 Proteins are polymers of amino acids

 Major structural components of many natural

foods
 Food analysts are interested in knowing the
total concentration, type, molecular structure
and functional properties of proteins in foods.
 Proteins represent a major source of
energy.
 Proteins contain essential amino-acids,
such as lysine, tryptophan, methionine,
leucine, isoleucine and valine, which are
essential to human health.
 Proteins are also the major structural
components of many natural foods, often
determining their overall texture, e.g.,
tenderness of meat or fish products.
 Proteins
are used as gelling agents,
emulsifiers, foaming agents and thickeners.
 isa complicated process because some food
components have similar physicochemical
properties.

For example, nitrogen could come from

non-protein sources like free amino acids,
nucleic acids, phospholipids, amino sugars,
vitamins, alkaloids, uric acid, urea, and
ammonium ions..
 1.Kjeldahl Method
 2.Dumas Method.
 3.Infrared Spectroscopy.
 4.Biuret Method.
 5.Lowry Method.
 6.Dye-Binding Methods.
 7.Bicinchoninic Acid Method.
 8.Ultraviolet 280nm Absorption Method.
 Kjeldahlmethod is the most common method
for the determination of organic nitrogen.
1. Kjeldahl Method
 A food digested with strong acid so that it
releases nitrogen which can be determined
by a suitable titration techniques
 Does not measure the protein content
directly
 Conversion factor is needed to convert the
measured nitrogen concentration to a
protein concentration
 Kjeldahl method is mainly based on an acid-
base titration.
 •The general procedure includes 3 steps:
 1.Digestion.
 2.Neutralization & Distillation.
 3.Titration.
 An accurately weighed amount of the sample
is placed in Kjeldahl’s flask + conc. sulfuric
acid (oxidizing agent) + catalyst
 digestion by heating until the flask contents
become clear indicating complete oxidation
of all organic matter → during digestion,
protein nitrogen is liberated and form non-
volatile ammonium sulfate.
 During digestion, H2SO4 oxidizes carbon and
hydrogen in the sample into CO2 and water.
 •This step is the most time-consuming step.
 To speed up the method, the following
measures are performed:
 1.Use of a catalyst [e.g. Copper (Cu),
Selenium (Se), Mercury (Hg)].
 2.Addition of anhydrous Na2SO4 to raise the
boiling point of H2SO4 and so increase the
temperature at which digestion occurs.
 After digestion, the digest is diluted with
water and then neutralized with NaOH which
converts the ammonium sulfate into ammonia
gas.
 (NH4)2SO4 + 2 NaOH → 2 NH3 + Na2SO4 + 2
H2O
 Then the produced ammonia is distilled into a
boric acid solution where ammonia is
converted into ammonium ion and boric acid
is converted into borate ion.
 NH3 + H3BO3 (boric acid) → NH4+ + H2BO3-
(borate ion)
C. Titration
 Titrate with standard sulfuric or hydrochloric acid
using a suitable indicator
 The concentration of hydrogen ions required to
reach the end-point is equivalent to the
concentration of nitrogen
 Once nitrogen content has been determined, it is
converted to a protein content using appropriate
conversion factor.
a conversion factor (F) is needed to convert
the measured nitrogen concentration to a
protein concentration. Most proteins contains
16% nitrogen, so the conversion factor of
6.25 (100/16 = 6.25) is used for many
applications where: [%protein = %N x 6.25].
However, this is only an average value, and
each protein has a different conversion
factor depending on its nitrogen content.
 Prtein content= %N * F
 Advantages:  Disadvantages:
 universal  does not give a measure of
 high precision true protein
 good reproducibility  different proteins need
different correction factors
 the use of concentrated
sulfuric acid at high
temperature
 time consuming
2. Enhanced Dumas Method
 Principles: a sample of known mass is combusted
in a high temperature to release CO2, H2O and
N2
 Nitrogen content is measured by separating N2
from CO2 and H2O using a column
 Advantages  Disadvantages
 Faster  High initial cost
 Doesn’t need toxic  Does not give a
chemicals or measure of true
catalysts protein
 Easy to use  Small sample size
 Samples can be make its difficult to
measured obtain a
automatically representative
sample
3. Methods using UV-Visible Spectroscopy
 Use either the natural ability of proteins to
absorb (or scatter) light or chemically or
physically modify proteins to make them absorb
(or scatter) light in the region.
 Calibration curve of absorbance versus protein
concentration must be built first
 Main difference: the chemical groups which are
responsible for the absorption or scattering of
radiation
Direct Measurement at 280 nm
 Principle: tryptophan and tyrosine absorb
ultraviolet light strongly at 280 nm
 Use the same w/length to measure protein
concentration
 Advantages: simple to carry out, non-
destructive and no special reagents are required
 Disadvantages: other substances may absorb at
280nm
Biuret Method
A violet-purplish color is produced when
cupric ions (Cu2+) interact with peptide
bonds under alkaline conditions
 Absorbance is read at 540 nm
 Advantages: no interference from
materials that absorb at lower w/lengths
and less sensitive to protein type
 Disadvantages: low sensitivity
Dye Binding Methods
 A known excess of a negatively charged dye
is added to a protein solution which are
positively charged (by adjusting the pH)
 Bound dye and protein form an insoluble
complex while the unbound dye remains
soluble
 The amount of protein present proportional
to the amount of dye that bound to it.
Other instrumental techniques
 Bulk physical properties
 Density – protein has the greatest
density. So increase in protein content
will increase density of food
 Refractive index – RI increases as protein
concentration increases
 Measurement of Adsorption of Radiation
 Infra-Red:protein absorb IR naturally
due to characteristic vibrations of certain
chemical groups
 NMR: measuring the area under a peak
in an NMR chemical shift spectra that
corresponds to the protein fraction
Advantages: Disadvantages:
 Non-destructive  Calibration curve must
 Little or no sample be prepared
preparation  Can only be used to
 Rapid and precise
analyze foods with
relatively simple
compositions
 Difficult to disentangle
the contribution that
the protein makes to
the overall
measurement from
that of the other
component
it helps to determine particular protein from
mixture of protein
1. Salting Out
 Proteins precipitate from aqueous solutions
when the salt concentration exceeds a
critical level
 Salt commonly used: Ammonium sulfate
2. Isoelectric Precipitation
 Isoelectricpoint (pI): pH where the net
charge on the protein is zero
 Proteins tend to precipitate at their pI
Different amino acid has different pI.
3. Denaturation of Contaminating
Proteins
 Proteinsare denatured and precipitated
when heated at high temperature or in
the high acid solution
B. Methods based on adsorption characteristics
 Involves the separation of compounds
by selective adsorption-desorption at a
solid matrix that is contained within a
column
 Separation can be carried out using
either an open column or high-
pressure liquid chromatography
1. Ion Exchange Chromatography
 Relies on the reversible adsorption-
desorption of ions in solution to a charged
solid matrix or polymer network
 Positively charged is called anion –
exchanger while negatively charged is called
cation – exchanger
 Two types of column are used
1. To bind protein of interest to the ion-exchange
column
2. To favor adsorption from the column
2. Affinity Chromatography
 Uses stationary phase that consists of a
ligand covalently bound to a solid
support
 The protein of interest binds to the
ligand in the column, and eluted using
another buffer solution
C. Methods Based on Size Differences
 Depends on the Stokes radius of a
protein
 Stokes radius is the average radius that a
protein has in solution
 Stokes radius increases in the following
order: compact globular protein <
flexible random-coil < rod-like protein
2. Ultrafiltration
 A protein solution is placed in a cell containing a
semi-permeable membranes and pressure is
applied to speed up the separation
 LMW protein pass through the membrane
whereas the LMW molecules remains in the
solution
 Use to concentrate a protein solution, remove
salt, exchange buffers or fractionate protein on
the basis of their size
3. Size Exclusion Chromatography
 Also known as gel filtration
 Column which is packed with porous
beads made of a cross-linked polymeric
material
 Molecules larger than the pores in the
beads are excluded and move quickly
through the column whereas the
movement of molecules which enter the
pores is retarded
D. Separation by Electrophoresis
 Relies on differences in the migration of
charged molecules in a solution when an
electrical field is applied across it
 It can be used to separate protein on the
basis of size, shape or charge