Protein Processing in Food and Bioproduct Manufacturing and Techniques for Analysis - Food and Industrial Bioproducts and Bioprocessing - page 102

Capillary electrophoresis
New developments in the field of capillary electrophoresis (CE) offer a powerful tool for the
rapid separation of proteins (Mattiasson and Hatti-Kaul, 2003; Huck and Bonn, 2008).
Separation of proteins by CE is based on electrophoretic migration driven by an electroos-
motic flow (EOF) and occurs in capillary tubes, filled with background electrolytes (BGE)
(Tagliaro et al ., 1998) following similar principles as for standard electrophoresis. CE is
characterized by high efficiency, short time of analysis, low consumption of samples
and running buffers, and possible connectivity to detectors originally designated for liquid
chromatography, including mass spectrometry. Examples of CE use in protein analysis
include successful quantification of 7 S and 11 S fractions in several soybean varieties
(Blazek and Caldwell, 2009), characterization of cereal proteins (Piergiovanni, 2007;
Salmanowicz, 2008 ; Salmanowicz et al ., 2008 ; Di Luccia et al ., 2009 ), and the analysis of
casein/caseinate addition in processed cheeses (Miralles et al ., 2006 ). Further information
on the use of CE to purify proteins and study their conformations can be found in the work
by Mattiasson and Hatti-Kaul (2003).

Thermal analysis by differential scanning calorimetry
Heat treatment is one of the most commonly used techniques in food processing, and is
frequently responsible for the transition of proteins from the native folded state to the
denatured unfolded state. Thermal denaturation of proteins involves a structural change
which affects the nutritional quality of foods (Plum, 2009).
Differential scanning calorimetry (DSC) is a suitable method for the characterization of
thermal and thermodynamic stabilities of the protein. DSC provides several tools for the
study of the thermal properties of proteins under controlled heating and cooling rates and
can be used to determine the apparent specific heat of proteins (O'Brien and Haq, 2004).
In brief, a DSC instrument comprises two cells fixed in an adiabatic chamber. One cell
contains the sample to be tested, the second cell contains a reference solution or an empty
DSC pan. The adiabatic chamber is maintained under pressure to avoid the evaporation of
the sample (Plum, 2009). A DSC-thermogram represents the plot of heat capacity difference
Δ C p (between the sample and the reference) as a function of temperature. Thermodynamic

parameters, such as T m ,

S, could be determined by the DSC curve analysis. T m is
the temperature at which the concentration of denatured and native forms of the protein are
equal. This specific temperature is also called the midpoint of the thermal transition.

Δ H and Δ

H
represents the enthalpy of thermal transition determined from the integration of the DSC
curve. The entropy (

Δ

S) of the thermodynamic transition of the protein may be calculated
from the integrated area under the curve of

Δ

G), which gives
an indication of the protein stability, can also be determined at any temperature from the
values of

Δ C p /T vs. T. The free energy ( Δ

S (O'Brien and Haq, 2004; Plum, 2009). Thermal and thermodynamic
properties of proteins analyzed by DSC are greatly affected by the experimental conditions
used, such as pH, salts, alcohols, and the presence of other food components (e.g., lipids,
polysaccharides) (Grinberg et al ., 2009 ).

Δ H and Δ

3.5.1.2 Biophysical methods

Biophysics offers a variety of accurate methods that enable kinetic resolution in the submil-
lisecond time scale as well as high protein structural resolution. This section summarizes
some of the most frequently used techniques for the biophysical analysis of proteins.