Food Proteins

Our Team Style
(Guide)
Scientist
Dairy Chemistry Division
National Dairy Research Institute,
Karnal, Haryana-132001
Dr. Priyanka Singh Rao
(Guide)
Scientist
Dairy Chemistry Division
National Dairy Research Institute,
Karnal, Haryana-132001
Dr. Richa Singh

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BISHAL BARMAN
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RONIT GOSWAMI

Proteins
 They are essentially polymers of amino acids linked by amide
linkages.
 They are highly complex polymers made up of 20 different amino
acids.
 At the essential level, proteins contain 50-55% carbon, 6-7%
hydrogen, 20-23% oxygen, 12-19% nitrogen and 0.2-3 % Sulphur
on w/w basis.
 Protein synthesis occurs in the ribosomes and after synthesis,
cytoplasmic enzymes modify some of the amino acid constituents
and are classified into homoproteins and conjugated proteins

Amino acids are the building blocks (monomers) of proteins. 20 different amino acids are used to synthesize proteins.
The shape and other properties of each protein is dictated by the precise sequence of amino acids in it.
Each amino acid consists of an alpha carbon atom to which it is attached
A hydrogen atom
An amino group (hence "amino" acid)
A carboxyl group (-COOH). This gives up a proton and is thus an acid (hence amino "acid")
One of 20 different "R" groups. It is the structure of the R group that determines which of the 20 it is and its spe
cial properties.
AMINO ACIDS

Parts of an amino acid
Alanine

Amino acid 3-letter
abbreviation
1-letter abbreviation
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamic acid Glu E
Glutamine Gln Q
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
All 20 Amino acids

1. Aliphatic amino acids
2. Hydroxy amino acids
3. Acidic amino acids
4. Amide amino acids
5. Basic amino acids
6. Sulfur-containing amino acids
7. Aromatic amino acids
8. Secondary amino acids
Groups of Amino Acids

1.Aliphatic Amino Acids
Glycine (GLY)
NH3
+
C COO
-
H
H
Alanine (ALA)
NH3
+
C COO
-
H
CH3
NH3
+
C COO
-
H
CH
CH3 CH3
Valine (VAL)

1.Aliphatic Amino Acids
Leucine (LEU)
NH 3
+
C COO
-
H
CH
CH 3 CH 3
CH 2
Isoleucine (ILE)
NH3
+
C COO
-
H
CH
CH3
CH2CH3

2.Amino Acids with Alcohol
Serine (SER)
NH 3
+
C COO-
H
CH2 OH
NH 3
+
C COO-
H
CHOH
3CH
Threonine (THR)

3.Acidic Amino Acids
Aspartic Acid (ASP) Glutamic Acid (GLU)
NH3
+
C COO
-
H
CH2
COO
-
NH3
+
C COO
-
H
CH2
COO
-
CH2

4.Amino Acids with Amides
Asparagine (ASN) Glutamine (GLN)
NH3
+
C COO
-
H
CH2
C NH2
O
NH 3
+
C COO
-
H
CH 2
CH 2
C NH2
O

5.Basic Amino Acids
Lysine (LYS) Arginine (ARG)
NH C NH
NH3
+
C COO
-
H
CH2
CH2
CH2
CH2
NH3
+
NH3
+
C COO
-
H
CH2
CH2
CH2
2
NH2
+
Histidine (HIS)
NH3
+
C COO
-
H
CH2
NHHN
+

6.Sulfuric Amino Acids
Cysteine (CYS H) Cystine (CYS-CYS)
NH3
+
C COO
-
H
CH2
SH
NH3
+
CH CH2 S S CH2 CH NH3
+
COO
-
COO
-
Methionine (MET)
NH3
+
C COO
-
H
CH3
CH2
CH2
S

7.Aromatic Amino Acids
Phenylalanine (PHE) Tyrosine (TYR)
NH3
+
C COO
-
H
CH2
NH3
+
C COO
-
H
CH2
OH H
NH3
+
C COO
-
H
CH2
N
Tryptophan (TRY)

8.Secondary Amino Acids
Proline (PRO) Hydroxyproline (HYPRO)
COO
-
CH2
CH2H2C
H2N
+
CH
CH
OH
COO
-
CH2H2C
H2N
+
CH

Amide Linkage
H3N
+
C C O
-
R1 R2O
H H
O
H3N
+
C C O
-
H O
R1
H3N
+
C C N C C O-
HH
R2 O
H2O
OR2
H H
H3N
+
C C N
+
C C O
-
R1
O
-
H
-
+
Amide Linkage

1.Primary Structure: The primary structure of a protein refers to the linear
sequence in which the constituent amino acids are covalently linked through amide
bonds, also known as peptide bonds.
STRUCTURES OF PROTEIN

H
H H
H
HH
OO OO
R
R
R
R
H
HN3
+
N-terminal amino acid C-terminalamino acid
( )n
C C N C C N C C N C C O-
Primary structure of a protein

Formation by hydrogen bonding between peptide bond
Small negative charged oxygen atom = d-
Small positive charged hydrogen atom = d+
Kinds of Secondary Structure:
1. a - Helix
2. Pleated sheets structure
A. Parallel
B. Anti-parallel
2.Secondary Structure

3.Tertiary Structure:- Aggregation of individual protein.
1. Hydrophobic attraction: the close association, attraction of hydrocarbon side-chains.
2. Ionic bond: between positively charged groups and negatively charged groups.
3. Hydrogen bonds
4. Disulfide bonds
A protein has size and shape as well as unique arrangement through hydrogen, ionic,
hydrophobic and disulfide bonds

4.Quaternary Structure
A protein has size and shape as well as unique arrangement of its polypeptide
chains. (Aggregation of several peptide chains to form a definite molecule by ionic
bond, hydrogen bond, and/or hydrophobic bond).

FOOD PROTEINS
They may be defined as those easily
digestible, non toxic, nutritionally adequate,
functionally usable in food products, available
in abundance and agriculturally sustainable.

Functions of Food Proteins
 Growth and Maintenance
 Causes Biochemical reactions
 Acts as a messenger
 Provides structure.
 Maintains proper pH inside our body
 Balances fluids
 Provides energy

Protein
Efficiency Ratio
Protein Quality
Determination
Biological Value
.
Protein
Digestibility
Amino Acid
Score
Digestible
Indispensabl
e Amino Acid
Score
Net Protein
Utilization

The PER was the first method adopted for
routine assessment of protein quality of food.
.
Protein Efficiency Ratio
It is actually the ratio of grams of body weight
to the grams of protein consumed.
PER=
𝐺𝑎𝑖𝑛 𝑖𝑛 𝑏𝑜𝑑𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔)
𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝐼𝑛𝑡𝑎𝑘𝑒(𝑔)
A high PER (>2.5) is assigned to proteins that
are useful in promoting growth.

Protein Efficiency Ratio of some food commodities
Product PER
Soybean 2.32
Cotton Seed 2.25
Egg 3.90
Chick Peas 1.68
Peanuts 1.65
Kidney Beans 0.88

Protein Efficiency Ratio
PER was found to be the within the range of 1.2-2.4 for plant proteins (including
pea flour, soy proteins, beans) and could be as low as 0.95 for wheat flour,
whereas animal protein were in the range of 3.1-3.7 (Sarwar et al., 1984; Cruz et
al., 2003

BV of a protein is an expression of a no. of nutritional
characters of food viz.
 The digestibility
 The availability of the digested product &
 The presence and amount of various essential amino
acids
.
Biological Value The Biological Value can be calculated by determining
the nitrogen of the food intake and then deducting the
urinary and fecal nitrogen excretion.
PER =
𝐷𝑖𝑒𝑡𝑎𝑟𝑦 𝑁− 𝑈𝑟𝑖𝑛𝑎𝑟𝑦 𝑁−𝐹𝑒𝑐𝑎𝑙 𝑁
𝐷𝑖𝑒𝑡𝑎𝑟𝑦 𝑁−𝐹𝑒𝑐𝑎𝑙 𝑁
* 100

Biological Value
 Animal Proteins have higher biological value because they have all the essential amino acids required in the
human body in comparison to plant protein which do not contain all the essential amino acids.
 Quinoa and Buckwheat are complete sources of protein from plant source.

Net Protein Utilization of a food is the percentage of
protein contained in the food which is retained by the
body after the food has been consumed.
.
Net Protein Utilization
It is actually used to describe the value or usefulness of
certain proteins in a diet.
As a value, NPU can range from 0 to 1 with a value of 1
indicating 100% utilization of dietary nitrogen as protein.
Foodstuffs such as eggs and Milk are rated as 1 on
NPU chart.
NPU =
𝑁𝑖𝑡𝑟𝑜𝑔𝑒𝑛 𝑟𝑒𝑡𝑎𝑖𝑛𝑒𝑑
𝑁𝑖𝑟𝑜𝑔𝑒𝑛 𝐼𝑛𝑡𝑎𝑘𝑒
∗ 100

Method adopted by FAO/WHO as the preferred
method for the measurement of protein value in
human nutrition.
.Protein Digestibility Corrected
Amino Acid Score (PDCAAS)
It is actually the method of evaluating the quality of
a protein based on both the amino acid
requirements and their ability to digest it.
It compares the amount of the essential amino
acids in the food to a reference pattern, based on
the essential amino acid requirements of a 2 to 5-
year old child to determine its most limiting amino
acid
Proteins are made of many amino acids and
PDCAAS evaluates a food’s protein quality by
comparing its amino acid composition to what our
bodies can use.

The highest PDCAAS value that any protein can achieve is 1.0. Generally casein, whey, soy,
and egg are considered good quality proteins and have PDCAAS scores of 1.00, while those of
tree nuts are a bit under 0.50 and wheat gluten even lower. The graph below compares some of
the common protein sources and range out there.
Protein Digestibility Corrected Amino Acid Score (PDCAAS)

DIAAS replaced PDCAAS after its introduction by
FAO in 2013.
.Digestible Indispensable Amino
Acid Score
It determines the amino acid digestibility at the end
of small intestine, providing a more accurate
measure of amount of amino acid absorbed by the
body and the protein’s contribution to amino acid
and nitrogen requirements.
• DIAAS <75% - Suboptimal
• >75% - <100% - Good Protein quality
• >100% - Excellent or high quality
The DIAAS methopd better reflects the true
nutritional value of dietary protein for humans than
does the PDCAAS nethod.

Some protein rich foods
PRODUCT PROTEIN
(per 100
gm)
Beef Jerky 30-40
Parmesan 32
Tuna Steak 32
Pumpkin Seeds 30
Turkey 30
Peanuts 25-28
Edam Cheese 27

PRODUCT PROTEIN
(per 100
gm)
Cheddar 25
Seitan 25
Beef 20-24
Chicken 24
Salmon 24
Stilton 24
Almonds 21

PRODUCT PROTEIN
(per 100
gm)
Sardines 21
Cod 20
Lamb 20
Mackerel 20
Pistachios 20
Pork Ion 17-20
Tempeh 20

PRODUCT PROTEIN
(per 100
gm)
Cashew Nuts 18
Mozzarella 18
Mussel 18
Chia seeds 17
Walnuts 15-17
Source:- https://www.coachmag.co.uk/nutrition/healthy-eating/3525/high-protein-foods-16-of-the-best

Recommended Dietary Allowance for proteins
 The Recommended Dietary Allowance (RDA) for protein is a modest 0.8 grams of protein
per kilogram of body weight.
Source:- https://nchstats.com/2010/03/03/adults%E2%80%99-daily-protein-intake-much-more-than-recommended/
AGE GROUP GRAMS OF
PROTEIN
NEEDED/DAY
Children ages 1-3 13
Girls ages 14-18 46
Boys ages 14-18 52
Women ages 19-70+ 46
Men ages 19-70+ 56

Animal Protein vs Plant Protein
Animal Protein Plant protein
90% Absorption 60-70% Absorption
95-100% Digestibility 85% digestibility
High Biological value Less biological value due
to limiting amino acids

Classification
.
Based on Composition
Based on Function
Based on essential amino acid
availability on the food
Based on Size and Shape
Based on Solubility
.Based on Biological Value

1. Based on Composition
Simple Proteins
 They only yield amino acids on
hydrolysis.
 Eg:-Albumins, Globulins,
Prolamins, Glutelin, Histones,
Protamine, Albuminoids
Compound Proteins
 These are simple proteins
combined with some non protein
substances known as Prosthetic
Groups.
 Eg:-Nucleoproteins,
Mucoproteins, Phosphoproteins,
Chromoproteins
Derived Proteins
 They are derived by partial to
complete hydrolysis from
Simple or Compound proteins
by the action of acids, alkalis
or enzymes.
 They are further classified into
Primary derived protein and
secondary derived protein.

Derived Proteins
Primary Derived
 They are formed by processes causing
slight changes in the protein molecule
and its properties
 There is little or no hydrolytic cleavage of
peptide bonds.
 Eg:-Coagulated proteins, Protean,
Metaproteins
Secondary Derived
 They are formed in the progressive
hydrolytic cleavage of the peptide bonds
of protein molecule.
 They are roughly grouped into Proteoses,
Peptones and Peptides according to their
average molecular weight

2. Based on Size & Shape
Globular Proteins
 They are spherical proteins and are one
of the most common types
 They are somewhat water soluble unlike
the Fibrous or membrane proteins.
 Eg:-Hemoglobin
Fibrous Proteins
 Fibrous or Scleroproteins are proteins with
an elongated shape and are insoluble in
water.
 They provide structural support for cells
and tissues.
 Eg:- Keratin and Collagen

3. Based on Solubility
1. Albumin
 These proteins are soluble
in distilled water, dilute
salt, acid and base
solutions.
 Eg:-lactalbumin, egg
albumin.
2.Globulin
 Insoluble in distilled
water, but soluble in
dilute salt, acid and
base solutions
 Eg:-serum globulins
and β-lactoglobulin in
milk, myosin and actin
in meat.
3. Protamine and
Histones
 These proteins are
highly soluble in
distilled water
 Protamine is soluble in
NH4OH, whereas
histones are insoluble in
NH4OH.
4. Glutelin
 These proteins are
insoluble in distilled
water and alcohol but
soluble in dilute acid and
base solution.
 Eg:-glutenin in wheat,
oryzenin in rice.

Based on Solubility
5. Prolamins
 These proteins are insoluble
in distilled water, but soluble
in dilute acid, dilute base
and 70-80% alcohol.
 Eg:-zein in corn, gliadin in
wheat
6. Sceloroproteins
 These proteins are insoluble in
most of the solvents like water,
dilute acid, dilute base, dilute
salt solution etc.
 They are generally fibrous
proteins serving structural and
binding purposes.
 Eg:- Collagen, elastin, keratin.

4. Based on Function
1.Catalytic
 They have the ability to
function within the living
cells as Biocatalysts
 These Biocatalysts are
called Enzymes.
 They enhance the
reaction rates a million
fold
 Eg:- Catalase
2.Regulatory
 Are polypeptides and
small proteins found
in relatively low
concentrations in
animal kingdom.
 Eg:- Growth
hormone, Insulin
3. Protective
 Has a protective
defense function by
combining with
foreign protein and
other substances
and fight with certain
diseases.
 Eg:- Immunoglobulin
4.Storage
 It is a major class of
proteins which has the
function of storing
amino acids as nutrients
and as building blocks
for the growing embryo.
 Eg:- Egg albumin,
Casein.

Based on Function
Transport proteins
5. Transport proteins
 Are capable of binding and
transporting specific types of
molecules through blood.
 Hemoglobin is a conjugated
protein composed of colorless
basic protein, the globin and
ferroprotoporphyrin or haem.
 It has the capacity to bind with
oxygen and transport through
blood to various
tissues.
6. Toxic proteins
 Some of the proteins are toxic
in nature.
 Ricin present in castor bean
is extremely toxic to higher
animals in very small
amounts.
 A bacterial toxin causes
cholera, which is a protein.
7. Structural proteins
 These proteins serve as
structural materials or as
important components of
extra cellular fluid.
 Examples of structural
proteins are myosin of
muscles, keratin of skin
and hair and collagen of
connective tissue.

Based on Function
Exotic proteins
Antarctic fishes live in -
1.9oC waters, well below the
temperature at which their
blood is expected to freeze.
These fishes are prevented
from freezing by antifreeze
glycoproteins present
in their body.
8. Contractile proteins
 Proteins like actin and
myosin function as
essential elements in
contractile system of
skeletal muscle
9. Secretary proteins
 Fibroin is a protein secreted
by spiders and silkworms to
form webs and cocoons.
10. Exotic proteins
 Antarctic fishes live in -1.9oC
waters, well below the
temperature at which their
blood is expected to freeze.
 These fishes are prevented
from freezing by antifreeze
glycoproteins present in
their body.

5. Based on Essential Amino acid availability
Complete
 All essential amino acids
are available in it needed by
the body in exact
proportion.
 Eg:- Meats, Poultry, Eggs
Incomplete
 Lacks one or amino acid
 Cannot build tissue without
help
 Eg:- Dried Beans, peas,
grains, Vegetables, nuts and
seeds.
Complementary
 Are combination of two or
more incomplete proteins that
supply all nine essential
amino acids
 Eg:- Grains + Legumes/
Vegetables, Nuts/Seeds +
Vegetable/Legumes

6.Based on Biological Value
High Biological Value
 Comes mainly from animal
foods such as meat, fish,
cheese, eggs and milk.
Low Biological Value
 Comes mainly from plant
foods such as peas, beans
and lentils, whole cereals
and nuts. These foods also
contain fibers and are low in
fat

Foaming
Emulsification
Crystallization
Viscosity
Amphoteric behavior
Ion Binding
Solubility
Isoelectric Point
Refractive Index
Protein Hydration/ Swelling
Gelation
Flavor binding
Absorption of UV
Physico- Chemical Aspects of Proteins

1.Protein Hydration / Swelling
 Water is an essential constituent of foods. The rheological and textural
properties of foods depend on the interaction of water with other food
constituents, especially with proteins and polysaccharides.
 In low- and intermediate-moisture foods, such as bakery and comminuted meat
products, the ability of proteins to bind water is critical to the acceptability of
these foods. The ability of a protein to exhibit a proper balance of protein–protein
and protein–water interactions is critical to their thermal gelation properties.
 There are two mechanisms whereby this swelling occurs.
 Donnan swelling:- which is reversible and caused by interactions between
ions and charged sites on the protein. To maintain electrical neutrality in the
swollen phase, small ions of opposite charge migrate from the solution to the
swollen phase. These excess ions in the swollen phase give rise to an osmotic
pressure which causes the swelling.
 Lyotropic swelling – which is irreversible and caused by non ionic reagents
which act by altering the water structure around the protein, interrupting the
hydrogen bonds and or through direct competition with internal hydrophobic
interactions.

2.Isoelectric point
 The isoelectric point, is the pH at which a molecule carries no net electrical
charge or is electrically neutral in the statistical mean.
 At isoelectric point protein will not migrate when an electric field is applied. At
isoelectric point its ionization is minimum – least soluble.
 Each protein have its own characteristic isoelectric point – due to difference in
amino acids make up. This character of protein often helps in its separation
 The major milk protein casein has an isoelectric point of 4.6.

3.Solubility
 The functional properties of proteins are often affected by protein solubility, and
those most affected are thickening, foaming, emulsifying, and gelling. Insoluble
proteins have very limited uses in food.
 Aggregation of proteins, which eventually leads to protein insolubility, in water
involves a balance between repulsive electrostatic interaction, which favors
solubilization, and attractive van der Waals and hydrophobic interaction, which
favors precipitation, between protein molecules.
 Protein solubility is minimal at the isoelectric point since at this pH the net charge on
the protein is zero and consequently electrostatic repulsive forces are minimal while
interaction between protein molecules is maximal.

3. Solubility
 Relationship between salt concentration and solubility is really complex.
 Globulins which are soluble in 5-10 % salt solutions, are insoluble in water while
albumins are readily soluble in both water and dilute salt solutions. However, in
concentrated salt solution ; all proteins become less soluble.
 The increase in solubility in dilute salt solution observed with globulins is known as
“Salting – in”. In dilute salt solution, salt molecules stabilize protein molecules by
decreasing the electrostatic energy between the protein molecules which increase
the solubility of proteins.
 The decreasing solubility of proteins at high salt concentration is known as
“Salting - out”. Protein molecules get dehydrated. The large number of salt ions
in the solution will get hydrated and organize water molecules around them, thus
reducing the water available for the protein molecules. Since protein solubility
depends on whether ‘clustering’ around the hydrophilic groups, the ‘dehydrated’
proteins will precipitate

4. Ion Binding
 As ampholytes, proteins can bind both anions and cations. Several ions will form
insoluble salts with proteins and this phenomenon is widely used to remove
proteins from solutions.
 E.g. Trichloro acetic acid (TCA) is used to separate protein nitrogen from non
protein nitrogen. It is possible to obtain interactions between proteins and charged
macromolecules such as alginates and pectates. These type of complexes have
great potential in the food.

5.Amphoteric Behavior
 Like amino acids, proteins are ampholytes, i.e. they act as both acids and bases. At al
but the extremes of pH, possess both positive and negative charged groups.
 Proteins behave like good buffers, due to their acid–base groups (amino and
carboxylic groups), with the highest resistance to change pH when the pH is close o
equal to their pKa.
 Owing to the presence of carboxylate groups of the acidic amino acids carboxylate
group at the end of the chain, most protein solutions are good buffers below pH 5.
 Similarly owing to the ε-amino groups of lysine, the guanidinium group of arginine and
the phenolic hydroxyl group of tyrosine, most proteins are good buffer at pH values
above 9.
 However at neutral pH values, most proteins have limited buffering capacity. This
buffering is of great importance in many living tissues.
 The demonstration presented here shows that a protein can neutralize both basic and
acidic solutions. Thus, a protein can act as a buffer. The amphoteric properties of a
protein can be represented by the following:-

6. Crystallization
 Protein crystallization is the process of formation of a regular array of individua
protein molecules stabilized by crystal contacts. If the crystal is sufficiently ordered, i
will diffract..
 The crystallization of protein may be obtained by addition of a salt such as ammonium
sulphate or sodium chloride and adjustment towards iso-electric pH.
 The addition of definite amount alcohol or acetone is occasionally advantageous. The
added substances and adjustment to isoelectric pH decrease the solubility of the
protein. The protein is also least dissociated at the isoelectric pH and crystallize best in
the form of protein salts.
Crystals of Lysozyme observed through polarizing filter

7. Emulsification
 Margarine, mayonnaise, spreads, salad dressings, frozen desserts, frankfurter
sausage, and cakes, are emulsion-type products where proteins play an importan
role as an emulsifier.
 In natural milk, a membrane composed of lipoproteins stabilizes the fat globules
When milk is homogenized, a protein film comprised of casein micelles and whey
proteins replaces the lipoprotein membrane.
 The emulsifying properties of food proteins are evaluated by several methods such as
size distribution of oil droplets formed, emulsifying activity, emulsion capacity (EC),
and emulsion stability.

8.Foaming
 Foams consist of an aqueous continuous phase and a gaseous (air) dispersed
phase. Many processed foods are foam-type products. Such as whipped cream, ice
cream, cakes, meringue, bread, souffles, mousses, and marshmallow.
 In most of these products, proteins are the main surface-active agents that help in
the formation and stabilization of the dispersed gas phase. The foaming property of
a protein refers to its ability to form a thin tenacious film at gas–liquid interfaces so
that large quantities of gas bubbles can be incorporated and stabilized.
 The foamability or foaming capacity of a protein refers to the amount of interfacial
area that can be created by the protein. It can be expressed in several ways, such
as overrun (or steady state foam volume) or foaming power (or foam expansion).
Egg white making a foam

9.Viscocity
 The consumer acceptability of several liquid and semisolid-type foods (e.g., gravies
soups, beverages) depends on the viscosity or consistency of the product. It greatly
affects the textural appeal, flavor and visual appeal.
 Protein solutions, do not display Newtonian behavior, especially at high concentrations
For these systems, the viscosity coefficient decreases when the shear rate increases
This behavior is known as “pseudoplastic” or “shear thinning”.
 In rheology, shear thinning is the non-Newtonian behavior of fluids whose viscosity
decreases under increase in shear strain. The pseudoplastic behavior of protein
solutions arises because of the tendency of protein molecules to orient their major axes
in the direction of flow.
 When flow is stopped, the viscosity may or may not return to the original value
depending on the rate of relaxation of the protein molecules to random orientation
Solutions of fibrous proteins, for example, gelatin and actomyosin, usually remain
oriented and thus do not quickly regain their original viscosity, whereas solutions o
globular proteins, for example, soy proteins and whey proteins, rapidly regain thei
viscosity when flow is stopped.

10. Flavor Binding
 Proteins themselves are odorless.
 Several proteins, especially oilseed proteins and WPCs, carry undesirable flavors
which mainly due to aldehydes, ketones, and alcohols generated by oxidation o
unsaturated fatty acids. Upon formation, these carbonyl compounds bind to proteins
and impart characteristic off-flavors.
 For example, the beany and grassy flavor of soy protein preparations is attributed to
the presence of hexanal.
 The flavor-binding property of proteins also has desirable aspects, because they can
be used as flavor carriers or flavor modifiers in fabricated foods but it should bind
flavors tightly, retain them during processing, and release them during mastication o
food in the mouth.
 In dry conditions, proteins binds flavors with van der Waals interactions, hydrogen
bonding and electrostatic interactions.
 In liquid or high moisture foods, proteins bind flavors
through hydrophobic region on the protein surface.
 Oil seed proteins and whey proteins carry undesirable
flavors and limit their food applications.

11. Gelation
 Protein gelation refers to transformation of a protein from the “sol” state to a “gel-
like” state. Heat, enzymes, or divalent cations under appropriate conditions facilitate
this transformation. All these agents induce formation of network structure.
 Most food protein gels are prepared by heating a moderately concentrated protein
solution. In this mode of gelation, the protein in a “sol” state is first transformed into
a “progel” state by denaturation.
 The progel state however is usually a viscous liquid state in which some degree of
protein denaturation and polymerization has already occurred.
 Also, in the progel state, a critical number of functional groups, such as hydrogen
bonding and hydrophobic groups that can form intermolecular noncovalent bonds,
become exposed so that the second stage, i.e. formation of a protein network, can
occur.
 When the progel is cooled to ambient or refrigeration temperature, the decrease in
the thermal kinetic energy facilitates formation of stable noncovalent bonds among
exposed functional groups of the various molecules and this constitutes gelation.
Sol Gel

12. Optical Activity
 The rotatory power of amino acid is affected by various factors which influence the
degree and the nature of the electrolytic dissociation of the amino acid. These
include
- The concentration of amino acid itself.
- pH of solution.
- The nature of solvent.
- The presence of electrolytes.
- The temperature.
 Optical rotation is an important property of proteins in which they differ widely.
Specific rotations of proteins obtained at 20°Cand using D line of sodium are
always negative and for globular proteins the values of [α]D 20] are usually within
the range of -30° to -60°.
 Denaturation of proteins produces marked increases in optical rotation.
Measurement of this property is a sensitive means of following denaturation.

13. Absorption of UV
 The absorption of ultra violet light with a wavelength of 280 nm is a characteristic
of proteins that depends on their content of the aromatic amino acids (tyrosine,
tryptophan and phenylalanine)
Aromatic amino acids such as tryptophan
tyrosine, and phenylalanine have absorbance
maxima at ∼ 280 nm. Each purified protein has a
distinct molecular absorption coefficient a
around 280 nm, depending on its content o
aromatic amino acids

14. Refractive Index
 The refractive index of protein solutions increases linearly with
concentration. The difference between the refractive index of a 1 %
protein solution and its solvent is called specific refractive increment
Most proteins have a refractive index increment of about 0.0018.

Reactions involved in processing and reactions with alkali
Denaturation Racemization
Deamidation
Heat treatment at alkaline pHDesulphuration

.
Disadvantages due to the reactions involved in
processing
Conversion of essential amino acids into
derivatives which are not metabolizable
Destruction of essential
amino acids in the product
Formation of Toxic
degradation compounds
Decrease in digestibility due to cross-linking

Denaturation
Normal Denatured
 Denaturation is a major change in the native structure that does not involve
alteration of the amino acid sequence
o Effect of heat usually involves a change in the tertiary structure, leading
to a less ordered arrangement of the polypeptide chains
o The temperature range in which denaturation and coagulation of most
proteins takes place is about 55°C to 75°C
o Casein and Gelatin are examples of proteins that can be boiled without
apparent change in stability.

Denaturation
o Denaturation is a phenomenon wherein a well-defined initial state of a protein formed under
physiological conditions is transformed into an ill-defined final state under non physiological conditions
using a denaturing agent.
o It does not involve any chemical changes in the protein.
o Denaturation has a negative connotation, because it indicates loss of some properties. Enzymes lose
their activity upon denaturation.
o Denaturation usually causes loss of solubility and some functional properties.

Denaturation ( Advantages )
 Proteinaceous anti-nutritional factors present in seeds and legumes are denatured and inactivated by mild
heat treatments.
 Partial denaturation of proteins at the air–water and oil–water interfaces improves their foaming and
emulsifying properties.
 Denatured proteins are more digestible than native proteins.
 These inhibitors impair efficient digestion of proteins and thus reduce their bioavailability.
 Certain proteinaceous toxins, e.g. botulism toxin and enterotoxins are inactivated.
Enterotoxin Botulism Toxin

Denaturation ( Disadvantages )
 Excessive thermal denaturation of soy proteins diminishes their foaming and emulsifying properties.
 In protein beverages, where high solubility and dispersibility of proteins is required, partial protein
denaturation during processing may cause flocculation and precipitation during storage of the product.
 Denatured protein losses its biological value.
 Most of the enzymes gets inactivated due to denaturation of proteins.

Denaturation agents
Physical Agents
 Acids and alkali
 Organic solvents
 Salts of heavy metals
 Chaotropic agents
 Detergents
 Altered pH
Chemical Agents
 Heat
 Violent shaking
 Hydrostatic pressure
 UV radiation

Desulfuration
 Thermal treatments of proteins or proteinaceous foods at high temperature and in the absence of any added
substances can lead to several chemical changes. Most of these chemical changes are irreversible and some
of these reactions result in the formation of amino acid types that are potentially toxic.
 Thermal treatments like sterilization at temperature above 115°C bring about the partial destruction of
cysteine and cystine residues and formation of H₂S, dimethyl sulfide and cystic acid; and H₂S other volatile
compounds produced contribute to the flavor of these heat treated foods.
 Sulphur containing amino acid like Cysteine and homo-Cysteine are deaminated by primary desulphuration
forming imino acid. It is then spontaneously hydrolyzed to α keto acid and NH3 is made free.

Deamidation
 Deamidation is a chemical reaction in which an amide functional group in the side chain of the amino
acids asparagine or glutamine is removed or converted to another functional group.
 This reaction takes place during heating of proteins at temperatures above 100°C. The ammonia released
comes mainly from the amide groups of glutamine and asparagine, and these reactions do not impair the
nutritive value of protein.
 Due to the unmasking of the carboxyl groups, the isoelectric points get affected and therefore the functional
properties of proteins are modified.

Racemization
 Severe heat treatment at temperatures above 200°C as well
as heat treatment at alkaline pH (e.g. in texturized foods)
invariably leads to partial racemization of L-amino acid
residues to D-amino acid residues.
 Since D-amino acids have no nutritional value, racemization
of an essential amino acid reduces its nutritional value by
50%.
 Racemization of amino acid residues causes a reduction in
digestibility because peptide bonds involving D-amino acid
residues are less efficiently hydrolyzed by gastric and
pancreatic proteases. This leads to loss of essential amino
acids that have racemized and impairs the nutritional value
of the protein.
 D-amino acids are also less efficiently absorbed through
intestinal mucosal cells and even if absorbed they can’t be
utilized in in-vivo protein synthesis.
Biological effects of racemization
o Decreases in the vitro digestibility of alkaline-
treated proteins
o Alkaline-treated casein was much more resistant
to enzymatic hydrolysis than untreated casein

Effect of heat treatment at alkaline pH:
 Heating of proteins at alkaline pH or heating above 200oC at neutral pH can result in β-elimination
reaction.
 The first stage of this reaction involves abstraction of proton from α-carbon atom resulting in formation of
carbanion.
 The carbanion derivative of cysteine, cystine and phosphoserine undergoes second stage of β-elimination
reaction leading to formation of dehydroalanine
 The resulting dehydroalanine residues are very reactive and react with nucleophilic groups such as ε-amino
group of lysine, thiol group of cysteine and delta-amino group of ornithine (degradation product of arginine)
 These reactions result in formation of lysinoalanine, lanthionine and ornithoalanine cross-links respectively
in proteins.
 Formation of protein-protein cross-links in alkali treated proteins decreases their digestibility and biological
value.

Maillard Reaction
 Maillard reaction (non-enzymatic browning) refers to a complex set of reactions
initiated by reaction between amines and carbonyl compounds, which, at elevated
temperatures, decompose and eventually condense into insoluble brown products
known as melanoidins.
 This reaction occurs not only in foods during processing but can also occur in
biological systems.
 In either case, proteins and amino acids generally provide an amino component
while reducing sugars, ascorbic acid and carbonyl compounds generated from
lipid oxidation provide the carbonyl component.

Maillard Reaction
1st Step:- formation of N glycoside
2nd Step:- After formation of N glycoside the immonium ion is formed and then isomerize, this reaction is called
Amadori rearrangement and forms a compound called ketosamine
3rd Step:-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.

Advantages and Disadvantages
 The positive contributions of the Maillard reaction are sensory attributes generation, such as
color, flavor, aroma and texture.
 The negative aspects are off-flavor development, flavor loss, discoloration, and loss of
protein nutritional value.
https://www.researchgate.net/publication/221925380_Maillard_Reaction_Products_in_Processed_Food_Pros_and_Cons

Significance
1. Production of color
Desirable as in coffee, chocolate bread crust, toast etc. Undesirable, as in milk & milk products
(khoa, condensed milk, milk powder etc.) and in many intermediate moisture products.
2. Production of flavor and off flavor
Flavor (odor) are due to formation of volatile products e.g. fission products and Strecker
aldehydes. Substances tasting sweet & bitter may be involved.
3. Antioxidant properties
 Maillard reaction products are reported by have antioxidant properties.
 This is thought to be due to formation of reductones, chelating of heavy metals, which may
otherwise act as a prooxidant.
4. Nutritional implications
 Intrinsic toxicity is due to nutritional properties of Maillard products and intermediates
 One of the important reasons for interest of food industry in Maillard browning is its relation
to nutrition.
 Considerations in this regards are reduction in nutritive value.
 Loss of essential amino acids - especially lysine.

Proteolytic Enzymes
 Processes involving proteolysis play important role in the production of many foods.
 Proteolysis can occur as a result of proteinases in the food itself, e.g., autolytic reactions in
meat, or due to microbial proteinases, e.g., the addition of pure cultures of selected
microorganisms during the production of cheese.
 The main subgroups formed are:
a. Peptidases:-(exopeptidases) that cleave amino acids or dipeptides
stepwise from the terminal ends of proteins.
b. Proteinases:-(endopeptidases) that hydrolyze the linkages within
the peptide chain and not attacking the terminal peptide bonds.
Eg:-Pepsin, trypsin

Types of proteolytic enzymes
Aspartic Endopeptidases
o Enzymes of animal origin, such as pepsin
and rennin active in the pH range of 2–4.
o At pH 6–7 rennin cleaves a bond of casein
causing curdling of milk.
o The pepsin-like enzymes are produced by
e.g. Aspergillus niger.
o The rennin-like enzymes are produced by
e.g. Aspergillus usamii.
Sulfhydryl proteases
o They are mostly of plant origin e.g. papain, ficin,
bromelain.
o The active sites of these plant enzymes contain a
cysteine and a histidine group that are essential for
enzyme activity. These enzymes catalyze the
hydrolysis of peptide, ester and amide bonds.
o Proteolytic enzymes (papain, ficin, bromelain)
prevent this type of haze(the combination of
polypeptide and tannin molecules in beer)by
reducing the polypeptide size.

Types of proteolytic enzymes
Serine endopeptidase
o Enzymes of this group, in which
activity is related to the pH range of
7−11, are denoted as alkaline
proteinases.
o Typical representatives from animal
sources are trypsin, chymotrypsin,
elastase and thrombin.
o Serine proteinases are produced by
great number of bacteria and fungi,
B. subtilis and Aspergillus flavus
Cysteine Endopeptidases
o Typical representatives of this group of
enzymes are: Papain, bromelain, ficin,
Streptococcus proteinase.
o The range of activity of these enzymes
is very wide and, depending on the
substrate, is pH 4.5–10, with a
maximum at pH 6–7.5
o Working mechanism is similar to Serine
Endopeptidase
Metalo Peptidases
o This group includes exopeptidases,
carboxypeptidases A and B,
aminopeptidases, dipeptidases and
prolinase, and endopeptidases from
bacteria and fungi, such as B.
subtilits, and Aspergillus oryzae.

Applications of Proteolytic Enzymes
.
The traditional applications of enzymes in the
seafood industry have been limited to very few
products (Fish protein hydrolysate, fish sauce or
cured herring). These processes are based on
endogenous proteases in the fish (Haard, 1992)
Sea Food Industry
.
Enzymatic milk coagulation
The food and nutrition board of the United States
national research council uses the term “rennet”
to describe all milk clotting enzyme preparations
(except porcine pepsin) used for cheese making.
The proteases produced by GRAS (Genetically
Regarded As Safe) cleared microbes such as
Mucor miehei, Bacillus subtilis, Endothia
parasitica are gradually replacing chymosin in
cheese making
Use of proteases to improve functional properties.
The insoluble heat denatured whey protein is
solubilized by treatment with immobilized trypsin.
Endo and exoproteases from Aspergillus oryzae
have been used to modify wheat gluten by limited
proteolysis. (Chen and Li, 1988).
Improve functional properties

THEORIES OF FORMATION OF TEXTURAL PROTEINS
Texturization
Spun fiber
Texturization
Extrusion
Texturization

Texturization
The globular protein is unfolded during texturization by
breaking the intramolecular binding forces. The resultant
extended protein chains are stabilized through interaction
with neighboring chains.
The protein produced for nutrition is currently about 20%
from animal sources and 80% from plant sources. Many
plant proteins have a globular structure and, although
available in large amounts, are used to only a limited
extent in food processing.
Suitable processes give products with cooking strength
and a meat-like structure. They are marketed as meat
extenders and can be used whenever a lumpy structure
is desired.
https://www.youtube.com/watch?v=GYl79Yt5jXo

Spun Fiber Texturization
A highly concentrated (~20% w/v) soy
protein isolate solution is adjusted to
pH 12–13 and aged until the viscosity
of the solution increases to 50,000–
100,000 centipoise as a result of
protein denaturation and certain alkali-
induced cross-linking reactions.
 This highly viscous “dope” is then pumped through a
spinneret, a device with a plate containing thousands of
micron-size holes.
The fibrous extrudate is passed through a bath
containing phosphoric acid and salt at pH 2.5 which
coagulates an becomes a fibrous mass.
 Next, it is "towed” through steel rolls where it is
compressed and stretched to enhance its strength.
The fiber is then passed through a
washing bath where excess acidity and
salt are removed. They are then passed
through a series of tanks containing fat,
flavors, colors, and binders depending
on the requirement
Then it is heated at 80°C–90°C to induce
gelation of the binder protein. Egg white is
often used as a binder because of its
excellent heat coagulation properties. The
final product is dried and sized.

Extrusion Texturization
2
3
4
5
1
After cooling, the protein polysaccharide matrix possesses a highly expanded dry structure. The porous material is
able to absorb 2 to 4 times its weight of water giving a fibrous, spongy structure with chewiness like meat. These
products are stable even under sterilization conditions.
The mixture is then extruded through a small diameter orifice into normal pressure environment. This results in flash
evaporation of the internal water with the formation of expanding steam bubbles leaving behind vacuoles in the
protein chunks.
Over a period of 20-150 s, the mixture is elevated to a temperature of 150-200oC. Under these conditions, the
mixture is transformed into a plastic viscous state, in which solids are dispersed. Hydration of the proteins takes
place after partial unfolding of the globular proteins.
The moisture content of the starting material is adjusted to 30-40% and the additives are incorporated. The
protein mixture is fed to the extruder where it is exposed to a high pressure(10,000 to 20,000 kPa)
Extrusion cooking was first introduced in food and feed processing in the late 1950s. Since then, the systems
involved have grown in popularity, efficiency and flexibility. Extrusion cooking technology is most used for cereal and
protein processing in the food industry and is closely related to the pet food and feed sectors.

Schematic representation of an extruder including its main parts and zones

Kjeldahl Method.
Dye Binding Method
Biuret Method
Lowry Method
Ultraviolet Method
Fluorescence Method
Protein Estimation Methods
Dumas Method

1.Kjeldahl Method - Nitrogen Determination
 The Kjeldahl method was developed over 100 years ago for determining the nitrogen contents in organic and
inorganic substances. Although the technique and apparatus have been modified over the years, the basic
principles introduced by Johan Kjeldahl still endure today.
 Kjeldahl nitrogen determinations are performed on a variety of substances such as meat, feed, grain, waste
water, soil, and many other samples.
 Various scientific associations approve and have refined the Kjeldahl method, including the AOAC International
(formerly the Association of Official Analytical Chemists), Association of American Cereal Chemists, American Oil
Chemists Society, Environmental Protection Agency, International Standards Organization, and United States
Department of Agriculture.

Kjeldahl Method - Nitrogen Determination
Digestion:- Digestion is accomplished by boiling a homogeneous sample in concentrated sulfuric acid. The end
result is an ammonium sulfate solution. The general equation for the digestion of an organic sample is shown
below:
 The Kjeldahl method may be broken down into three main steps: digestion, distillation, and
titration.
Organic N H2SO4 →
(NH4)SO4 + H2O + CO4 other sample matrix byproducts
Distillation: Excess base is added to the digestion product to convert NH4 to NH3 as indicated in the following
equation. The NH3 is recovered by distilling the reaction product.
ammonium
sulfate
heat
ammonia
gas
(NH4)2SO4 2NaOH → 2NH3 Na2SO4 2H2O

TITRATION
 Titration quantifies the amount of ammonia in the receiving solution.
 There are two types of titration—back titration and direct titration. Both methods indicate the ammonia present in the
distillate with a color change.
 In back titration the ammonia is captured by a carefully measured excess of a standardized acid solution in the
receiving flask. The excess of acid in the receiving solution keeps the pH low, and the indicator does not change
until the solution is "back titrated" with base.
ammonia
standard
sulfuric acid
acid
excess
ammonium
sulfate
sulfuric
acid
2NH3 2H2SO4 → (NH4)2SO4 H2SO4
(no color change)

In direct titration, if boric acid is used as the receiving solution instead of a standardized mineral acid, the chemical
reaction is:
ammonia
sulfate
measured
excess
acid
measured
sodium
hydroxide
ammonium
sulfate
(NH4)2SO4 H2SO4 2NaOH →
Na2SO4 +(NH4)2
SO4 +2H2O
(color change occurs)
ammonia
gas
boric
acid
ammonium-
borate complex
excess
boric acid
NH3 H3BO3 → NH4 H2BO-
3 H3BO3
(color change occurs)

 The boric acid captures the ammonia gas, forming an ammonium-borate complex. As the ammonia collects, the
color of the receiving solutions changes.
ammonium-
borate
complex
sulfuric
acid
ammonium
sulfate
boric
acid
2NH4 H2BO-
3 H2SO4 (NH4)2SO4 2H3BO3
(color change occurs in reverse)
The boric acid method has the advantages that only one standard solution is necessary for the determination and
that the solution has a long shelf life.
Calculation:
Gram nitrogen/ gram of sample =
*(ml of sample - ml of blank)  N (normality) of standard acid  0.014g/meq
weight of sample
* ml of hydrochloric acid required to titrate sample solution.

Dye Binding Method
Principle: At low pH, basic groups of protein are (+) charged. These will
quantitatively bind a (-) charged dye.
What are these basic amino acids with positive charge at low pH?

Dye Binding Method
N = N
HO
SO3
-
Acid Orange 12: Procedure:
 Mix protein, dye, buffer at pH = 2.
 Filter or centrifuge the mixture.
 Measure the absorbance of filtrate.

Factors Influencing Dye Binding determination:
1. Temperature
2. Non-proteins.
3. Buffer systems.
4. Protein quality.
Dye Binding Method

Biuret Method
Principle: Cu++ in alkaline solution form complex with peptide bonds - give pinkish-purple color.
Measure the intensity of color at 540 nm.
Aat540nm
% Protein (Kjeldahl))

Lowry Method
Cu++ in alkaline solution to form complexity with protein.
Cu++ catalyzes oxidation of phenol group of tyrosine with phosphomolybdic-phosphotungstic
acid.
Absorbanceat750nm
g of protein (Kjeldahl)m

Ultra-violet Absorption (UV) at 280 nm
 Chromophoric side chains of aromatic amino acids (Tyrosine, Tryptophan).
 Absorption at 280 nm. “Non-destructive means to determine protein”.
 Calculation of protein concentration based upon absorption

Fluorescence Method
Tyrosine and Tryptophan are fluorescent compounds.
Excites the amino acids at 280 nm.
Measure emission at 348 nm.
Advantage: more sensitive than UV absorption.

 Fluorescence spectroscopy (fluorometry or Spectro fluorometry), is a type of electromagnetic
spectroscopy which analyzes fluorescence from a sample.
 It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain
compounds and causes them to emit light of a lower energy, typically, but not necessarily, visible light. This
shift to longer wavelength is called the Stokes shift.
 Devices that measure fluorescence are called fluorometers or fluorimeters.
Fluorescence Method

Dumas Method
The method is based on the combusting of a sample at 1000°C in the presence of oxygen, whereby the
carbon and nitrogen are converted to CO2 and NOx respectively. Both gasses are separated by
chromatography and measured in a thermal conductivity cell.

Advantage of Dumas over Kjeldahl
 The Dumas method has the advantage of being easy to use and automated.
 It is also considerably faster than the Kjeldahl method, taking a few minutes per measurement, as compared to
an hour or more for Kjeldahl.
 It also does not make use of toxic or harmful chemicals or catalysts. The Kjeldahl method uses concentrated
sulfuric acid and a catalyst for digestion of samples. When the use of mercury and cadmium in the laboratory
was banned in most countries during the 1990’s, many laboratories evaluated the Dumas method as an
alternative and numerous comparative studies have been made.

Bibliography
 Fenemma’s Food Chemistry (5th edition)
 H.D Belitz, W Grosch, P Schieberle (4th edition)
 E-course of Food Chemistry
 PF Fox, Mcsweeny

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