized in identical
ways. Following removal of the α-amino nitrogen by transamination, the resulting carbon
“Skeleton” is then degraded

There are four stages in the metabolism of amino acids are (1) transamination, (2) deamination
(3) ammonia formation and transport, and (4) synthesis of urea by urea cycle.
8.2.2.1. Transamination by transaminases
Transamination interconverts a pair of amino acids and a pair of keto acids, generally and α
amino acid and a β -keto acid. Most amino acids undergo transamination. Exceptions are lysine,
threonine, proline and hydroxy proline. 

Since transaminations are freely reversible, transaminases or aminotransferases can function

both in amino acid catabolism and biosynthesis. Pyridoxal phosphate resides at the catalytic site
of all transaminases. Two transaminases viz Alanine-pyruvate transaminase and glutamate a
-ketoglutarate transaminase present in mammaliantissues catalyse the transfer of amino group
from most amino acids.
8.2.2.2.Oxidative Deamination

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The α -amino groups of most amino acids ultimately are transferred to α -ketoglutarate by
transamination, forming L-glutamate. Release of this nitrogen as ammonia is then catalyzed by
L- glutamate dehydrogenase that uses either NAD + or NADP + as oxidant.

L- glutamate dehydrogenase

Glutamate + NAD(P)+ ↔ α -ketoglutamate + NAD (P) H + H+ + HN3

8.2.2.3. Ammonia formation and Transport

1. Ammonia Transport by L-amino acid oxidases

L-amino acid oxidase is present in liver and kidney tissue. These autoxidizable flavoproteins
oxidize amino acids to an α-imino acid that adds water and decomposes to the corresponding α-
keto acid with release of ammonium ion. The reduced flavin is reoxidized directly by molecular
oxygen, forming hydrogen peroxide (H 2 O 2 ), which is split to O 2 and H 2 O by the enzyme
catalase present in many tissues, especially liver.

2. Formation of ammonia and transport by Glutamine synthetase 

While ammonia is constantly produced in the tissues, it is rapidly removed from the circulation
by the liver and converted to glutamate, glutamine, and ultimately to urea. Formation of
glutamine is catalyzed by glutamine synthetase. Synthesis of the amide bond of glutamine is
accomplished at the expense of hydrolysis of one equivalent of ATP to ADP and Pi.

3.Action of Glutaminase and asparaginase Hydrolytic release of the amide nitrogen of

glutamine as ammonia, catalyzed by glutaminase, strongly favours glutamate formation.
Glutamine synthetase and glutaminase thus catalyze inter conversion of free ammonium ion
and glutamine. An analogous reaction is catalyzed by L-asparaginase. These enzymes occur in
liver, kidney and gills and also in red and white muscles.

Synthesis of Urea by urea cycle (Ornithine Cycle or Krebs – Henseleit Cycle)

            Urea is the major end product of nitrogen catabolism. Urea is formed from ammonia, carbon
dioxide, and aspartate, synthesis of one mol each of ammonium ion and the a -amino nitrogen of
aspartate. The synthesis of urea has several steps.

(i) Synthesis of carbamoyl phosphate:

            The biosynthesis of urea begins with the condensation of carbon dioxide, ammonia, and ATP to
form carbamoyl phosphate, a reaction catalyzed by carbamoy1 phosphate synthase. Formation of
carbamoy1 phosphate requires 2 mol of ATP.(Fig8.2.2).One ATP serves as a source of phosphate.
Conversion of the second ATP to AMP and pyrophosphate, together with the coupled hydrolysis of
pyrophosphate to orthophosphate, provides the driving force for synthesis of the amide bond and the
mixed acid anhydride bond of carbamoy1 phosphate.
(ii) Synthesis of citrulline

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            In the next step, carbamoy1 phosphate donates its carbamoy1 to ornithine to form citrulline and
release phosphate in a reaction catalyzed by ornithine transcarbamoylase, a Mg2+ requiring enzyme. The
citrulline thus formed leaves the mitochondria and passes into the cytosol of the liver cells.

(iii). Synthesis of Arginosuccinate

            The second amino group is introduced in the form of L asparate, which in turn acquired it form L.
glutamate by the action of aspartate  transaminase. The transfer of the second amino group to citrulline
occurs by a condensation reaction between the amino group of aspartate and the carbamoy1 carbon
citrulline in the presence of ATP to form arginosuccinate, catalyzed by the enzyme arginosuccinate
synthetase.

(iv) Action of  arginosuccinate lyase

            In the next step, arginosuccinate is reversibly cleaved by arginosuccinase
to form free arginate and fumarate. The fumarate so formed returns to the pool of
citric acid cycle intermediates.

(v) Action of arginase
 In the last reaction arginase cleaves arginate to yield urea and ornithine.
Ornithine thus generated enters the mitochondria again to initiate another round of
urea cycle. The overall equation of the urea cycle is

2NH4+  + HCO3- + 3ATP4- + H2O     →    Urea + 2ATP3- + 2Pi- + AMP- + PPi3- +

H+

            The urea cycle brings together two amino and HCO3- to form a molecule of
urea which diffuses from the liver cells into the blood thence to be excreted into the
urine by the kidneys.

Thus the toxic ammonia is converted into harmless urea in ureotelic animals.
Two molecules of ATP are required to make carbamoy1 phosphate and one is
required to make arginosuccinate in which one molecule of ATP undergoes a

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pyrophosphate cleavage to AMP and pyrophophosphate, which may be hydrolyzed
to yield two orthophosphates.

 The ultimate cost of one molecule of urea is four ATP molecules. The daily
output of urea through urine is 20-30g. A less quantity is excreted through sweat.
The excretion of urea is proportional to the total protein metabolism.

Chapter 3: Protein synthesis

During protein synthesis the mRNA nucleotide sequence is translated into the sequence of amino acids
of the specified protein. The translation starts near the 5' terminal with the formation of the
corresponding amino terminal of the protein molecule. In eukaryotic organisms, the mRNA translation
occurs in the cytoplasm. Protein synthesis can be described in three phases; initiation, elongation, and
termination.

8.3.1.1. Initiation

Initiation of protein synthesis requires that an mRNA molecule be selected for translation by a
ribosome. Once the mRNA binds to the ribosome, the latter finds the correct reading frame on
the mRNA, and translation begins. This process involves tRNA, rRNA, mRNA, and at least 10
eukaryotic initiation factors (eIFs), some of which have multiple (three to eight) subunits. Also
involved are GTP, ATP, and amino acids. Initiation can be divided into four steps;

(1) Dissociation of the ribosome into its 40S and 60S subunits

(2) Binding of a ternary complex consisting of met-tRNA, GTP, and eIF-2 to the 40S ribosome to
form a preinitiation complex

(3) Binding of mRNA to the 40S preinitiation complex

(4) Combination of the 40S initiation complex with the 60S ribosomal subunit to form the 80S
initiation complex

1) Dissociation of the ribosome

Two initiation factors, eIF-3 and eIF-IA, bind to the 40S subunit. This favors dissociation of the
80S ribosome into its 40S and 60S subunits and prevents ressociation.

2)Formation of the 40S preinitiation complex

Formation of the 40S preinitiation complex involves the binding of GTP by eIF-2. This binary
complex then binds to met-tRNA, a tRNA specifically involved in binding to the initiation codon
AUG. This ternary complex binds to the 40S ribosomal subunit to form the 40S preinitiation
complex, which is stabilized by as sociation with eIF-3 and eLF-1A.

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3) Formation of the 40S initiation complex

The 5' terminals of most mRNA molecules in eukaryotic cells are “capped,”. This methyl-
guanosyl triphosphate cap facilitates the binding of mRNA to the 40S preinitiation complex. A
cap binding protein complex, eIF-4F, binds to the cap through one of its subunits. Then eIF-4F
and eIf-4B bind and probably reduce the complex secondary structure of the 5'end of the 
mRNA through their respective ATP ase and helicase activities.

4) Formation of the 80S initiation complex

The binding of the 60S ribosomal subunit to the 40S initiation complex involves the hydrolysis of
the GTP bound to eIF-2 by eIF-5. This reaction results in the release of the initiation factors
bond to the 40S initiation complex. These factors are then recycled and lead to the rapid
association of the 40S and 60S subunits to from the 80S ribosome. At this point, the met-tRNA
is on the p site of the ribosome, ready for the elongation cycle to commerce.

8.3.1.2 Elongation

Elongation, a cyclic process, involves several steps catalyzed by proteins called elongation
factors (eEF). These steps are (1) binding of aminoacyl - tRNA to the A site, (2) peptide bond
formation, and (3) translocation.

In the complete 80S ribosome formed during the process of initiation, the A site is free. The
binding of the proper aminoacyl-tRNA in the A site requires proper codon recognition Elongation
factor eEF-1a forms a complex with GTP and the entering aminoacyl-tRNA. This complex then
allows the aminoacyl-tRNA to enter the A site with the release of eEF-1a-GDP and phosphate.

Peptide Bond Formation: The a-amino group of the new aminoacyl-tRNA in the A site caries out
a nucleophileic attack on the esterfied carboxyl group of the peptidyl-tRNA occupying the P site.
This reaction is catalyszed by a protein component, peptidyltransferase, of the 60S ribosomal
sub-unit. This enzymatic activity may be performed by some RNA component o the ribosome-
perhaps the 23S rRNA.

Translocation: Upon removal of the peptidyl moiety from the tRNA in the P site, the discharged
tRNA quickly dissociates from the P site. Elongation factor 2(3EF-2) and GTP are responsible
for the translocation of the newly formed peptidyl tRNA at the A site into the empty P site, The
GTP required for eEF-2 is hydrolyzed to GDP and phosphate during the translocation process.
The translocation of the newly formed peptidyl-tRNA and its corresponding codon into the P site
then frees the A site for another cycle of aminoacyl-tRNA codon recognition and elongation.

8.3.1.3. Termination

In comparison to initiation and elongation, termination is a relatively simple process. After

multiple cycles of elongation culminating in polymerization of the specific amino acids into a
protein molecule, the nonsence or terminating codon of mRNA (UUA, UAG, UGA) appears in

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the a site. Normally, there is no tRNA with an initiation codon capable of recognizing such a
termination signal. Releasing factors (eRF) are capable of recognizing that a termination single
resides in the A site. The releasing factor, in conjunction with GTP and the peptidyl transferase,
promotes the hydrolysis of the bond between the peptide and the tRNA occupying the P Site. 

Thus, a water molecule, rather than an amino acid, is added. This hydrolysis releases the
protein and the tRNA from the P site. Upon hydrolysis and release, the 80S ribosome
dissociates into its 40S and 60S subunits, which are then recycled. Therefore, the leasing
factors are proteins that hydrolyze the peptidyl-tRNA bond when a nonsense codon occupies
the A site. 

The mRNA is then released from the ribosome, which dissociates into its component 40S and
60S subunits, and another cycle can be repeated.
In animal cells, many proteins are synthesised from the mRNA template as a precursor
molecule which is then modified to form the required active protein.

Unit 9: Nutritive values of proteins

Chapter 1: Nutritive values of proteins

9.1.1. Introduction
Food proteins provide amino acids for degradation into carbon skeletons to synthesize glucose for energy
and amino acids for the synthesis of other proteins. The diet contains a variety of different animal and
plant proteins. The nutritional quality or biological value of a given protein depends upon two factors: (i)
its content of essential amino acids and (ii) its digestibility. On the basis of their nutritional quality the
proteins has been classified into (1) complete protein (2) partially complete and (3) incomplete protein.

1. Complete proteins: Complete proteins maintain life and provide for normal growth of the young when
used as the only protein in the diet of experimental animals. Proteins of meat, poultry, fish, eggs and milk
are complete proteins and are of high biological value because they contain all essential amino acids.
Gelatin is an exception. Some proteins in legumes, cereals and nuts are also complete proteins e.g.
glycinin of soybean, glutenin of wheat and glutelin of corn.

2. Partially complete:  Partially complete proteins maintain life but fail to support normal growth. e.g.
gliadin of wheat, hordein of barley.

3. Incomplete proteins: Incomplete proteins neither maintain life nor support growth. Most vegetable
proteins are incomplete and are of low biological value(e.g. rice, wheat, corn, beans).Proteins differ
considerably in the relative proportion of amino acids they contain and hence their biological value. Some
proteins contain a complete set of essential amino acids. There are 10 essential amino acids which are
need in food in take and the body cannot synthesis them at rates at which they are needed for growth. The
nonessential amino acids are those acids that can be synthesized in the body from a suitable carbon

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source, amino groups from other amino acids and consequently do not have to be supplied in a ready
made form diet.
Essential amino acids: The 10 essential amino acids are methionine, tryptophan, threonine, valine,
isoleucine, leucine, phenylalanine, lysine, arginine and histidine and are essential for growth in
children. Arginine and histidine are not essential for maintenance in adults. The remaining amino acids
are called non-essential or dispensable as they can be synthesized in animals from other compounds.
They are glycine, cysteine, alanine, serine, proline, tyrosine, aspartic acid, asparagine, glutamic
acid and glutamine. Cereals contain lower value of lysine while pulses contain lower value of
methionine. Combining plant proteins from different sources ensures a more complete complement of
essential amino acids. 
9.1.3. Digestibility
Digestibility is defined as the proportion of food nitrogen that is absorbed after ingestion. The nutritive
value of proteins depends upon its digestibility. Animal proteins are the most available for digestion
and  absorption and the proteins of legumes are the least available because of their incomplete
digestibility. Digestibility depends upon the fiber content present in the food item and also in the
remainder portion of diet. The average digestibility of some food proteins are presented in Table 9.1.

Table 9.1 Average Digestibility of some food proteins

                           Food group             Digestibility coefficient

             Animal foods                                97            
             Cereals                                          85
             Fruits                                             85
             Vegetables                                    83
             Legumes, dried                             78

Plant proteins, particularly those of wheat and other grains, are not completely hydrolyzed during
digestion because the protein-rich portions of the grains are surrounded by protective husks of cellulose
and other polysaccharides that are not hydrolyzed by intestinal enzymes. Since only free amino acids can
be absorbed from the intestine, not all the amino acids of most plant foods are biologically available.
Requirement of proteins and amino acids
When the intake and outgo of nitrogen are approximately same, the individual is said to be
in nitrogen equilibrium. The minimum nitrogen intake that will allow the state of nitrogen equilibrium is
considered minimum nitrogen (protein) requirement. Average food proteins have 16% nitrogen. To
estimate the protein content of foods, the nitrogen content of food will be first estimated and the nitrogen
values will be multiplied by 6.25, the protein conversion factor.

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The nitrogen balance of the body depends on the following factors such as the physiological state
of the individual, adequacy of energy intake from carbohydrates and fats and the capacity to adjust to
levels of intake.

Negative nitrogen equilibrium: When the intake and outgo of nitrogen are less, the individual is said to
be in Negative nitrogen equilibrium

Positive nitrogen equilibrium: When the intake and outgo of nitrogen are less, the individual is said to
be in Negative nitrogen equilibrium
RDA for proteins
For an adult (18-35) about 10-12% of total calories from proteins in diet are considered adequate.
The requirement for infancy, childhood, during pregnancy and lactation are more. More proteins are
needed under conditions of stress. Increased intake is needed after surgery or injury, wasting illness, or
while recovering from extensive burns.

For normal persons the minimum requirement of proteins intake is about 0.8g/Kg of body weight
for maintenance of nitrogen balance and the recommended daily intake is 1.0g/Kg of the body weight.

FAO reference protein

            The protein requirements were reported by a committee for FAO in 1957. For this a ‘reference
protein’ was used which was similar to proteins found in milk, egg and meat. A comparison of essential
amino acids of reference protein is presented in Table 2.

Table.9.1.5.1 Essential amino acids in reference protein and milk, egg proteins

Essential Amino acids per 100gm of protein

amino acid Reference Cow’s Human Egg
protein milk milk
Isoleucine 4.2 6.4 6.4 6.8
Leucine 4.8 9.9 8.9 9.0
Lysine 4.2 7.8 6.3 6.3
Phenylalanine 2.8 4.9 4.6 6.0
Methionine 2.2 2.4 2.2 3.1
Threonine 2.8 4.6 4.6 5.0

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Tryptophan 1.4 1.4 1.6 1.7
Valine 4.2 6.9 6.6 7.4
The FAO committee has also suggested that the ‘reference protein’ may also be used for
appraisal of the protein (amino acid) value of diets and for indicating appropriate methods of
supplementing diets deficient in protein (amino acids).

The minimum amounts of essential amino acids needed by adults are given in Table 3.

Table 9.1.5.2. Minimum amounts of essential amino acids needed by adults

Essential Women Men (g)

amino acid (g)
Isoleucine 0.45 0.70
Leucine 0.62 1.10
Lysine 0.50 0.80
Methionine 0.29 1.10
Phenylalanine 0.22 1.10
Threonine 0.31 0.50
Tryptophan 0.16 0.25
Valine 0.65 0.80
Food sources for the supply of proteins and amino acids.
Proteins are widely distributed in nature but only a few foods provide sufficiently higher quantity
of proteins. Eggs, fish, meats, nuts and poultry have protein ranging from 12-29%. Cereal contributes 1-
2% of proteins. Fruits and vegetables are low sources of protein foods. Syrups, oils and fat contain no
protein. Animal foods have high protein values and also have good quality. Soya bean and some nuts
contain protein of high quality.

4. The supplementary value of protein

Foods differ in the nutritive value of the protein they contain. Each food, with the exception of
gelatin, contains more than one kind of protein, each with a characteristic number and concentration of
amino acids. The proteins of some foods contain limited amount of certain essential amino acids,
generous amounts of others. Therefore a combination of foods in which the amino acid contained in the
proteins supplement each other is of higher nutritive quality than either alone. There are possibilities of
combining foods so that amino acids may supplement each other. For e.g. Lysine is a critical or limiting
amino acid of cereals. Similarly methionine is limited in pulses and is the critical or limiting amino
acid of their protein. They need to be supplemented by animal proteins.
Deficiency of Protein:
            Protein- energy malnutrition results from inadequate intake of protein and/or calories. Two forms
of child malnutrition, often occurring together, are Marasmus and Kwashiorkor.

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a. Marasmus: Marasmus (from Greek, “to waste”) is the term applied to chronic deficiency of calories in
children. Marasmus is caused by a diet deficient in both protein and calories. (Total calorie
deprivation). Marasmus occurs in famine areas when Infants are weaned from breast milk and given
inadequate bottle feedings of these watery gruels of native cereals or other plant foods, usually deficient
in both calories and protein. Marasmus is characterized by arrested growth, extreme muscle wasting,
weakness and anemia. It is usually complicated by multiple deficiencies of vitamins and minerals. Calorie
deficiency in early childhood, even if ultimately alleviated with an ample diet, leaves a permanent deficit
in body growth.

b. Kwashiorkor: Chronic protein deficiency in children is called “Kwashiorkor”, an African word that

means ‘weaning disease”. Kwashiorkor is caused by a diet inadequate in protein in the presence of
an  adequate calorie intake consisting primarily of carbohydrates. Since the protein content of plant foods
is generally low and proteins are also low in quality, a serious shortage of “good” protein often occurs in
areas where population growth is especially great.

 The growth of protein-deficient children is retarded, they become anemic, and the tissues become
watery and bloated because of the low serum protein levels, which upset the normal distribution of water
between tissues and the blood. Moreover, the liver, Kidneys, and pancreas undergo severe degeneration.
There are defects in cellular immunity. The mortality rate of kwashiorkor is very high. There is a
permanent physiological deficit. Protein deprivation in early childhood also leaves a deficit in learning
and mental ability.

Chapter 2. Methods for the determination of nutritional quality of proteins

Methods for the determination of nutritional quality of proteins

 The nutritional quality of proteins can be determines in the following methods
1. Protein Efficiency Ratio [PER]: This index is not based on nitrogen balances studies. It is, therefore,
less precise than BV / NPU, but it is technically easier to derive and use.
PER is defined as the change in body weight relative to the amount of protein eaten. It is usually
measured in laboratory rats kept under standardized diet conditions.
                  Weight gain in grams
PER =                                               x 100
    Dietary protein in grams
For example, if a rat is given a standard diet nutritionally adequate in all respects containing 2 g of casein
per day as the only source of  protein and his weight gain is found to be 5 g per day, then the PER of case
in would be 5/2 = 2.5. Whole egg has a PER of 3.8, while gelatin has 0 PER.
2. Net Protein Utilization [NPU]: NPU is an index that takes into account the relative digestibility of
proteins. Because even the best mixture of amino acids will be less available for use in the body it is
packaged in a protein that is only partially digested. Therefore,
NPU = BV x digestibility.
           
               N retained             Dietary N - [Urinary N + Faecal N]

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NPU =                     x 100                                                            x  100

                 Dietary N                             Dietary N  
Proteins are generally easy to digest. Most proteins are 90% or more digestible. Thus is most cases, NPU
approximates the BV.

3. Chemical Score: The protein is completely hydrolyzed and its amino acid composition measured   and
compared with that of egg or milk protein as a standard. This chemical score of a protein indicates the
potential value of the protein.
                                     mg of amino acid per g of test protein
Chemical Score    =    100x
                                               mg of amino acid per g of reference protein
             Animal proteins, for example, those of milk, fish, beefsteak, and eggs rank high (>80) in both
chemical score and biological value. At the other end of the scale, the proteins of rice or wheat have a low
chemical score (75) because they are deficient in one or more essential amino acids. However, they also
have lower biological value, since they are incompletely digested. Thus a much larger amount of these
plant proteins must be consumed to provide the minimum daily requirement of all the amino acids. All
the essential amino acids must be available simultaneously for body protein synthesis.
4. Digestibility Coefficient
Nutrients present in the food are not completely available to the human body. Large portions of the
nutrients are excreted in the faeces because of being not digested in the alimentary tract. Hence, the
digestibility of the food is defined as the portion of a food or nutrient which is not recovered in faeces, the
portion which has been absorbed by the animal. Calculation of the digestibility of a whole food is of no
use hence it is calculated for each nutrient present in it.
Digestibility Coefficient (DC) =   Nutrients eaten (g) – Nutrient in feces x 100
                                                  ---------------------------------------------
                                                            Nutrient eaten (g)
.The digestibility coefficients normally determined are the apparent digestibility coefficients since the
nutrients found in the faeces contain small proportion of nutrients from the previously utilized food in the
form of mucosal debris, unspent enzymes ate.
5. Pepsin digestibility
Pepsin digestibility assesses the in vitro  digestibility of the protein in fluids containing the proteolytic
enzyme pepsin and the release of amino acids are measured. In one method, test proteins are first digested
with pepsin and then with pancreatin. In another method proteins are digested with three enzymes namely
pancreatic trypsin, chymotrypsin and porcine intestinal peptidase under standard assay conditions.
Unit 10: Fish Muscle Proteins

Chapter 1: Fish MuscleProteins, Chemical changes in muscle during contraction

10.1.1.Introduction

Fish meat can be compared with other animal foods such as livestock meat, eggs and milk.
Marine products have conditions which differ greatly from those of livestock meat as far as
harvesting, storing and processing are concerned.

Fish Meat Composition

            The edible portion of fish is roughly 45-50% of the whole fish body weight but varies according to
shape, age and whether caught before or after spawning. With elliptically shaped fish such as skipjack
and salmon, it is over 60% and with large headed or bellied fish like cod and Pollack and of flat shaped
fish like sole it is 35-40%. A comparison of the proximate composition of fish meat and live stock meat

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are presented in table 10.1. A comparison of proximate composition of meat from various sources are
given in table 10.2
Table10.1.1 Proximate composition of fish meat and livestock meat
Type of Moisture Protein Lipid Carbohydrate Ash
meat % % % % %
Fish meat 66-84 15-24 0.1-22 1-3 0.8-2
Livestock 65-80 16-22 1.5-13 0.5-13 1%
meat
           
Table10.1.2
Type of Water Protein Lipid Ash Proximate
meat composition
  of meat
from
Red meat various
Beef 70.62 20.78 6.16 1.02 sources ( %
Pork 72.34 21.07 5.88 1.04 of edible
Lamb 73.42 20.29 5.25 1.06 portion)
Poultry
Chicken 74.76 23.09 1.24 1.02
Turkey 74.12 24.60 0.65 1.02
Fish
Cod 81.22 17.81 0.67 1.16
Tuna 68.09 23.33 4.09 1.18
Poultry meat contains smaller amounts of lipid. The amount of crude fat in fish meat varies
according to the species, age, part of body, pre-or post spawning season and the food condition.
Contradictory correlations are noticed between crude fat and water content in the same fish species.
During the season in which the fat content is low, the water content is high. Similarly during the season in
which fat content is high,   the water content is low. Of the same fish species caught in the same season
and fishing ground, bigger fish has higher amount of fat than smaller once. Great amount of highly
unsaturated fatty acids are present in the fish meat.
            There is marked similarity between the amino acid composition of muscle protein of fish and
livestock meat. In many of the unutilized deep sea fishes, the crude fat content is mostly low and the
water content high. Reports state that amino acid compositions of low value fishes of white meats are
similar to those in fish caught commercially.
10.1.3. Colour of fish meat
Colour of fish meat
          Fish muscle can be divided into two groups according to colour, viz ordinary muscle (white
meat) and dark muscle (red meat) (Figure 10.1).
                       
                  Fig10.1.3.1 Macroscopic arrangement of fish muscle
White meat forms the major portion of fish muscle and the dark muscle lies along the sides of the
body under the skin and near the vertebrae (Fig.10.2).The shape and volume of red muscle vary
according to the fish species. 

                 Fig  10.1.3.2  Types of dark meat in various fish species

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  A: Cod, B: Mackerel, C: Frigate mackerel
Proximate composition of dark and white meat of some food fishes is presented in Table 10.1.3.1.

Table 10.1.3.1 Proximate composition of dark and white meat of some fishes
Fish species Kind of meat Moisture % Crude protein % Crude fat %
Tuna D 66.4 22.9 6.7
W 68.5 22.9 4.5
Sardine D 70.0 15.9 12.8
W 72.0 23.1 2.9
Mackerel D 54.2 14.9 29.7
W 65.5 21.2 13.1
Herring D 57.8 15.5 28.2
W 74.0 22.0 13.0
Cod D 77.8 18.6 2.5
W 78.4 19.9 0.5
Halibut D 62.0 11.3 27.3
W 77.7 14.5 7.0
10.1.4. Structure of skeletal muscle of fish
             There are three types of muscles: (i) striated muscles (ii) smooth muscles and (iii) the heart
muscle. Striated muscles constitute the livestock meat or fish meat. Smooth muscles constitute viscera
and the tissue of mollusks. The heart muscle is an intermediate structure between the other two. Livestock
meat and fish meat are basically striated muscles which are formed by groups of muscle fibers with
striations.

Individual skeletal muscles vary greatly in size and morphology. They consist of a parallel
arrangement of elongated, multinucleated cells called myofibrils or muscle fibers.
10.1.5. Structure of Muscle fiber

Muscle fiber, smaller unit of muscle, are bound together by connective tissue (endomysium),
and covered with myocommata. Muscle fibers lie parallel to one another within myotomes,
divided by the membrane myocommata. A cross section of the fish body shows that the
myotomes of the back and belly are arranged in concentric circles.

Length and diameter of muscle fiber differ according to the fish. The muscle fiber of horse
mackerel (weight: 100g, length: 19cm, pH of meat: 6.22) is 50-70mm in diameter and 5-6mm in
length. The diameter of dark muscle fiber of horse mackerel is 1/3 to 1/7 of that of ordinary
muscle. There are more connective tissues around the muscle fibers in dark muscle than in
ordinary muscle. The striations are observed distinctly in fish muscle fiber. The width of striation
of horse mackerel muscle is 0.4-0.8mm and the space between striations is 0.6-0.8mm.

10.1.6. Protein Composition of fish

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The protein of fish muscle tissue can be divided into the following three groups based on
solubility to sarcoplasmic, myofibrillar, and stroma protein. The sarcoplasmic protein is located
in the muscle plasma. The myofibrillar protein is present in the myofibrils and the stroma protein
is present in the connective tissue. A similar composition is found in fish and livestock meat. The
livestock meat contains more stroma than fish meat. The protein compositions of fish, shellfish
and animal muscle tissue are presented below.

Table10.1.6.1 Protein Composition of Fish, Shellfish and Meat

 
  Sarcoplasmic Myofibrillar proteins Stroma

Type of Meat proteins protein

 

% of total proteins
Cod 21 76 3
Carp 23-25 70-72 5
Flatfish 18-24 72-79 3
Mackerel 32 58 7
Lactariussp. 26 77 7
Prawn 29.85 65-70 70

(Penaeus indicus)
Meat 16-28 39-68 16-28
10.1.6.1. Sarcoplasmic proteins

Sarcoplasmic protein contains many kinds of proteins called myogen (myoglobin, globulin and
enzymes) constituting 21-25% of the total protein. These proteins are soluble in neutral salt
solutions of low ionic strength. The content of sarcoplasmic protein in fish meat varies with fish
species, but generally higher in pelagic fish such as sardine and mackerel, and lower in
demersal fish like snapper and plaice. It is disposed off during the water washing and pre-
processing of fish. The protein of this fraction is suited to distinguish between different fish
species as all the different species have their characteristic band pattern when separated by the
isoelectric focusing method.

10.1.6.2. Myofibrillar protein

Myofibrillar protein forms myofibril, which contains myosin, actin, tropomyosin, troponin and
actinin. These proteins cover 66-76% of the total protein of the fish meat. These proteins are
soluble in neutral salt solutions of high ionic strength (<0.5M). They make up the contractile
apparatus responsible for muscle movement. The structure of fish protein is easily changed by
changing the physical environment. The solubility characteristics are greatly affected freezing
frozen storage. Treatment with high salt solution denatures proteins and the native structure is
irreversibly altered. These proteins play an important role in coagulation and gel forming when
fish meat is being processed. Fish meat contains a larger percentage of myofibril protein than
mammalian skeletal muscle. The amino acid composition of fish muscle is approximately the
same as that of the protein of mammalian muscle.

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10.1.6.3. Stroma Protein

Stroma protein is the protein, which forms connective tissue. It is made up of collagen and
elastin. It constituted approximately 3% of the protein of teleost fishes and about 10% of the
elasmobranches. It cannot be extracted by water, acid or alkaline solution and neutral salt
solution of 0.01-0.1M concentration. Fish meat separates easily from the myomere when
heated. This phenomenon is caused by gelatinization of collagen in thin myocommata which
combines the myomere. Elastin is very resistant to moist heat and cooking does not affect
elastin in connective tissue. Lately the existence of elastic fibers in the cell has been discovered.
Dark meat contains more stroma protein than white meat, but less sarcoplasmic protein. White
meat contains more connectin while the connectin of dark meat is half that of white meat.

10.1.7. Structure of Myofibrils

Individual muscle fiber is made up of many myofibrils running parallel to each other which are 1-
2µm thick. These are the basic unit of muscle contraction. The individual myofibrils are
separated by a fine network of tubules, the sarcoplasmic reticulum which contains the
sarcoplasm. The myofibrils are cross striated. An alternate dark A-bands and light I-bands are
present . The A band has a light region in the centre

10.1.7.1. Myosin subunit

There is some variation of myosin subunits of fish and rabbit muscles. Rabbit myosin molecules
consist of two heavy chains with a molecular weight of 200,000 each, and several light chains
with a molecular weight of about 20,000 each When myosin is treated with urea, alkali or
guanidine-HCL the light chains are dissociated from the heavy chains. The myosin of fresh
water fish when observed using SDS polyacrylamide gel electrophoresis, showed the presence
of one heavy chain and three light chains. There are differences between molecular weights of
the light chains in different fish species.

Table 10.1.7.1.Molecular weights of myosin light chains of different fish species

Species Molecular weight

LC1 LC2 LC3
Rabbit 2.5x104 1.85x104 1.4x104
Tilapia 2.5x104 1.75x104 1.4x104
4
Carp 2.5x10 1.75x104 1.6x104

When light chains are dissociated from, heavy chains, the actin binding ability and ATPase
activity disappear. When myosin is digested by trypsin or chymotrypsin for a short period,
myosin is divided into two components; a rapid sediment component called H-meromyosin
(HMM), and slow sediment called L-meromyosin (LMM). When HMM is treated with trypsin or
chymotrypsin or papain, it is divided into a head and neck part. The head part is called S 1 and
the neck part S 2 . The HMM has ATPase activity and actin binding ability, but LMM does not
have any biological functions. The ATPase heat stability of HMM follows the heat stability of
myosin ATPase. It is considered that the unstable structure of fish myosin exists in S 1 part of
HMM. HMM of tilapia is not homogeneous, it consists of two main components of sedimentation
constant 5.7S and 4.7S and a small quantity of 3.2S.

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10.1.7..2. Actin

Actin is the main component of thin filament making up 15-30% of the total protein of the
contractile apparatus. Actin monomer has a globular shape and is designated as G-actin. It
exists in the form of a double stranded helix in which the G-actin beads are stabilized by
tropomyosin fibrils. 

Purified actin from various fish species shown no difference in molecular weight from that
obtained by SDS-polyacrylamide gel electrophoresis. Sedimentation equilibrium method and 3-
methylhistidine contents show a molecular weight form 46,000-48,000, similar to rabbit G-actin.
The value estimated by SDS-polyacrylamide electrophoresis is 43,000. Trout and rabbit G-actin
contains five SH-groups.

10.1.7.3.Tropomyosin and Troponin

Tropomyosin and Troponin regulate the muscle contraction. Tropomyosin is a highly elongated
molecule. It is assumed to be a double stranded helix. The molecular weight of tropomyosin is
68,000 and it has two sub unit chains. It is the most heat stable and is easily purified. Purified
tropomyosin from carp, cod, lamprey, shark, ray, cuttlefish, and scallop showed a remarkable
similarity in amino acid composition. The tropomyosin lacks tryptophan and has a small amount
of proline. As tropomyosin is a heat stable protein it can be extracted from heat treated fish
products, also making it possible to identify the material fish of the product by SDS-
electrophoretic pattern of tropomyosin.

Troponin is a necessary protein for tropomyosin to act as a relaxation factor for muscle.
Troponin is formed from 3 components with a different biological function. The three
components are: troponin-T which combine with tropomyosin; troponin-I which inhibits ATPase
of actomyosin and troponin-C which combines with Ca++ ions. In carp troponin, the molecular
weights of troponin –T is 30,000, troponin-I is 21,000 and troponin-C is 19,000. In shark
troponin, molecular weight of troponin C is the same as in carp, however, troponin I is 30,000
and troponin T is 58,000.

10.1.7.4. Actomyosin

Actin and myosin when extracted from fish meat with a salt solution form actomyosin. The
activity of Mg2+ ATPase of actomyosin is markedly higher than that of myosin, while
Ca2+ ATPase activity of actomyosin is the same as that of myosin because as this ion does not
influence the activity of Ca2+ ATPase. In the case of actomyosin extracted from rainbow trout
and Alaska Pollack, Ca2+-ATPase activity is 1/5 of that of tilapia.

Chapter 2: Chemical changes in muscle during contraction

10.2.1. Chemical changes during muscle contraction and relaxation

Muscle contraction is accomplished by a sliding together or telescoping of the inter digitating

thick and thin filament array without and detectable shortening of the filaments themselves. The
force causing the thick and thin filaments to slide past one another is generated by the cross-
bridges or myosin heads that project outward from the surface of the thick filament. Although it
is still unclear how the chemical energy in the ATP molecule is converted to the mechanical

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energy of muscle contraction, a great deal is known about the anatomical and biochemical
events that accompany this energy transduction. 

It is possible to divide the events that occur during muscle contraction into two general
categories (1) those events that occur in the thin filament and (2) those events that occur in the
thick filament. 

10.2.2.Changes in Thin Filaments during Muscle Contraction

A motor nerve impulse reaching the muscle cell passes along the sarcolemma and into the T-
tubules. Passage of an electrical signal along the extra cellular T-tubule causes the adjoining,
intracellular sarcoplasmic reticular membranes to momentarily lose their ability to retain the
calcium that they have accumulated against a concentration gradient. This Ca2+ therefore floods
into the interior of the cell, and intracellular free Ca2+ concentration rises from approximately 10-
8
M in resting muscle to 10-5 to 10-6M.

Because troponin-C has a binding constant near 5x10-6M for Ca2+, an intracellular
Ca2+ concentration of 10-5 to 10-6M is sufficiently high to permit binding of Ca2+ to troponin-C. In
the absence of Ca2+, troponin- C binds to troponin–T and troponin-I, and troponin-I binds loosely
to troponin-T, but firmly to actin. In the presence of Ca2+ troponin-T binds strongly to
tropomyosin, troponin-C binds strongly to troponin-T and troponin-I, and troponin-I binds to
troponin-T but loses its affinity for actin.

In resting muscle, the tropomyosin stand is located out of the groove of the double-stranded
actin filament in a poison where it physically block or at least sterically interferes with the
myosin-binding site of actin. Troponin is necessary to hold tropomyosin in this out position, and
a firm interaction between troponin-I and actin is evidently necessary to maintain this out
position of tropomyosin.

When troponin-C binds Ca2+, the firm linkage between troponin-I and actin is weakened, and
troponin can no longer hold the tropomyosin strand in the out or “off” position. Then the
tropomyosin strand rolls back into the groove of the double-stranded actin filament, and the
myosin-binding site on actin is exposed. Myosin binds to actin, contraction ensues and
continues until the Ca2+ is removed from the troponin-C and troponin-I binds to actin and forces
the tropomyosin strand back out of the groove of the actin filament to block the myosin-binding
site of actin.

Turning muscle contraction on and off in vertebrate striated muscle is accomplished by moving
the tropomyosin strand in (on) and (off) of the groove in the double-stranded actin filament.

10.2.3.Change in Thick Filaments During Muscle Contraction

During muscle contraction, myosin cross-bridges extend outward, attach to the thin or actin
filament and then swivel or rotate so that the tip of the cross-bridge undergoes approximately a
10nm translocation, and pushes the actin filament toward the center of the sarcomeres. At the
end of this stroke, the spent cross-bridge detaches from the thin filament, is reoriented, and is
ready to repeat the cycle. During a single twitch of a muscle fiber, each myosin cross-bridge
may perform many cycles of attaching to actin, swiveling and then dissociating from the actin

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filament, but only 10-20% of the total cross-bridges in a single sarcomeres are ever attached to
thin filaments at any given instant during contraction.

The remaining cross-bridges are in various other stages of the cross-bridge cycle. Because
cross-bridges interact asynchronously with actin, the thick and thin filaments slide past each
other at a smooth, uniform rate during muscle contraction rather than with a “jerky”, ratchet-like
motion that would be produced by synchronous cross-bridge actin.

ATP hydrolysis is clearly involved in providing energy for muscle contraction in the presence of
Mg2+, myosin binds and splits ATP very rapidly Myosin molecule passes through at least five
conformation states during hydrolysis of ATP in the presence of Mg2+. One of these
conformational states is free myosin. Binding of ATP very quickly produces a second
conformational state of myosin. Immediately after the conformational changes resulting from
binding of ATP to myosin, the bound ATP induces an additional conformation state designated
as M.ATP. Hydrolysis of the bound ATP in this third conformational state occurs at an
intermediate rate and produces a fourth conformation form.

In living resting muscle, almost every myosin cross-bridge is “energized” and contains one
molecule each of ADP and inorganic phosphate, the hydrolysis products of ATP. “Energized”
here refers to some unknown conformational state of myosin in which the energy of ATP is
stored; this state is represented by Pi.M.ADP. In resting muscle, the energy in energized myosin
is dissipated slowly through step 4 of the transient state mechanisms. The energized cross-
bridge is unable to bind to actin because the thin filament is “turned off”, i.e., tropomyosin is
blocking the myosin-binding site on actin.

Muscle contraction is triggered by a release of Ca2+ from the sarcoplasmic reticular membranes,

and intracellular free Ca2+ concentrations rise to approximately 10-6M. This enables the troponin-
C polypeptide to bind Ca2+, and the thin filament is “switched on” by the series of events.
Because the ATP bound to myosin has already been split to ADP and Pi and because neither
ADP nor Pi are very effective dissociators of the actin-myosin complex, the myosin cross-bridge
interacts with actin in the thin filament immediately after the actin is unblocked in the “switching
on” process.

Interaction with actin not only accelerates the rate-limiting step in ATP hydrolysis by myosin.
That is, force for contraction may be generated at the point of the actin-myosin interaction rather
than within the myosin cross-bridge itself. Once the myosin cross-bridge has rotated or
swiveled, involving release of the products of ATP hydrolysis in the transient state Kinetic
mechanism for ATP hydrolysis, follow rapidly. Only after ADP has been released from the spent
cross-bridge the myosin head can bind a new molecule of ATP. ATP is a potent dissociator of
the actin-myosin complex, binding of a new molecule of ATP immediately dissociates the
myosin cross-bridge from the actin filament. At this point, however, the myosin cross-bridge is
angled with respect to the actin filament, and even it if were to rebind to the actin filament, it
could not swivel and push the filament toward the center of the sarcomeres. Binding of ATP and
the resulting dissociation of the myosin cross-bridge from actin must be followed by a
reorientation of the angled or spent cross-bridge back to the state.

Energy for this reorientation of myosin cross-bridges originated from hydrolysis of ATP,
determination of the rate constants in the transient state mechanism for ATP hydrolysis by
myosin. It has shown that by far the largest free energy change in this mechanism occurs at
step 2 immediately after ATP is bound to the myosin head and before it is hydrolyzed to ADP

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and Pi. Indeed, actual hydrolysis of ATP releases only-1600 cal/mole of free energy. Therefore,
it seems that the energy of ATP is released in the form of a protein conformational change
during the binding of ATP to the myosin cross-bridge and that hydrolysis of ATP to ADP and Pi
is necessary only to allow the myosin cross-bridge to bind to actin whenever actin is available,
thus, dissociation of the spent cross-bridge from actin and reorientation to prepare it for a new
cycle.

The cyclic series of events continues uninterrupted until Ca2+ is rebound into the sarcoplasmic
reticular membranes. Thin filament is “turned off” so that actin is no longer available to bind
myosin cross-bridges or until muscle ATP is exhausted and no ATP is available to bind to the
spent myosin cross-bridge and dissociate it from actin. In the latter situation, all myosin cross-
bridges stop attached to actin at an angled position. 

The nature of the binding of myosin cross-bridges to actin probably has immense effects on
possible implications of the molecular architecture and biochemical properties of myofibrillar
proteins in fish meat quality.

Unit 11: Chemistry of taste, flavour, and odour

Chapter 1: Chemistry of taste, flavour, and odour

Flavour plays an important and indispensable role in modern food. Flavor is the sensation produced by a
material taken in the mouth, perceived principally by the senses of taste and smell, and also by the general
pain, tactile, and temperature receptors in the mouth. Flavor also denotes the sum of the characteristics of
the material which produces that sensation.
The flavour of a food are potentially limitless. The flavor of the food can be altered with natural or
artificial flavorants, which affect these senses.
1. Natural Food Flavors: Natural flavoring substances are obtained from plant or animal raw materials,
by physical, microbiological or enzymatic processes. They can be either used in their natural state or
processed for human consumption. The natural flavorants are first extracted from the source substance by
solvent extraction, distillation, or using force to squeeze it out. The extracts are then usually purified and
subsequently added to food products to flavor them.
The natural flavours present in food are listed in the following table 11.1.1.
Table 11.1.2 Classification of Natural Food Flavors
Flavor types Examples
1.Fruit flavor grapefruit, orange
i. Citrus-type flavors apple, raspberry, banana
ii.  Berry-type flavors
 

2. Vegetable flavors lettuce, celery

3. Spice flavors cinnamon, peppermint
i.Aromatic onion, garlic
ii. Lachrymogenic Pepper, ginger
iii. Hot
 
4. Beverage flavors juices, milk
i. Unfermented flavors wine, beer, tea
ii. Fermented flavors soft drinks
iii. Compounded flavors

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5. Meat flavors lean beef
i.Mammal flavors fish, clams
ii Sea food flavors
 
6.Fat flavors olive oil, coconut fat, pork fat, butter fat
7.Cooked flavors Beef bouillon
i.Broth Legume, potatoes
ii.Vegetable Marmalade
iii. Fruit
 
8. Processed flavors Ham
i. Smoky flavors Processed meat products
ii. Broiled fried flavors Coffee, snack foods, processed, cereals
iii. Roasted, toasted, baked flavors
 

Artificial food flavorants or flavorings

An artificial flavorant is a substance that gives another substance flavor, altering the
characteristics of the solute, causing it to become sweet, sour, tangy, etc.
e.g. Different flavors due to the use of different scents or fragrances in artificially flavored jellies, soft
drinks and candies, which are made of bases with a similar taste.
Artificial flavorings are focused on altering or enhancing the flavors of natural food product such as
meats and vegetables, or creating flavor for food products that do not have the desired flavors such as
candies and other snacks. Most types of flavorings are focused on scent and taste. Few commercial
products exist to stimulate these senses, since these are sharp, astringent, and typically unpleasant flavors.
These are of two types
i. Nature-identical flavoring substances: These are flavoring substances that are obtained by synthesis
or isolated through chemical processes, which are chemically and organoleptically identical to flavoring
substances naturally present in products intended for human consumption.
ii. Artificial flavoring substances: Flavoring substances not identified in a natural product intended for
human consumption, whether or not the product is processed. These are produced by fractional
distillation and additional chemical manipulation of naturally sourced chemicals or from crude oil or coal
tar. They are chemically different but in sensory characteristics are the same as natural ones.
Most artificial flavors are specific and often complex mixtures of singular naturally occurring flavor
compounds combined together to either imitate or enhance a natural flavor (Table11.1.2). These mixtures
are formulated to give a food product a unique flavor and to maintain flavor consistency between
different product batches or after recipe changes. The known flavoring agents include thousands of
molecular compounds, and the flavorist can often mix these together to produce many of the common
flavors. Many flavorants consists of esters, which are often described as being "sweet" or "fruity".
Table 11.1.2 Type of Artficial Flavorants
Type of Flavour   Artificial flavourant
Buttery Diacetyl
Banana Isoamy acetate
Bitter almond Benzaldehyde
Cinnomon Cinnamic aldehyde
Fruity Ethyl propionate

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Grape Methyl anthranilate
Orange Limonene
Pear Ethyl,2,4decadienoate
Pineapple Allyl hexanoater
Sugar,cotton candy Ehtyl maltol
Vanilla Ethylvanillin
Winter green Methyl salicylate
11.1.4 Taste

The acceptability of any food product greatly depends on the impression of taste when it is eaten. The
sense of taste is really a combination of two of our senses, taste and smell. Both of these senses respond
to certain chemicals. Taste is a complex mixture of flavours and aroma, or smell. The receptors for the
human sense of taste are located on the tongue and on the soft palate.

There are just five stimuli to which these receptors respond. These are:sweet (as in sugar), sour (as in
acidic substances like lemon juice), bitter (strong coffee or quinine in tonic water) ,salt (table salt) and
umami (monosodium glutamate, savouries, soya sauce, crisps).

Bitter taste buds are found at the back of the tongue. Sweet/salty taste buds are in the front of the tongue,
sour taste buds are on both sides whereas the middle of the tongue has very few taste buds at all.
Receptors for umami are thought to be spread evenly across the surface of the tongue (Fid.11.1.2.1.).
The sense of smell also makes up a big part of how well one 'taste' food. Flavour molecules in the
food enter the air in the nose and are detected by millions of receptors that feed information to the brain.
Chewing helps to transfer more odour from the mouth to the back of the nose.  The area which is sensitive
to smell is located at the back of the nose where several million receptor cells per square centimeter
respond to thousands of chemicals in the food. Sight plays an unexpectedly important role in the
perception of flavours. The taste of a colourless, shapeless food is extremely difficult to recognise. Visual
"clues" to enable one to identify taste and flavour accurately.  The brain interprets signals from taste,
smell and even vision before turning them into an impression of the food's taste. Different people will
find different tastes nice or unpleasant. Flavourings are added to food products to give, enhance or
intensify taste and flavour.

          While salt and sugar can technically be considered flavorants that enhance salty and sweet tastes,
usually only compounds that enhance umami, as well as other secondary flavors are considered and
referred to as taste flavorants. Artificial sweeteners are also technically flavorants.

1. Sweet taste: Sweet taste is almost universally regarded as a pleasurable experience. Foods rich in
simple sugars are commonly associated with sweetness. However, excessive sugar intake is linked with a
number of health problems including tooth decay, obesity and diabetes. There are other natural and
artificial compounds that are sweet at lower concentrations and are used as non-caloric sugar
substitutes.e.g saccharin.

2. Sour taste: It is the taste that detects acidity. The sourness of substances is rated by comparing to
dilute hydrochloric acid which has a sourness index of 1. By comparing the sourness index of other
compounds are determined.  For tartaric acid it is 0.7, citric acid, 0.46 and carbonic acid, 0.06. Acids
responsible for sour taste in food are presented in table 11.1.2.1.

Table 11.1.2.1 Acids responsible for sour taste in food

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     Acid Food
Acetic acid In vinegar- gives sour taste and distinctive smell
Ascorbic acid
In oranges and green peppers- gives a crisp, slightly sour taste.
(Vitamin C)
Citric acid In citrus fruits - gives them their sour taste
Fumaric acid Used as a substitute for citric and tartaric acid
Lactic acid In various milk or fermented products - give them a rich tartness
Malic acid In apples- gives them their sour/tart taste
Phosphoric acid Used in all Cola drinks to give an acid taste
Tartaric acid In grapes and wines - gives them a tart taste

3. Bitter: Bitterness is the most sensitive of the tastes. It may be unpleasent, sharp or disagreeable.Coffee
and unsweetened cocoa, marmalade, bitter melon, beer, cirus peel chicory and lemons are examples of
common bitter food. Quinine is known for its bitter taste and is used in tonics. 

4. Salty: It is a taste produces by the presence of sodium ions. The other ions of the alkali metal group
also taste salty but less salty when compared to sodium. The saltinessof a substance is rated relatetive to
sodium chloride which has an index of 1. Potassium chloride has a saltiness index of 0.6 and is used as a
principal salt substitute.

5. Umami (savory) flavorants

Umami or "savory" flavorants, more commonly called taste or flavor emnhancers are largely based
on amino acids and nucleotides. These are typically used as sodium or clacium salts.

i. Glutamic acid salts: This amino acid's sodium salt, monosodium glutamate (MSG) is one of the most
commonly used flavor enhancers in food processing. Mono and diglutamate salts are also commonly
used.

ii. Glycine salts: A simple amino acid that is usually used in conjunction with glutamic acid as a flavor
enhancer.

iii. Guanylic, acid salts: Nucleotide salts that is usually used in conjunction with glutamic acid as a
flavor enhancer.

iv. Ionosinic acid salts and 5'-ribonucleotide salts: Nucleotide salts created from the breakdown of
AMP. Due to high costs of production, it is usually used in conjunction with glutamic acid as a flavor
enhancer.

v. Organic and inorganic acids: Certain organic and inorganic acids can be used to enhance sour tastes,
but like salt and sugar these are usually not considered and regulated as flavorants under law. Each acid
imparts a slightly different sour or tart taste that alters the flavor of a food.

Color as flavour enhancer

The colour of food can affect flavor. For example, adding more red color to a drink increases its
sweetness with darker colored solutions being rated 2–10% higher than lighter ones even though it had
1% less sucrose concentration.

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11.1.6.Substances of taste of fish and shellfish
Substances of taste and flavor of fish and shellfish

Taste results from the combination of basic flavors such as sweet, sour, bitter, salt and umami. Sweet
flavor in fish and sea products is derived from sugars, amino acids and organic acids.
Fish flavors are mainly characterized by the volatile compounds in fish. Main groups of fish odors are
species related fresh fish odor, microbial spoilage odor, oxidized odor, environmentally derived odor and
processing odor. Off odours and flavours in fish or sea products arise from contamination from
metabolites, from the spoilage of protein, or from oxidation products of the oil itself.
Fresh fish flavor
The delicate aroma and taste of very fresh prime quality fish in quite different from that in most
commercially available seafood. Sweet flavor in fish and sea products is derived from sugars, amino acids
and organic acids.
Some compounds are produced due to the action of enzymes on compounds present in fish. IMP formed
from AMP  by the action of AMP deaminase. It contributes to a delicious taste. Inosine nucleotides are
acted by nucleoside phosphorylase and  inosine nucleosidase  and form hypoxanthine which contributes
to the bitter taste.

Iced fish when stored, results in bacterial spoilage. During frozen storage changes in flavor and odor
result from alterations in lipid components. The fresh flavor is prevalent during the first few days after
catching. After that oxidation products and micribialmetabolites dominate the aroma of fish. The
compounds associated with fresh fish flavours are mostly C6, C8 and C9 carbon aldehydes, ketones and
alcohols derived from the unsaturated fatty acids characteristic of fish by lipoxygenases. C8 compounds
such as 1-octen-3-ol, 1-octen-3-one, 1-cis-5-ctadiene-3-ol, 1-cis-5-octadien-3-one occur in most types of
fish and sea food and contribute heavy plant like odors and metallic –like flavors. Oxidation of body meat
lipids damages the odour and flavour of fresh, cooked, stored (refrigerated and frozen) or reheated meat
resulting in rancid or warmed over flavor.
11.1.6.2.Seaweed flavor

i) DMS is a major component of green seaweed aroma and green seaweed contains a greater
content of an enzyme that decomposes DMPT to DMS. DMS also contributes to the aroma of
red and brown seaweeds.

ii) Long chain, saturated aldehydes are formed enzymatically from stearic, oleic, linoleic and
linolenic acids. Long chain aldehydes-forming enzyme (LAFE) appears to be present in
seaweeds but not terrestrial plants. The reaction involves formation of a hydroperoxide adjacent
to the fatty acid carboxyl followed by decarboxylation to form long chain aldehydes. Lipid
oxidation and hydrolysis of oxidised lipid also form relatively short chain aldehydes (eg. (2E)—
nonenal) that contribute to seaweed flavor.

iii) Halogen containing compounds are important to the aroma of red seaweeds.
Bromoperoxidases have been identified in several species of red algae but it is not clear
whether they are involved in postharvest transformation of Haloforms. Red seaweed also
contains greater quantities of sulfate-adenyltransferase, the initial enzyme in the inorganic
sulfate assimilation pathway. Brown seaweed contains an unidentified enzyme that catalyzes
release of large quantities of sulfuric acid and an offensive odor after harvest.

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11.1.6.3. Stale fish flavor

Stale fish flavor

 Much of the spoiled odor and taste of post harvest fish is due to bacterial enzymes. However,
endogenous enzymes are important catalysts of spoiled fish aroma in some species.

i) Ammonia off-odour:  Meat of prerigor elasmobranches may develop a strong ammoniacal odor and
bitter taste. This is associated with the hydrolysis ofurea (1-2.5% fwb) to ammonia and carbon dioxide
by urease. The problem can be avoided by washing the flesh in water or cooking to remove  urea.
Deamination of amino acids also results in ammonia off-odour.

ii) Fishy off-odour.  Varying amounts of TMAO have been found in all tissues of saltwater fish as well
as in some freshwater species. Trimethylamine oxide reductase catalyzes formation of trimethylamine
(TMA) from TMAO. TMA is responsible for the fishy off-odour.

iii) Stringent aftertaste due to enzymatic reactions:  Stringent aftertaste and other quality defects in the
flesh of struggling tuna have been linked to endogenous calpains. Blackberry odor in herring, a flavor
defect caused by DMS, has been linked to the rapid degradation of DMPT, which as mentioned above,
and may be enzyme catalyzed. Rapid formation of ammonia in shrimp has also been partly attributed to
endogenous deaminases.

iv) Enzymic or non-enzymic oxidative rancidity:  Endogenous phospholipases and possible lipases are
clearly involved with the deterioration of flavor in some seafood products during frozen storage.
Free fatty acids from phospholipids may be responsible for off-flavors and the hydrolytic reaction may
facilitate enzymic or non-enzymic oxidative rancidity.

Unit 12: Food additives

Chapter 1: Food additives-types and their chemical nature-Enzymes, vitamins and amino

acids

12.1.1. Food additives

Food additives are substances added to food to preserve flavour or improve its taste and
appearance. Some additives have been used for centuries; for example, preserving food by pickling (with
vinegar), salting, as with bacon, preserving sweets with sugar or using sulphur dioxide as in some wines.
With the advent of processed foods in the second half of the 20th century, many more additives have been
introduced, of both natural and artificial origin.
Regulation
To regulate the usage of these additives, and inform consumers, each additive is assigned a
unique number. Initially these were the "E numbers" used in Europe for all approved additives. This
numbering scheme has now been adopted and extended by the Codex Alimentarius Commission to
internationally identify all additives, regardless of where they are approved for use.
E numbers are all prefixed by "E", but countries outside Europe use only the number, whether the
additive is approved in Europe or not. Example: Acetic acid is written as E260 on products sold
in Europe, but is simply known as additive 260 in some countries.

Types of food additives

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1. Emulsifiers

2. Antimicrobial agents or Preservatives

3. Sequestrant

4. Flavor enhancers

5. Sweetener

6. Antioxidants

6. Colour additives

1.      Emulsifiers

 An emulsifier is a substance which stabilizes an emulsion by increasing their kinetic

stability. Emulsifiers allow water and oils to remain mixed together in an emulsion. There are two types
of emulsions.

1.      Oil-in-water emulsion: It contains small droplets of oil that are dispersed in water.

2.      Water-in-oil emulsion: It has small droplets of water that are dispersed in oil. Usually the
water and oil will not mix and the emulsifier, or emulsifying agent, keeps the mixture stable and
prevents the oil and water from separating into two layers.

One class of emulsifiers is known as surface active substances, or surfactants.

Use: Emulsifiers are among the most frequently used types of food additives. They are used for many
reasons. Emulsifiers can help to make a food appealing. The example of the mayonnaise without the
emulsifier shows how unappealing it would be if the oil and water separated before it was used.
Emulsifiers have a big effect on the structure and texture of many foods. They are used to aid in the
processing of foods and also to help maintain quality and freshness. In low fat spreads, emulsifiers can
help to prevent the growth of moulds which would happen if the oil and fat separated.

Examples of food emulsifiers:

Egg yolk (where the main emulsifying chemical is lecithin), honey, and mustard, where a variety of
chemicals in the mucilage surrounding the seed hull act as emulsifiers; proteins and low-molecular weight
emulsifiers are common as well. Soy lecithin is another emulsifier and thickener. In some cases, particles
can stabilize emulsions as well through a mechanism called Pickering stabilization. Both mayonnaise and
Hollandaise sauce are oil-in-water emulsions that are stabilized with egg yolk lecithin.

Table 12.2.1 Foods that Commonly Contain Emulsifiers

Biscuits Toffees Bread

Extruded snacks Chewing gum Margarine / low fat spreads
Breakfast cereals Frozen desserts Coffee whiteners
Cakes Ice-cream Topping powders

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Desserts / mousses Dried potato Peanut butter
Soft drinks Chocolate coatings Caramels

Antimicrobial agents or Preservatives

Antimicrobial agents or preservatives prevent or inhibit spoilage of food due to fungi, bacteria and other
microorganism.

Microbes will grow quickly when they are in the right conditions; warm, moist, correct pH and a supply
of food to grow on. Preservation tries to alter the conditions to slow or stop the microbe growth. When
this is not possible, or convenient, preservatives may be added to stop the food from going 'off'. Different
microbes are sensitive to different types of preservatives and so a wide range of preservatives are in use
today.

List of some important preservatives

i) Sorbic acid and its salts (E200-203): Sorbic acid and its salts are naturally occurring substances and
they are among the most important food preservatives. Sorbic acid gives no taste or flavour to products
and it is effective over a wide range of foods and beverages. Sorbic acid is used in beverages, dairy
products, fish and seafood, fat-based products, fruit and vegetable products, baked goods and
confectionery products.

ii) Benzoic acid and its salts (E210-E213): A widely used preservative. It is only used in acidic
situations which include non-alcoholic beverages, products prone to spoilage by bacteria and fruit-based
products.

iii) Sulfur dioxide and sulfites (E220-E228): These preservatives are multifunctional food ingredients
which act as preservatives, antioxidants and colour stabilizers. They have a much more pronounced
antibacterial effect than other preservatives and are therefore used when control of bacterial growth is
essential. Sulfur dioxide is used in a wide range of products including packet soup, dried bananas and
apricots, tinned crabmeat, sausage meat, beer, wine, quick frozen chips and jams

iv) Potassium nitrite and sodium nitrite (E249 and E250): They act as preservatives, stabilizers and
flavours. Potassium and sodium nitrite are particularly important in the preservation of cured meat
products.

v) Propionic acid and its salts (E280-E283): The propionates are also naturally occurring preservatives.
They work better in the more alkaline conditions of bakery products and may be used, for example, to
delay the green mould growth on bread

12.1.4.Sequestrant

A sequestrant is a food additive whose role is to improve the quality and stability of the food
products. Sequestrants form chelate complexes with polyvalent metal ions, especially copper,
iron, and nickel, which serve as catalysts in the oxidation of the fats in the food. Sequestrants
are a kind of preservative.

Common sequestrants

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1. Calcium disodium ethylene diamine tetra-acetate (E385) 
Sodium and calcium salts of EDTA are commonly used in many foods and beverage

2. Glucono delta- lactone (E575)

Glucono delta-lactone (GDL) E575a is a naturally-occurring food additive used as a

sequestrant, an acidifier, or a curing, pickling, or leavening agent. 3. Sodium gluconate (E576)

4. Potassium gluconate (E577)

5. Sodium tripolyphosphate

6. Sodium hexamataphosphate (E452i)

Sodium hexametaphosphate (SHMP) is a hexamer of composition (NaPO3)6. Sodium

hexametaphosphate of commerce is typically a mixture of polymeric metaphosphates, of which
the hexamer is one, and is usually the compound referred to by this name. It is more correctly
termed sodium polymetaphosphate. SHMP is used as asequestrant and has applications with in
a wide variety of industries, including as a food additive in which it is used under the E
number E452i .

7. Sodium tripolyphosphate

It is prepared by heating a stoichiometric mixture of disodium phosphate, Na2HPO4 and

monosodium phosphate, NaH2PO4 under carefully controlled conditions.

2Na2HPO4 + NaH2PO4 → Na5P3O10 + 2H2O

It is used in various applications such as a preservative for seafood, meats, poultry and pet
foods. In foods, STPP is used to retain moisture. Many governments regulate the quantities
allowed in foods, as it can substantially increase the sale weight of seafood in particular.

12.1.5 Flavor enhancers

Flavor enhancers

Flavor enhancers enhance a food's existing flavors. They may be extracted from natural sources (through
distillation, solvent extraction, among other methods) or created artificially.

i) Acidulants: Acidulants are additives that give a sharp taste to foods. They also assist in the setting of
gels and to act as preservatives. Many natural foods are acidic. For example, oranges, lemons, apples,
tomatoes, cheese and yoghurt contain natural acids, such as citric acid, that give them their
characteristically sharp taste.  Some important acidulants are acetic acid, citric acid, fumaric acid, lactic
acid, malic acid, phosphoric acid and tartaric acid

ii) Bases:  Bicarbonates, particularly bicarbonate of soda, are used as mild alkalis whilst phosphates are
used for their buffering action as well as for their characteristic taste..

Sweetener

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The desire for sweet taste is inborn. The use of honey dates back to 2000 BC but it is sugar which has
been the sweetener of choice for centuries.

Natural sweetener: Sugar is a most important natural sweetener. It gives the sensation of sweetness and
provides a source of energy. Bulk sweeteners are indigestible and include sugar alcohols, such
as sorbitol, mannitol, and xylitol. Some are found in natural foods while others are manufac tured.

1. Nutritive Sweeteners
i. Sugars. Sugars (saccharides) are widespread in nature and are the building blocks of carbohydrates -
monosaccharides, disaccharides and polysaccharides.

Monosaccharide: The monosaccharides, glucose, fructose, and galactose are composed of just one
sugar molecule, and are known as the “simple sugars.” Glucose and fructose are abundant in fruits,
honey, and processed foods. Galactose is found only in milk.

Disaccharide: Disaccharides are formed from two simple sugar units that are chemically attached, and
include sucrose, lactose and maltose.

Sucrose comes from sugar beets or sugar cane, and is more commonly known as table sugar. Sucrose is
composed of the two simple sugars, glucose and fructose. It is the most abundant sugar in nature,
important for its palatability, availability, low cost, and simplicity of production. Additional products
from the refinement process of sucrose are molasses, brown sugar, and confectioners’ sugar.

Lactose also is referred to as milk sugar. Lactose is made of the two simple sugars glucose and galactose.

Maltose, two glucose units, is the result of the fermentation of the starch in grains by yeast or enzymes,
as in bread-making or brewing.

Polysaccharide: The polysaccharide family includes starch, cellulose, pectin, and glycogen. These
complex carbohydrates are chains of glucose molecules. Starch, cellulose, and pectin are found in plants.
Glycogen is the storage form of glucose for humans and animals.

The simple sugars and many foods with large amounts of simple sugars provide energy (calories), but
contain few other nutrients. They may replace other foods that are high in vitamins, minerals and other
important nutrients in the diet. However, while fruits are sweet because of the sucrose and fructose
they naturally contain, they are excellent sources of vitamins and fiber. Starch is abundant in nutrient-

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rich foods such as vegetables, grains, beans, and potatoes. Carbohydrates, regardless of the form, are
important for providing energy to the body.

ii. Corn Syrup. Corn syrup is a glucose derivative of corn starch, popular in the brewing, canning, and
baking industries because it lends texture and body to these types of products.

High-fructose corn syrup (HFCS) takes the processing of corn syrup one step further, by converting much
or all of the glucose to fructose. The resulting product is sweeter than sucrose, allowing less of it to be
used. HFCS is the main nutritive sweetener in the soft drink industry.

iii. Sugar Alcohols. Sugar alcohols are sometimes used as a substitute for sucrose. Mannitol, sorbitol,
and maltitol occur naturally in fruits. Xylitol is a normal intermediate product in the metabolism of
carbohydrates in fruits and vegetables. Sugar alcohols add bulk and texture to food such as chewing
gum and hard candies. Because they are metabolized by the body more slowly than sucrose, they are
useful in foods for people following special diets, such as a diabetic diet.

2. Artificial sweetener: Excessive sugar intake is linked with a number of health problems including
tooth decay, obesity and diabetes. Alternatives to sugar have therefore been developed which provide the
sweetness without the energy content. Saccharin was discovered in 1878 but it was not until the 1950's,
when consumers became interested in low calorie foods, that synthetic sweeteners came into significant
use to replace sugar with a sweet tasting, non-calorific or artificial sweeteners.

Another reason is for using these synthetic sweeteners in foods and drinks is for diabetics. Artificial
sweeteners allow diabetics to have sugar-free but sweet-tasting foods. High intensity sweeteners include
the artificial sweeteners and need to be added in very small amount.

12.1.6.1 Relative sweetness of natural and artificial sweeteners

Sugar Relative sweetness

1. Natural sugars (Sucrose 100)
Lactose 16
Maltose 32
Glucose 74
Sucrose 100
Invert sugar 130
Fructose 173
Sugar (stevia sugar) 100-300
Sorbitol 67
2. Artificial sweeteners (No. of times as sweet as sucrose)
Cyclamate 30

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Aspartame 160-200
Dulcin 250
Ace sulfame K 200
Saccharin 300-500
Sucralose 600

Sugar alcohols are used to sweeten chewing gum and other confectionery products. High
intensity artificial sweeteners are a boon for diabetic and obese individuals. Use of saccharin is permitted
in carbonated water, soft drink concentrates, supari, and paan masala. Artificial sweeteners, such as
saccharin, aspartame, or ace sulfame may be sold as tabletop sweeteners.

Table 12.1.6.2. Foods that typically contain alternative sweeteners

Item Types
Drinks Carbonated, non- carbonated, milk-based and alcoholic
Baked goods Cakes, buiscuits, muffins
Breakfast cereals All brands of breakfast cereals
Confectionery Sweets including chewing gum
Desserts, fillings and Ice-cream, sweet whipped cream
toppings
Processed fruit and Jams, jellies, baked beans, canned fruit
vegetable products
Medicines Syrups, salad dressings and condiments
Antioxidants

Cotrol of undesirable oxidation reactions in food is usually achieved by achieved by employing

processing and packaging techniques that exclude oxygen or involve the addition of appropriate chemical
agents called antioxidants.

Ascorbic acid is employed to prevent enzymic browning of the cut surfaces of fruits and vegetables. Here
it acts as a reducing agentby transferring hydrogen atoms back to quinines that are formed by enzymatic
oxidation of phenolic compounds. In closed systems ascorbic acid readily react with oxygen and serve as
oxygen scavenger.

Sulfites are readily oxidized oxidized in food systems to sulfonates and sulfate and function as effective
antioxidants in foods such as dried fruits. A number of synthetic compounds are used as anti oxidants. E.g
Butylated hydroxyl anisole(BHA), Butylated hydroxyl toluene(BHT), propyl gallate(PG)etc.
12.1.8. Colour additives in food

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Colour additives are added to food to replace colors lost during preparation, or to make food
look more attractive. Colours that are allowed to be used in foods are strictly tested. Some
common food colour additives are shown in the table

Table 12.1.8.1.Common food colour additives

E No Name Description and color formed Foods

E100 Curcumin Orange-yellow colour that is extracted Curry, fats and oils,
from the roots of the turmeric plant processed cheese.
E101 Riboflavin Riboflavin is also known as vitamin Sauces, processed cheese
B2. It can be obtained by fermenting and foods with added
yeast or synthesised artificially. In vitamins such as bread.
foods, it is used as an orange-yellow
colour.
E102 Tartrazine Yellow coloured synthetic azo dye. Is no longer widely used.
This colouring sparks controversy as Now rarely used in curries
some groups suggest it causes and some ready-meals
behavioral problems in children
E160a Beta-carotene Orange-yellow colour found in plants Soft drinks, margarine,
such as carrots, tomatoes and butter, yoghurt
oranges
E150a Plain caramel Dark brown to black colour. The most Cola drinks, confectionery,
common colouring. 90% of all baked-foods, ice cream,
colouring used is caramel. Obtained chocolate, beers, vinegar
by the heating of sugars and whisk y
E123 Amaranth Dark purple coloured synthetic colour. Powdered soup, jam, ice
Similar in colour to blackcurrants cream, instant gravy
12.2.1.Introduction
A dietary supplement, also known as food supplement or nutritional supplement , is a preparation
intended to provide nutrients, such as vitamins, minerals, fiber, fatty acids or amino acids s, that are
missing or are not consumed in sufficient quantity in a person's diet. Some countries define dietary
supplements as foods, while in others they are defined as drugs. Supplements containing vitamins or
dietary minerals are included in the Codex Alimentarius Commision, a guidebook on food safety
sponsored by the United Nations
Dietary supplements, often containing vitamins, are used to ensure that adequate amounts of
nutrients are obtained on a daily basis, if optimal amounts of the nutrients cannot be obtained through a
varied diet. Scientific evidence supporting the benefits of some vitamin supplements is well established
for certain health conditions, but others need further study. In some cases, vitamin supplements may have
unwanted effects, especially if taken before surgery, with other dietary supplements or medicines, or if
the person taking them has certain health conditions. Dietary supplements may also contain levels of
vitamins many times higher, and in different forms, than one may ingest through food.
12.2.9.1. Protein supplement
Protein supplement
Whey protein is the most commonly used type of protein used for supplementation in food. It is absorbed
by the body very quickly and easily. It contains high levels of all the essential amino acids, branched-
chain amino acids and has the highest content of the amino acid, cystein which is important for the
biosynthesis of glutathione; which has extremely important immune boosting properties for the body.
Casein protein (or milk protein) is the richest in glutamine, an amino acid that aids in recovery ], and has
casomorphuin which helps the body to absorb the amino acids over a long time.

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Soy protein contains all essential amino acids, and is an alternative protein for vegetarians. Soybeans also
contain isoflavones, a type of phytoestrogen, which have a weak estrogenic activity.
Egg white protein is a lactose- and dairy-free protein. Hemp seed contains complete and highly-digestible
protein and hemp oil is high in essential fatty acid.
12.2.9.2 Amino acids
Amino acids
Amino acids are the building blocks of protein, the body breaks consumed protein into amino acids in the
stomach and intestine. Amino aids are added as additive in some food preparation Glutamine is the most
abundant amino acid found in human muscle and is supplemented because supplement manufacturers
claim the body's natural glutamine levels are depleted during anaerobic. It is argued that bodybuilders
should supplement with glutamine, as deficiency may lead to a weakened immune system and wasting of
muscle tissue. It is sold as a micronized, instantly soluble powder. Some studies have shown there to be
no significant effect of glutamine on bench press strength, knee-extension torque or lean muscle mass
when compared to controls taking a placebo, though another study found that glutamine is beneficial in
raising T-helper/suppressor cell ratio in long distance runners.
12.2.9.3.Essential Fatty Acids

For a healthy diet Essential Fatty Acids are necessary nutrients especially while bodybuilding.
Bodybuilders often go on such a low fat diet that they become fat deficient. There are foods and
supplements available to fulfill these needs e.g.low-fat fish, salmon, trout, or mackerel.
Polyunsaturated vegetable oils, such as corn, sunflower, and safflower oils, can provide linoleic
acid. Soybean oil is the only oil that contains linolenic acid. Flax seed oil, which can also be
found in walnuts and pumpkin seeds, is the ideal source of α- linolenic acid.

12.2.9.4.Enzymes
. Enzymes
Digestive enzymes function in the digestive tract. Protease breaks down protein, lipase breaks down fat
and amalyase breaks down starch (into simple sugars), lactase breaks down lactose (milk sugar).
Pancreas produce many digestive enzymes that the body uses (protease, lipase, amylase). However, if the
pancrease malfunctions, it does not produce the enzymes necessary for metabolism. The pancreatic
insufficiency is at the root of many degenerative diseases, including cancer. Food allergies are formed as
a result of insufficient proteases being secreted by the pancreas. Some of the bigger food molecules may
elicit an allergic response. The nomal production of pancreatic enzymes is enough to prevent food
alleries. If the pancreas does not produce sufficient enzymes, supplemental enzymes from plant sources
can be administered.
Bromelain from pineapple and papain from papaya are both proteases used as supplemental enzymes.
These plant enzymes break down protein molecules into amino acids. Bromelain has additional use as an
anti-inflammatory, and helps to reduce clot formation in the arteries.
Some species of aspergillus (a fungus) produce the enzymes amylase, lipase, and cellulase. Hence these
aspergillus species are especially cultured for the commercial production of these enzymes for dietary
supplementation.
Unit 13: Energy values and energy requirements

Chapter 1:Energy values, Energy requirements and their estimation

Energy is provided by oxidation of the bulk organic nutrients. The first requisite of an adequate
diet is a source of energy, provided by oxidation of the three bulk nutrients: carbohydrate, fat and protein.

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(1) Carbohydrate: Carbohydrates are the major source of energy. Carbohydrate rich foods are abundant
and cheap compared with fats and protein. Each gram provide 4kcal of energy. They naturally form major
part of the diet in most of the world. Carbohydrates provide at least 70% and up to 90% of the total
caloric intake. In affluent countries carbohydrates furnish only 45% of the total daily caloric intake.
Starches furnish nearly all carbohydrate intakes and very little sucrose is consumed. In affluent countries
over 40% of the dietary carbohydrate is furnished by sucrose and other refined sugars and the remainder
by the polysaccharide starch.

(2) Fat: Fats provide calories and essential fatty acids. Triacylglycerols constitute about 98 percent of
total dietary lipids, the remaining 2 percent consists ofphospholipids and cholesterol and its esters. At
room temperature triacylglycerols of animal origin, which contain a relatively large proportion of
saturatedfatty acids, are usually solid and those of plant origin which contain a greater fraction of
unsaturated fatty acids are usually liquid.

On oxidation in the tissues triacylglycerols by metabolism, provide energy 9g/Kg. Since fats tend
to remain in the stomach longer than carbohydrates and are digested more slowly they also have greater
satiety value than carbohydrates.

(3) Proteins: The diet contains a variety of different animal and plant proteins. Food proteins   provide
essential amino acids which are not synthesized in the body. On oxidation in the tissues amino acids by
metabolism energy is produced. The energy equivalence of proteins used for calculating the caloric values
of food are 4 kcal/g. 
Estimation of Energy value in food

Energy content of food is measured in terms of calories. In nutrition the term kilocalorie (Kcal) is
used in calculating energy requirements. It is defined as the amount of heat required to raise the temp of 1
Kg of water through one degree centigrade (from 15 o to 16oC) and 1000 times of calories (cal) used in
physics and chemistry.

i) Estimatiom of energy value of food using bomb calorimeter

A weighed amount of food is ignited electrically inside a pressure-resistant bomb filled with
excessoxygen under pressure (Fig.13.1.1). Combustion of the food causes an increase in the temperature
of the known weight of water in the outer chamber. Since 1.0kcal of H 2O through 1ºC, the heat evolved
by combustion of the food is easily calculated.

The amount of energy released by the

oxidation                                                                                                                                                            
                      of carbohydrates, fats, and proteins is determined by burning weighed samples in an
atmosphere of oxygen in a bomb calorimeter and measuring the total amount of heat produced. Pure
carbohydrates yield on the average of about 4.1 kcal / g, fats about 9.35 kcal/g, and proteins about 5.68

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kcal/g. The caloric value of specific foods, such as bread, potatoes, meat, fruit, and so on, can also be
determined by combustion in a calorimeter.
   
ii) Estimation of energy value of food using by chemical analysis or from nutrition tables: Another
way is to determine the weight of carbohydrate, fat, and protein in a given sample of the food by chemical
analysis; the weight of each is then multiplied by the caloric equivalents.

Nutrition labels or other sources of information to determine the fat, sugar and energy content per serving
of various food and drinks can also be used.

When oxidized in the body, foods are not completely digested and absorbed; and hence yield an
amount of heat not equal to the heat released when they are oxidized in a calorimeter. N2 formed by the
oxidation of protein gives 1.25 K.cal which is not available for the energy utilization. Hence the energy
equivalence of food used for calculating the caloric values of food are 4 kcal/g for carbohydrates and
proteins; and 9 kcal / g for fat.

Table13.1.1  Caloric values of food (K. cal)   

No Factors CHO Fat Protein

1 Available energy in food 4.1        9.35   5.68
2 Digestibility 98 95 92
coefficients  %
3 Physiological fuel 4.0 9.0 4.0
value                         
 
The amount of energy released by the
oxidation                                                                                                                                                            
                      of carbohydrates, fats, and proteins is determined by burning weighed samples in an
atmosphere of oxygen in a bomb calorimeter and measuring the total amount of heat produced. Pure
carbohydrates yield on the average of about 4.1 kcal / g, fats about 9.35 kcal/g, and proteins about 5.68
kcal/g. The caloric value of specific foods, such as bread, potatoes, meat, fruit, and so on, can also be
determined by combustion in a calorimeter.
   
ii) Estimation of energy value of food using by chemical analysis or from nutrition tables: Another
way is to determine the weight of carbohydrate, fat, and protein in a given sample of the food by chemical
analysis; the weight of each is then multiplied by the caloric equivalents.

Nutrition labels or other sources of information to determine the fat, sugar and energy content per serving
of various food and drinks can also be used.

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When oxidized in the body, foods are not completely digested and absorbed; and hence yield an
amount of heat not equal to the heat released when they are oxidized in a calorimeter. N2 formed by the
oxidation of protein gives 1.25 K.cal which is not available for the energy utilization. Hence the energy
equivalence of food used for calculating the caloric values of food are 4 kcal/g for carbohydrates and
proteins; and 9 kcal / g for fat.

Table13.1.1  Caloric values of food (K. cal)   

No Factors CHO Fat Protein

1 Available energy 4.1        9.35   5.68
in  food
2 Digestibility 98 95 92
coefficients  %
3 Physiological fuel 4.0 9.0 4.0
value                         
 
Energy requirements
The energy allowances recommended are designed to provide sufficient energy to promote growthin
infants and children and to maintain constant weight and good health in adults. The factors that influence
energy requirements are age, body weight, physical activity, climate and altered physiological status such
as pregnancy and lactation.  In the case of  energy RDA represents only the average daily  expenditure of
an individual.
 
Factors that determine the total energy requirements are basal metabolic rate, physical activities
and specific dynamic action.
Basal Metabolic Rate- BMR:
(1) BMR is the largest fraction of the total energy requirements.

(2) The basal metabolic rate represents the amount of energy needed when the body is at complete rest.
(3) The metabolism under such a condition is referred to as the basal metabolism occurring when the
subject is comfortably relaxed in bed at physical and mental rest in the post absorptive state about
fourteen hours after food.
(4) Under these conditions the heat generated by the body is utilized only to permit vital activities and
maintain body heat at 37oC.
Measurement
The measurement of BMR is done either directly from the oxygen consumption for 2-6 min from
the graph obtained with Benedict-Roth apparatus or the Du Bois apparatus or by an indirect method by
analyzing the expired air and estimating the oxygen consumption and CO 2 output. The total heat
production is then determined. Since greater part of heat is radiated away it has been assumed that the
production of heat from the body is largely dependent on the surface area of the body.

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BMR is expressed as the no. of  Kcal liberated per square meter of body per hr. K.cal/sq.m/hr. and the
average value for an adult man is about 40 K.cal/sq.m/hr while that of an adult women is 37
K.cal/sq.m/hr. It increases from 52 to -55 K.cal/sq.m/hr. in the I year. Then decrease slowly as age
advances.

The energy needed for BMR can be calculated by measuring the O 2 consumption for a certain period. The
consumption of every 1 lt. of O2 produces a heat equivalent of 4.82 K.cal.

A very crude method that can be used to compute BMR is using an arbiter value of 1kcal/Kg of body
weight for 24 hrs.

In clinical practice it is customary to express BMR in relation to surface area i.e. kcal/hr/square meter of
body surface. The surface area is calculated using the following formula
A=W 0.425 x H 0.725 x 71.84
Where A is surface area in m2, W is weight in Kg and H is height in meters.

Equation for prediction of BMR proposed by ICMR expert group for Indians (1989) is presented in table
13.2.1.

Table 13.2.1 Equations for prediction of BMR (kcal/24h):

  Age              (years Equation for Prediction of BMR

) Proposed by ICMR expert
group for Indians (1989)
Male 18-30 14.5xB.W.(kg)+645   
  30-60 10.9xB.W.(kg)+833   
     >60 12.6xB.W.(kg)+463
Female 18-30 14.0xB.W.(kg)+471   
  30-60 8.3xB.W.(kg)+788     
      >60 10.0xB.W.(kg)+565
Source:  National Institute of Nutrition  Report (2009)
            B.W. = Body weight
The BMR factors for occupational and non-occupational
Physical activities      

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Mechanical efficiency is given by the ratio of useful work done to the total potential energy of the
fuel used. Energy is used for the maintenance of mechanical, electrical, synthetic and osmotic work done
by the cells. By metabolic reactions, chemical energy is liberated and part of the energy liberated is
converted to energy rich compounds and the remaining part is liberated as heat and used for the
maintenance of body temperature. The energy output is so well balanced that the body weight in adults
generally remains constant.

Energy is needed to carry out physical work. The energy requirement depends upon the type of activities
involved. Physical activity ratio (PAR) is expressed as the ratio of the energy cost of an individual
activity per minute to the cost of the basal metabolic rate (BMR) per minute. Table 13.2.2 shows  PAL
Values proposed by ICMR Expert Group (2009), compared to the figures proposed by FAO/WHO/UNU
Consultation, 2004.

able13.1.2.  PAL Values proposed by ICMR Expert Group (2009), compared to the figures roposed
by FAO/WHO/UNU Consultation, 2004

Activity ICMR FAO/WHO/UNU

2009      Consultation, 2004
Sedentary 1.53 1.40 -  1.69
Work
Moderate   1.8     1.70 – 1.99
Work
Heavy Work 2.3    2.0 – 2.40*

Source:  National Institute of Nutrition  Report (2009)

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Energy requirement for some activities are given in table 13.2.3. Physical activity ratio (PAR) is
expressed as the ratio of the energy cost of an individual activity per minute to the cost of the basal
metabolic rate (BMR) per minute.

Table 13.1.3. Total energy expenditure under some activites

PAR values for some activities*

Average PAR
Activity Male Female
Sleeping 1.0 1.0
Sitting quietly 1.2 1.2
Reading 1.3 1.5
Standing 1.4 1.5
Dressing 2.4 3.3
Walking slowly 2.8 3.0
Walking briskly 3.8 3.8
Cycling 5.6 3.6
Running – sprint 8.2 8.3
Running- long distance 6.3 6.6
Basketball 7.0 7.7
Football 8.0 -
Swimming 9 -
Source:  National Institute of Nutrition  Report (2009)
                        
                                                       Energy cost of an activity per minute
*Physical Activity Ratio (PAR) = ----------------------------------------------
                                                      Energy cost of basal metabolism per minute
Specific Dynamic Action: 
            The increased heat production brought about by the ingestion of food itself is called the specific
dynamic effect or calorigenic effect. It represents the additional calories necessary for ATP production
when nutrients are oxidized by the tissues.. It is 30% for protein metabolism,13% for fats and 5% for
carbohydrates. When foods are taken in a mixed diet, the SDR is not the total of the SDA of each food
when fed separately.
Measurement of energy requirement
The energy requirement can be measured directly by calorimetry or indirectly by measuring the
heat out put from the amount of O2 consumed and the CO2 evolved.

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1. Direct calorimetry
The energy released by combustion of food stuffs can be measured directly in the bomb
calorimeter designed by Berthelot which is a robust metal case sealed with O 2 under pressure and
surrounded by water inside an insulated container. The food stuff is ignited by an electron spark and the
change in the temperature of the surrounding water caused by the combustion of the food stuff gives a
measure of the heat produced which is expressed in Kilocalories.

The average amount of heat produced on combustion of the three important basis varieties of
food stuffs in the bomb calorimeter is 4.1, 9.35 and 5.68 Kilo cal./g of carbohydrate, fat and protein
respectively. When the food is oxidized in the body, the oxidation is almost complete for carbohydrates
and lipids. However, the protein are not completely oxidized in the body and yield nitrogenous excretory
products, like urea, creatinine, uric acid and ammonium salts after their overall metabolism and contain
oxidisable carbon and hydrogen. When foodstuffs are ingested the carbohydrates, fats and proteins are not
digested and absorbed totally.  Hence their calorific value in the body is only 4, 9 and 4 Kilo cal/g of
carbohydrate, fat and protein respectively.

2. Indirect calorimetry

            In indirect calorimetry, heat out put is calculated from the amount of O 2 consumed and the
CO2 evolved. The amount of O2 utilized per unit of time is proportional to the energy liberated but the
quantity of O2 required for oxidation varies slightly with the type and combination of food stuffs being
oxidized. The ratio of vol. of CO2 produced to vol. of O2 used is measured. This ratio is referred to as the
respiratory quotient ( RQ).

1.RQ of Carbohydrate: The conplete oxidation of glucose is as follows

C6H12O2  + 6O2                                              6CO2 + 6H2O + energy

                                      6CO2              6
             RQ               =  ------      =   ---       = 1
                                      6O2                   6

2. RQ of fat - tristearin: The oxidation of tristearin is as follows

           
               2C57H110O6 + 163O2                        114CO2+110H2O

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            RQ                  =  114/163     = 0.7
Fats have lower R.Q value because the oxygen content of their molecule in relation to carbon content is
very low.

3. RQ of  Protein:  Proteins vary in their chemical structure and hence their RQ is very difficult to
calculate. Indirect measurements give the RQ of 0.8 for most of the proteins.

4. RQ of Mixed diets: For a mixed diet the measurements give a RQ of 0.85. The RQ under basal
conditions are estimated to be 0.82. The value of R.Q provides an approximate clue to the nature of the
food stuffs being metabolized in the body. During decreased CO 2 elimination as in metabolic acidosis due
to diabetes, kidney failure or alcoholism the R.Q is 0.8
Daily calorie requirement
            It is estimated that the calorie requirement of a sedentary man is bout 2,500 K.cal per day. The
total calorie requirement are generally assessed by calculating, on the basis of BMR, surface  area, the
energy required to perform the usual patterns of activity during the day. Correction for the energy value
lost in the preparation of food and during digestion and absorption are made to the extend of 10% of the
total energy value. The pattern of work usually varies and even the food intake of an individual alters
from day to day. The daily recommended calorie allowances of the NRC are given in the following table
3.

Suitable adjustments are desirable in these for various factors such as for age, stress periods of
pregnancy, lactation/ growth etc.

Table 13.2.4 Energy requirements of Indians at different ages

Age Group  Category Body weights  Requirement (kcal/kg/day)

(kcal/day)     
Man Sedentary work                      60 2320    39
        Moderate work 60 2730                                   46
  Heavy work 60 3490 58
Woman Sedentary work           55 1900 35
  Moderate work            55 2230                     41

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  Heavy work 55 2850 52
  Pregnant woman 55 + GWGb          + 350  
  Lactation 55 + WGc  + 600  
0-6m + 520
6-12m         
Source:  National Institute of Nutrition  Report (2009)
GWG – Gestational Weight Gain. Energy need in pregnancy should be adjusted for actual 
            bodyweight, observed weight gain and activity pattern for the population.
WGc – Gestational Weight gain remaining after delivery

Over Nutrition: Obesity is the result of caloric over nutrition. It increase the risk of cardiovascular
diseases, hypertension, and diabetes, is quite simply the result of caloric in excess of body needs. It
usually begins in childhood or adolescence, and the longer it is allowed to persist, the less likely that it
can be controlled. Sensible dietary intake and exercise are the most assured way to control obesity.
Hormonal imbalance will also lead to obesity (e.g Thyroid hormone).

Under nutrition: Two forms of child under nutrition, often occurring together

are marasmus and kwashiorkor ( from Greek ‘to waste’).

Marasmus:  Marasmus is the term applied to chronic deficiency of calories in children. It is caloric

starvation. It occurs in areas when infants are weaned from breast milk and given inadequate bottle
feeding of thin watery gruel of native cereals or other plant products usually deficient in both calorie and
protein. This occurs in infants and children who are in the active stages of growth when both calorie and
proteins are needed in plenty. Marasmus is characterized by arrested growth, extreme muscle wasting,
weakness and anemia. It is complicated by deficiencies of vitamins and minerals. Calorie deficiency in
early childhood leaves a permanent deficit in the body even if alleviated with ample diet later.

Kwashiorkor:  Kwashiorkor (an African word meaning ‘Weaning disease’) is a chronic disease in

children due to protein deficiency. It occurs when there is little in take of animal protein and more of
plant proteins that are low in quality. The growth of protein deficient children is retarded, they become
anemic and the tissue become watery and bloated due to low serum protein levels, which upsets normal
distribution of water between the tissue and blood. The liver kidneys and pancreas undergo severe
degeneration. The mortality rate of kwashiorkor is very high. There is also permanent physiological
deficit. and a deficit in mental ability.
           
The deficiency of both calories and protein is called protein-calorie malnutrition
Unit 14: Water, electrolyte and acid base balance
14.1.1.Introduction
Water is the major component of all living organisms.  It constitutes 60-70% or more of the
weight of most living things, and it pervades all portions of every cell. Water is the universal solvent and
dispersing agent, as well as a very reactive chemical compound. Water is an essential constituent of all
cell structures and is the medium in which all the chemical reactions of a cellular metabolism take place.
Biologically active structures of macromolecules are spontaneously formed only to aqueous media. It is

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an active partner of molecular interactions, participating directly in many biochemical reactions as a
substrate or a product.  Its high heat capacity allows water to act as a heat buffer in all
organisms.  Regulation of water contents is important in the maintenance of homeostasis in all living
systems.

Water is a major component of every body cell, tissue and organ constituting about 50-70%.
Approximately 90% of the water present in the human body is present in a cell the basic unit of the
body.  It plays an important role in almost every body function. It is distributed throughout the body as
the major component of the intracellular and extracellular fluid.
13.1.1.1.Function of Water
                                  
Functions of water
1.       Temperature regulation
Water regulates the body temperature, as the cooling and heating is distributed through
perspiration. A certain amount of water can cause maximum cooling by evaporation; so that
body temperature does not increase.
2.       Transportation of oxygen and nutrients through the blood
It helps transport oxygen, nutrients and waste products in and out of cells. It is necessary for
all digestive, absorption, circulatory and excretory functions as well as for the utilization of
the water-soluble vitamins.

3.  Necessary component of chemical reactions

     All cells and organs functions that make up the entire anatomy and physiology depend on
water for their functioning.A number

3.       Regulation of metabolism
Water is needed to separate (by a process called hydrolysis) a phosphate group from
adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to get energy as illustrated
by the following equations.

         ATP+ H2O = Energy + ADP + Inorganic Phosphate

         GTP + H2O = Energy + GDP + Inorganic Phosphate

All energy from food is used to generate ATP or GTP before its energy can be used by the
human body, which makes water crucial to all energy usage by the human body
4.       Aid in elimination of waste through urine and feces

Water helps to alleviate constipation by moving food through the intestinal tract and
thereby eliminating waste. Water is also very important for removing toxins from the human
body.  The body has four major ways of removing toxins which are: Bowels, Urination,
Perspiration, and Processing of toxins by the liver

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The first three of these four methods directly excrete water from the body.  When
dehydrated, the body will try to save water by minimizing the use of the first three functions
and will force the liver to assume as much of the workload as possible.  This extra work will
place a heavy burden on the liver which still has other functions in addition to detoxification. 
Even then, the liver by itself will not be able to do all that work very efficiently and toxins will
build up rapidly
5.       Lubrication of joints
Water serves as a lubricant. Water forms the fluids that surround the joints, pleura,
conjuntiva and peritoneum to prevent friction.
6.       Major component of body fluids
      Water forms the base for saliva, mucus and other body fluids. Water is the containing
medium for electrolytes and all other ions throughout the human body. Some of these ions
help to form electrical pathways for nerve functions.
7.       Shape and stability of cell.
Water helps to maintain proper muscle tone by giving muscles their natural ability to
contract and by preventing dehydration. It also helps to prevent the sagging skin that usually
follows weight loss - shrinking cells are buoyed by water which plumps the skin and leaves it
clear, healthy and resilient.
8.       Important to fitness and fat loss

            Water is important to fitness and fat loss for several reasons. Water fills us up without
adding any calories. Dehydration will degrade a person’s ability to exercise and burn
calories. Dehydration will reduce protein synthesis which is needed to build or repair
muscles.

Distribution of water in the body

Nearly 45 litres of water is present in 70 Kg adult male. Of this 30 L is found in intra cellular
fluids including bone and rest (15 L) is present in extracellular fluids. Water distribution among various
extracellular fluids is 8.5 L interstitial fluid, 3.0 L in plasma and 4.5 L in trans cellular fluids like
secretions of respiratory, gastrointestinal tract, skin, ear, nose, vitreous humor of the eye and
cerebrospinal fluid.

Since fat is water insoluble, water content of body alters according to fat present in the body. In
obese people water constitutes low percentage (55-65%) of body weight. In lean people water constitutes
high percentage (70-75%) of body weight. Females have low water percentage (65%) because of
relatively high percentage of fat compared to males. The daily water intake and water output of an adult
leading sedentary life is presented in table 14.1.1

Table 14.1.1 The daily water intake and water output of an adult

Water intake mL/day Water output mL/day

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Drinking water       :1350
Food water             :  900
Metabolic water     :  450
 
                             :2700
Urine                :1400
Skin                  :700
Lungs               :500
Fecal water      :100
                         :2700

Factors affecting water intake and water output

1. Environment influences water intake and water output of an individual. In hot weather, water output
decreases (urine) and water intake increases. Water intake is less in cold climate and water output (urine)
is more.

2. In disease like diabetes and renal diabetes, output of urine is more. Water loss is more in diarrhea and
vomiting and these losses can be fatal in infants.
13.1.1.4.Maintenance of water balance
Maintenance of water balance
Fluid intake (thirst) and urine volume are involved in water balance maintenance. They play crucial role
in body water homeostasis. When the water is deficit, it stimulates thirst centre in hypothalamus to cause
thirst and at the same time another centre in hypothalamus is stimulated to release antidiuretic hormone
(ADH). More fluid is taken because thirst centre is activated. ADH increases reabsorption of water in
kidney. Thus water deficit is compensated. When the body contains excess water the reverse process
occurs i.e. the thirst centre is inhibited drinking ceases and in the absence of ADH reabsorption of water
in kidney decreases. These mechanisms come into action with deficit or excess of 200-300 ml in body
water.The total body water in a healthy 70 Kg man varies by no more than 500 ml under normal
physiological conditions.

Disorders of water balance

1. Dehydration (water depletion). It is due to deficiency of water. It occurs in vomiting, diarrhea, diabetes
incipidus and in lesions of hypothalamus.

2. Edema. It is due to excess water in body. It may leads to edema. It occurs in water intoxication,
excessive administration of intravenous fluids, increased secretion of ADH, protein deficiency, cancer
14.1.5.Requirement of water
Requirement of water
A person should consume approximately 2 to 3 liters of fluid each day. Being 2% dehydrated can
seriously degrade physical and mental functions. Being 15% dehydrated is likely to be lethal.
Requirement of water may change according to a person’s medical condition, exercise habits, and living
environment (high altitude location or extremely hot area), day-to-day activities (e.g. -water requirements
are increased when flying or during strenuous exercise). Besides drinking water, a lot of fluids are
supplied by the foods that are eaten.

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Water supplied by food

Many fruits and vegetables have high water content. Fruit juice also contributes fluid to our diet,
as do other beverages such as coffee and soda, though in small amounts. 20% of the daily fluid need is
met from the foods eaten. Fruits tend to have more water, whereas something dry like toast would have
much less. The other 80% of fluid needed comes from the beverages drunk or liquid foods eaten.

Plain water intake provides water without adding any calories. Healthy beverages include 100%
fruit juices, vegetable juices and low-fat milk. They contain some calories but also offer lots of vitamins
and minerals. Vegetable juices can be high in sodium. Sugar-sweetened soft drinks usually contain no
nutritional value, and they may have a lot of calories that can lead to excess weight gain if regularly
consumed. Artificially sweetened diet soft drinks have no nutritional value as they contain no sugar.

Other beverages that aren't good choices, if excess calorie intake is to be avoided, include shakes,
malts, ice cream sodas and frozen sugary coffee drinks. They all contain lots of calories from sugar and
from fat.

ELECTROLYTES

Electrolytes are the smallest of chemicals that are important for the cells in the body, body  fluid,
and blood to function and allow the body to work. Electrolytes such as sodium, potassium, and others are
critical in allowing cells to generate energy, maintain the stability of their walls, and to function in
general. They generate electricity, contract muscles, move water and fluids within the body, and
participate in myriad other activities. Physiological processes like membrane potential, neuromuscular
excitability, nerve impulse transmission, HCl secretion and gas transport are dependent on ICF and ECF
electrolyte composition. Blood clotting, enzyme catalysis, bone formation and muscle contraction are
dependent on electrolytes.

Distribution of electrolytes
Sharp differences exist in the distribution of anions and cations in the ICF and ECF. Na+ is the major
cation of extracellular fluid whereas K+ predominates in ICF. Similarly Cl is the major anion in ECF
whereas organic anions predominate in ICF.

Important electrolytes in ICF:

:
Cations: Na+, K+, Ca2+, Mg2+                             
Anions : Cl–, HCO3–, PO43– , SO42–

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Important electrolytes in ECF:

Cations: Na+, K+, Ca2+, Mg2+
Anions; Cl–, HCO3–, PO43–, SO42–,
Electrolytes of blood plasma : The important anions in blood plasma are bicarbonate,

chloride, phosphate, sulfate, iodide and fluoride.

The concentration of electrolytes in the body is controlled by a variety of hormones, which are
manufactured in the kidney and the adrenal glands. Sensors in specialized kidney cells monitor the
amount of sodium, potassium, and water in the bloodstream. The body functions in a very narrow range
of normal. Hormones like renin (made in the kidney), angiotensin (from the lung, brain and heart),
aldosterone (from the adrenal gland), and antidiuretic hormone (from the pituitary) that keep the
electrolyte balance within those normal limits. They also stimulate the thirst mechanism when the body
gets dehydrated.

1. Sodium (Na): Sodium is most often found outside the cell, in the plasma (the non-cell part) of the
bloodstream. It is a significant part of water regulation in the body, since water goes where the sodium
goes. Sodium is an important electrolyte that helps with electrical signals in the body, allowing muscles
to contract and relax and the brain to work. It is half of the electrical pump at the cell level that keeps
sodium in the plasma and potassium inside the cell.

Conditions of Sodium Imbalance

1. Hypernatremia: Hypernatremia is associated with dehydration, and instead of having too much
sodium, there is too little water. This water loss can occur from illnesses with vomiting or diarrhea,
excessive sweating from exercise or fever, or from drinking fluid that has too high concentrations of salt.

2. Hyponatremia: Hyponatremia is caused by water intoxication (drinking so much water that it dilutes

the sodium in the blood and overwhelms the kidney's compensation mechanism) or by a syndrome of
inappropriate anti-diuretic hormone. It can be associated with illnesses like pneumonia, brain diseases,
cancer, thyroid problems and some medication.

Normal blood sodium level- 135 - 145 milliEquivalents/liter (mEq/L), or

                                              135 - 145 millimoles/liter (mmol/L).

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2. Potassium ( K+): Potassium is the major positive ion (cation) found inside of cells. The proper level of
potassium is essential for normal cell function. Among the many functions of potassium in the body are
regulation of the heartbeat and the function of the muscles.

Hyperkalemia: Increased potassium is known as hyperkalemia. Potassium is normally excreted by the

kidneys, so disorders that decrease the function of the kidneys can result in hyperkalemia. Certain
medications may also predispose an individual to hyperkalemia.

Hypokalemia: Hypokalemia or decreased potassium, can arise due to kidney diseases; excessive loss due
to heavy sweating, diarrhea or vomiting or, eating disorders, certain medications, or other causes.

A seriously abnormal increase in potassium hyperkalemia or decrease in potassium hypokalemia can

profoundly affect the nervous system and increases the chance of irregular heartbeat (arrhythmias),
which, can be fatal.

Normal blood potassium:   3.5 - 5.0 milliEquivalents/liter (mEq/L) or

            - 5.0 millimoles/liter (mmol/L).

Calcium: Normal plasma range is 9-11 mg/dl. Its level decreases in rickets

Chloride: Chloride is the major anion (negatively charged ion) found in the fluid outside of cells and in
the blood. It forms the negatively charged part of certain substances  such as table salt (sodium chloride
or NaCl) when dissolved in liquid. Sea water has almost the same concentration of chloride ion as human
body fluids. Chloride also plays a role in helping the body maintain a normal balance of fluids. Significant
increases or decreases in chloride can have deleterious or even fatal consequences.

Hyperchloremia (Increased chloride): Elevations in chloride may be seen in diarrhea, certain kidney

diseases, and sometimes in overactivity of the parathyroid glands.

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 Hypocholeremia (Decreased chloride): Chloride is normally lost in the urine, sweat, and stomach

secretions. Excessive loss can occur from heavy sweating, vomiting, and adrenal gland and renal disease

Normal serum range: 98 - 108 mmol/L.

Bicarbonate: The bicarbonate ion acts as a buffer to maintain the normal levels of acidity (pH) in
blood and other fluids in the body. Bicarbonate levels are measured to monitor the acidity of the
blood and body fluids. The acidity is affected by foods or medications that we ingest and
the function of the kidneys and lungs. The chemical notation for bicarbonate on most lab reports is
HCO3- or represented as the concentration of CO2.
 

Normal serum range: 22-30 mmol/L.

Phosphate:  It is involved in maintenance of plasma pH. It is component of phosphate buffer system.

Normal phosphate level in plasma ranges from 2-4 mg/dl.

Maintenance of electrolyte balance

1. For normal function of body electrolytes concentrations of body fluids must be controlled. Many
mechanisms operate to control body electrolyte balance. One such mechanism is sodium pump. It
maintains low intracellular level of Na+ and high extracellular level. Hormone aldosterone maintains
electrolyte balance by acting on kidney. It increases Na+ absorption and K+ excretion by kidney.

2. Diet, water and salt intake influences the concentration of electrolytes in body fluids.

3. Kidney maintains plasma bicarbonate concentration. Kidney also maintain electrolyte balance     by
excreting salts or by retaining salts depending on diet and environmental condition.

ACID-BASE BALANCE

          Acid-base balance is concerned with the proper balance between acids and bases or pH.  There is a
normal pH value in each body compartment (i.e. extracellular fluid, plasma, intracellular fluid etc).
Intracellular pH is difficult to measure and may vary in different types of cells and in different parts of

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cells. pH of the plasma is controlled at 7.4. Changes in plasma pH reflect pH changes in other
compartments. When the source of pH change is intracellular the plasma pH change will be in the same
direction as the intracellular pH change but of lesser magnitude. When the primary change is in the
extracellular fluid the magnitude of any intracellular change will be less than the extracellular change.  In
a normal healthy person the blood pH ranges from 7.35-7.45. Throughout ones life this blood pH remains
constant.

           The human body is very sensitive to its pH level, so strong mechanisms exist to maintain it.
Outside the acceptable range of pH, proteins are denatured and digested, enzymes lose their ability to
function, and death may occur.

During metabolic processes both acids and bases are formed. Under normal conditions they are
neutralized by specific systems involved in maintenance of pH level. Under pathological conditions
excessive amounts of acids or bases may accumulate in body fluids and tissues leading to disturbances in
acid base balance.
Importance of Acid base maintenance
 
Proper pH is required for the optimal action of enzymes and for the transport of molecules within the
body and between cells and its surroundings. Proper pH is required for the maintenance of structure of
nucleic acids, proteins, coenzymes and various metabolites. Acidosis and alkalosis are two important
disorders of acid base balance.

Mechanism of regulation
Three different systems are involved in the maintenance of stable blood pH level. They are:
1. Buffer systems of blood plasma, tissue fluids and cells like erythrocytes.
2. Lungs.
3. Kidneys.
By the combined action of these systems constant H+ concentration is maintained in the body

Buffer systems
They are responsible for the maintenance of pH of plasma, ICF, ECF and tissues of the body. For the
good understanding of role of buffer in the regulation of body pH, some physical chemistry of buffer is
required.
Buffers of blood plasma
i) Bicarbonate buffering system:
It is present in greater concentration and plays major role in regulating pH of blood with in normal limits.
Even though the pK of H2CO3 is 6.1, the HCO 3–/H2CO3 function as major buffer at pH 7.4 by
maintaining ratio of 20 : 1 for conjugate base (HCO3–) to weak acid (H2CO3).
The pH of blood remains 7.4 as long as this ratio is maintained. Increase or decrease in   pH due to entry
of acids or bases into blood is met by adjustment in this ratio. Any alteration in the ratio for prolonged
time leads to disturbances in acid base balance.

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ii) Ammonia: The kidneys have several powerful physiological mechanisms to control pH by the
excretion of excess acid or base. In responses to acidosis, tubular cells reabsorb more bicarbonate from
the tubular fluid, collecting duct cells secrete more hydrogen and generate more bicarbonate, and
ammoniagenesis leads to increased formation of the NH 3 buffer. In responses to alkalosis, the kidney may
excrete more bicarbonate by decreasing hydrogen ion secretion from the tubular epithelial cells, and
lowering rates of glutamine metabolism and ammonia excretion.
iii) Proteins: Proteins are the most important buffers in the body. They are mainly intracellular and
include haemoglobin. The plasma proteins are buffers but the absolute amount is small compared to
intracellular protein. Protein molecules possess basic and acidic groups which act as H+ acceptors or
donors respectively if H+ is added or removed.
(b) Phosphate: Phosphate buffer (H2PO4- : HPO42-) is mainly intracellular. The pK of this sytem is 6.8 so
that it is moderately efficient at physiological pH's. The concentration of phosphate is low in the
extracellular fluid but the phosphate buffer system is an important urinary buffer.
Unit 15: Assessment of quality in food

Chapter 1: Assessment of quality in food by instrumental and Chemical methods

15.1.1.Introduction
Food is made of chemicals. These chemicals are all potentially significant, as they determine the
nutritional value, eating properties and suitability for use in particular products and processes.   Estimation
of chemical composition or proximate composition of food will give the details of nutritional value of
food. The proximate composition of food that is estimated is the moisture, protein, fat, carbohydrate and
ash content. The quality characteristics involve the assessment of changes to volatile bases, nucleotide
and lipid oxidation products.
3. Incomplete proteins: Incomplete proteins neither maintain life nor support growth. Most vegetable
proteins are incomplete and are of low biological value(e.g. rice, wheat, corn, beans).Proteins differ
considerably in the relative proportion of amino acids they contain and hence their biological value. Some
proteins contain a complete set of essential amino acids. There are 10 essential amino acids which are
need in food in take and the body cannot synthesis them at rates at which they are needed for growth. The
nonessential amino acids are those acids that can be synthesized in the body from a suitable carbon
source, amino groups from other amino acids and consequently do not have to be supplied in a ready
made form diet.

2. Partially complete:  Partially complete proteins maintain life but fail to support normal growth. e.g.
gliadin of wheat, hordein of barley.
14.1.2.Instrumental Method
 
 
I. Moisture content
a)Air oven drying
   Owing to its convenience, air-oven drying is the most common and widely used method for
routine moisture determination in standard laboratories around the world. Because the principle
of oven drying is based on weight loss, the sample needs to be thermally stable and should not
contain a significant amount of volatile compounds.
 
b) Infrared lamp drying    
  The sample to be analyzed is placed under an infrared lamp and its mass is recorded as a
function of time. The water molecules in the food evaporate because they absorb infrared

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energy, which causes them to become thermally excited. One of the major advantages of
infrared drying methods is that moisture contents can be determined rapidly using inexpensive
equipment, e.g., 10-25 minutes. This is because the IR energy penetrates into the sample,
rather than having to be conducted and convected inwards from the surface of the sample. To
obtain reproducible measurements it is important to control the distance between the sample
and the IR lamp and the dimensions of the sample. IR drying methods is widely used in industry
because of its speed and ease of use.
15.1.3. Proteins in foods

The quantification of total protein in food products can be performed directly, or by

determination of the total organic nitrogen followed by conversion of total nitrogen to crude
protein content using a suitable conversion factor. An approximate value of the protein content
can be obtained by multiplying the nitrogen content by 6.25, a factor corresponding to an
average protein nitrogen content of 16%.

The Kjeldahl Method (Kjeldhal distillation apparatus): The Kjeldhal method maintains its
position as the most frequently used technique for the determination of organic nitrogen in food
products. During the decomposition step, dehydration and carbonisation of organic matter
occurs, combined with oxidation of the liberated carbon to carbon dioxide. Organic nitrogen is
transformed to ammonia, which is retained in solution as ammonium sulfate. Ammonia is
liberated from the digest by distillation for 10 min. in the presence of alkali. The liberated NH3 is
collected in a boric acid solution. It then is titrated against a very dilute H2SO4 and the Nitrogen
content is determined. The protein content is then calculated by multiplying the N value with
6.25 (protein conversion factor).

The Lowry's Method (Spectrophotometer): The Lowry's method is based on a reduction of

the Folin-Ciocalteau reagent, composed of a mixture of phosphomolybdic and phosphotungstic
acid, by oxidation of tyrosine, tryptophan, and to a lesser extent, cystine, cysteine, and histidine
residues on the polypeptide side chains of protein. The oxidation-reduction reaction is
accompanied by the formation of a characteristic blue color with absorption maximum at 745-
750 nm. Copper chelates in the peptide structure, which facilitates the electron transfer from the
amino acid functional groups to the mixed acid chromatogen. The method is particularly suitable
for the estimation of small amounts of protein in solution.

Protein reacts with Folin-Ciocalteau reagent to give a coloured complex. The colour thus formed
is due to the reaction of the alkaline copper with protein and the reduction of phosphomolybdate
by tyrosine and tryptophan present in the protein. The intensity of the colour, depends on the
amount of these compounds in the protein, which is then measured using a spectrophotometer

15.1.4. Lipids from foodstuffs (Soxhlet apparatus)

Lipids should first be extracted either for total lipid estimation or for further analysis to be
performed. In all cases, lipids should be isolated quantitatively, free of nonlipid contaminants
such as proteins and in their native state for accurate further analyses. For this purpose, care
must be taken to ensure that lipolytic enzymes are deactivated. Precautions must also be taken
to avoid or at least to minimise auto- or enzymic oxidation of polyunsaturated fatty acids.

The analysis of crude fat content present in food sample involves refluxing extraction of fat
using ether in a Soxhlet apparatus. After evaporating the ether, the crude fat obtained will be
dried and weighed

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15.1.5.Ash contents (Using Muffle furnace)

Ash is the inorganic residue from the incineration of organic matter. Total ash content is a useful
parameter of the nutritional value of many foods and feeds. The ash content is determined from
the loss of weight that occurs during complete oxidation of the sample, at a high temperature
(usually 500-600oC) in a muffle furnace through volatilization of organic materials.

The determination of ash content is of value in the analysis of food for various reasons. The ash
content can be regarded, as a general measure of quality in certain foods often is a useful
criterion in identifying the authenticity of a food. The ash analysis has been chiefly used for the
determination of adulteration of certain foods. A high-ash figure suggests the presence of an
inorganic adulterant, and this condition is advisable to determine the acid-insoluble ash. High
levels of acid-insoluble ash indicate the presence of sand or dirt in the sample.

15.1.6.Chemical Methods

Chemical methods that may measure, freshness quality are estimation of (1) total volatile base
nitrogen (TVB-N) (2) trimethylamine (TMA) and (3) nucleotide degradation products (Inosine
mono phosphate (IMP), hypoxanthine etc.) and (4) lipid oxidation products (peroxides,
aldehydes etc.). Other chemical methods include estimation of indole, biogenic amines and
histamine .

15.1.7.Total volatile base nitrogen (TVB-N)

Spoilage organisms convert many nitrogenous compounds into volatile bases. Urea present in
elasmobranches is degraded to ammonia by bacterial action. The TVB-N developed during the
storage of unfrozen fish is ammonia and trimethylamine, which are characteristics of flavor and
odour changes.

Volatile bases are liberated when the sample is treated with K 2 CO 3 . The liberated NH3 is
absorbed in acids and the quantity absorbed is determined by the titration of excess of acids
against a standard alkali.

The measurement of these compounds is an indirect method of assessment of freshness quality

of seafood. This method is not capable of identifying the early stages of deterioration of
freshness quality because TVB-N values are not linearly related to the length of time (days), if
the species being evaluated was stored in ice and it cannot be used to predict the storage life of
that species. However, these values are useful for assessing quality during later stages of
storage, and therefore can be routinely used as a standard method for assessing the quality
changes in chilled, frozen, dried and canned seafood.

Levels of TVB-N in fish and fishery products

Item Maximum admissible limit

Fresh fish 35-40 mg/100 g

Frozen fish Not greater than 30 mg / 100g

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Salted & sun dried fish Not greater than 100 –200 mg / 100g

Canned seafood Not greater than 20 mg / 100g

15.1.8.Trimethylamine (TMA)

Trimethylamine (TMA) is formed from trimethylamine oxide (TMA-O) which in turn is converted
into dimethylamine (DMA) and formaldehyde

Values of TMA depend upon fish species, the time of year at which caught, location of the
catch, stage of spoilage, type of processing and / or storage and method of analysis. The
estimation of TMA is suitable for evaluating samples of medium to poor freshness quality of fish
stored in ice and in frozen condition.

TVB-N and TMA-N can be accurately measured by Conway micro diffusion method and
distillation method.

10-15 mg TMA-N / 100 g are usually regarded as the limits of whole round and chilled fish.
Levels above 15 mg TMA-N or 1.08m moles/tma/100g of fish are considered to be an indication
of spoilage of fish.

The fishy odour of TMA plus lipid is generally detectable when TMA level reaches 4 - 6 mg
n/100 g, which is before the actual spoilage point.

15.1.9.Nucleotide degradation products

The ATP of fish muscle breakdown either during the death struggle or subsequently. This
breakdown results in liberation of inosine 5’-monophosphate (imp) and then during storage, is
converted into hypoxanthine in a progressive degradation by dephosphorylation, and the action
of ribonuclease enzymes (fig.2). Concentration of free hypoxanthine present in the chilled stored
muscle of most species is as a consequence of these series of reactions. IMP is known to
contribute to the pleasant flavour of fresh fish. Its degradation to hypoxathine is a factor in the
progressive loss of desirable flavour and in the development of bitter ‘off’ flavour. Consequently,
the measurement of nucleotide degradation products may serve as useful indices of quality.

Estimation of Hypoxanthine : A protein extract of fish muscle is made using perchloric acid. A
portion of it is neutralised and the perchlorate is removed as its insoluble potassium salt. The
hypoxanthine in an aliquot of the neutral solution is converted to uric acid by the enzyme
xanthine oxidase and measured by its absorption at 290nm.

Most of the currently available chemical indices measure only bacterial spoilage; whereas the
assay of hypoxanthine also measures certain initial autolytic phases of deterioration. However, it
must be remembered when using any measurement based upon a nucleotide degradation and
hypoxanthine accumulation. In general, it can be assumed that if little or no hypoxanthine is
present, the fish is fresh. However, in good quality fish its value should not exceed 5 m mol/g.

Estimation of nucleotides : The ATP is converted to adenosine diphosphate (ADP), adenosine

monophosphate (AMP), inosine monophosphate (IMP), inosine, xanthine (HXR) and
hypoxanthine. These nucleotides can be separated by means of anion-exchange

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chromatography and measured using a spectrophotometer and k value is calculated which is
also used to assess the quality of fish.

HX + HXR

K value (%) = ---------------------------------------------------------- X 100

ATP + ADP + AMP + IMP + HXR + HX

K % of maximum 20 is acceptable for consumption of raw fish.

15.1.10Indole

Indole is an useful index of the freshness quality of shrimp during its non-frozen storage. Indole
is the decomposition product formed by the break down of tryptophan in the protein of muscle
by microbial enzymes. It has a fecal odour and is highly volatile.

Estimation of indole involves steam distillation followed by extraction of indole in CHCl3, colour
development using Ehrlich’s reagent, stripping down colour formed in acidic phase and
measuring in spectrophotometer or spectroflurometer at 290nm.

This method may be used to determine if raw shrimp had undergone high temperature abuse.
The cause of indole formation in raw shrimp is storage temperature rather than storage time.

Shrimps with less than 25micrograms/100g indole are organoleptically accepted.

15.1.11.Lipid oxidation products

1. Free fatty acids

The free fatty acid value is an indication of the extent of hydrolytic rancidity. During storage, fat
or fatty material may be hydrolysed by lipases to liberate free fatty acid and glycerol. Lipases
may be present in sources from which fat is extracted or produced by microorganisms during
storage.

The fat is extracted using chloroform, methanol and isopropanol in the ratio of 2:1:2 and then
titrated with a standard alkali using an indicator. The amount of free fatty acid present is
neutralised by the alkali and then calculated as the acid number. Acid number is defined as the
number of mg of potassium hydroxide required to neutralise the free fatty acid present in 1g of
fat of the sample.

The FFA content is determined by titration with a standard alkali. Acceptable free fatty acid
values must be established for each species, as the maximum limits will vary depending upon
the type of fish.

2. Peroxide value

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The peroxide value gives an indication of the extent of oxidative rancidity. It is a measurement
of the amount of oxygen absorbed at double bonds in unsaturated fatty acids contained in fish
oil, and is defined as the number of milli-equivalents of peroxide per kilogram of oil.

The unsaturated fatty acids present in fish fat undergo oxidation during storage and form
peroxides. These peroxides react with potassium iodide and liberate iodine in acid medium, the
estimation of which gives the amount of peroxide present.

- CH-CH = CH-CH - + O2 I 2-CH-CH-CH-CH +K1 + →

||

O-O

The liberated iodine is estimated using standard sodium thiosulphate solution with starch as
indicator. Estimation of peroxide value is used as an indicator of quality of fat.

Fresh oil usually has peroxide values of less than 1 meg/kg and may increase upto 1 0meg/kg.
Values of the order of 10 to 20 meg/kg usually indicate rancidity. The most common method is
based on iodometric titration that measures the iodine liberated from potassium iodide by the
peroxide present in the oil.

3. Thiobarbituric acid value

The TBA test measures the malonaldehyde produced during fat oxidation. It gives an indication
of the extent of oxidative rancidity. As spoilage progresses, there is a steady increase in the
TBA number.

The malonaldehyde and its derivatives can be measured through reaction with thiobarbituric
acid that produces a red chromogen, which can be measured spectrophotometrically at 538 nm.

Fish of good quality will have a TBA value of less than 2, and poorer quality than 2 will probably
smell and taste rancid. Heat processing, such as canning, causes a large decrease in TBA
value, so it is not a useful indicator of rancidity in canned products.

4. Iodine value

The iodine value is a measure of the degree of unsaturation. The glycerides of unsaturated fatty
acids react with a definite amount of iodine, which adds across the double bond. The iodine
value is often used to classify an unknown oil or fat into a particular class by determining the
degree of unsaturation for example, cod liver oil have an iodine value between 155 and 170.

5. Saponification value

This value refers to the number of milligrams of potassium hydroxide necessary to completely
saponify 1 gram of fat. This value gives an estimate of the average molecular weight of the fat.

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