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28 Part 1: Principles/Food Analysischromatography, electrophoresis, or immunology or a combina-
tion of these methods (VanCamp and Huyghebaert 1996).
Electrophoresis is defined as the migration of ions (electri-
cally charged molecules) in a solution through an electrical field
(Smith 1998). Although several forms of this technique exist,
zonal electrophoresis is perhaps the most common. Proteins
are separated from a complex mixture into bands by migrat-
ing in aqueous buffers through a polyacrylamide gel with a
pre-determined pore size (i.e., a solid polymer matrix). In non-
denaturing/native electrophoresis, proteins are separated based
on their charge, size, and hydrodynamic shape, while in dena-
turing polyacrylamide gel electrophoresis (PAGE), an anionic
detergent sodium dodecyl sulfate (SDS) is used to separate pro-
tein subunits by size (Smith 1998). This method was used to
determine the existence of differences in the protein subunits
between control coffee beans and those digested by an Asian
palm civet as well as between digested coffee beans from both
the Asian and African civets (Marcone 2004). These protein sub-
units differences lead to differences in the final Maillard brown-
ing products during roasting and therefore flavor and aroma
profiles. During the analysis of white and red bird’s nests, Mar-
cone determined that SDS–PAGE might be a useful analytical
technique for differentiating between the more expensive red
bird nest and the less expensive white bird (Marcone 2005). Ad-
ditionally, this technique could possibly be used to determine
if the red nest is adulterated with the less expensive white nest.
Using SDS-PAGE analysis, Marcone was the first to report the
presence of a 77 kDa ovotransferrin-like protein in both red
and white nests, being similar in both weight and properties to
ovotransferrin in chicken eggs. In isoelectric focusing, a modifi-
cation of electrophoresis, proteins are separated by charge in an
electrophoretic field on a gel matrix in which a pH gradient has
been generated using ampholytes (molecules with both acidic
and basic groups, that is, amphoteric, existing as zwitterions in
specific pH ranges). The proteins migrate to the location in the
pH gradient that equals the isoelectric point (pI) of the protein.
Resolution is among the highest of any protein separation tech-
nique and can separate proteins with pI differences as small as
0.02 pH units (Chang 1998, Smith 1998). More recently, with the
advent of capillary electrophoresis, proteins can be separated on
the basis of charge or size in an electric field within a very short
period of time. The primary difference between capillary elec-
trophoresis and conventional electrophoresis (described above)
is that a capillary tube is used in place of a polyacrylamide gel.
The capillary tube can be used over and over again unlike a gel,
which must be made and cast each time. Electrophoresis flow
within the capillary also can influence separation of the proteins
in capillary electrophoresis (Smith 1998).
HPLC is another extremely fast analytical technique having
excellent precision and specificity as well as the proven ability
to separate protein mixtures into individual components. Many
different kinds of HPLC techniques exist depending on the na-
ture of the column characteristics (chain length, porosity, etc.)
and elution characteristics such as mobile phase, pH, organic
modifiers, etc. In principle, proteins can be analyzed on the
basis of their polarity, solubility, or size of their constituent
components.Reversed-phase chromatography was introduced in the 1950s
(Howard and Martin 1950, Dierckx and Huyghebaert 2000) and
has become a widely applied HPLC method for the analysis
of both proteins and a wide variety of other biological com-
pounds. Reversed-phase chromatography is generally achieved
on an inert packed column, typically covalently bonded with a
high density of hydrophobic functional groups such as linear
hydrocarbons 4, 8, or 18 residues in length or with the relatively
more polar phenyl group. Reversed-phase HPLC has proven
itself useful and indispensable in the field of varietal identifica-
tion. It has been shown that the processing quality of various
grains depends on their physical and chemical characteristics,
which are at least partially genetic in origin, and a wide range of
qualities within varieties of each species exist (Osborne 1996).
The selection of the appropriate cultivar is an important decision
for a farmer, since it largely influences the return he receives on
his investment (Dierckx and Huyghebaert 2000).
Size-exclusion chromatography separates protein molecules
on the basis of their size or, more precisely, their hydrodynamic
volume, and has in recent years become a very useful separa-
tion technique. Size-exclusion chromatography utilizes uniform
rigid particles whose pre-determined pore size determines which
protein molecules can enter and travel through the pores. Large
molecules do not enter the pores of the column particles and
are excluded, that is, they are eluted in the void volume of the
column (i.e., elute first), whereas smaller molecules enter the col-
umn pores and therefore take longer to elute from the column.
An application example of size-exclusion chromatography is the
separation of soybean proteins (Oomah et al. 1994). In one study,
nine peaks were eluted for soybean, corresponding to different
protein size fraction, with one peak showing a high variability
for the relative peak area and could serve as a possible differen-
tiation among different cultivars. Differences, qualitatively and
quantitatively, in peanut seed protein composition were detected
by size-exclusion chromatography and contributed to genetic
differences, processing conditions, and seed maturity. In 1990,
Basha demonstrated that size-exclusion chromatography was an
excellent indicator of seed maturity in peanuts as the area of one
particular component (peak) was inversely proportional to in-
creasing peanut seed maturity, which also remained unchanged
toward later stages of seed maturity (Basha 1990). The peak
was present in all studied cultivars, all showing a mature seed
protein profile with respect to this particular protein, which was
subsequently named Maturin.LIPID ANALYSIS
Compared to most other food components, lipids are a group
of relatively small, naturally occurring molecules containing
carbon, hydrogen, and oxygen atoms, but with much less oxy-
gen than carbohydrates. This large group of organic molecules
includes fats, waxes, cholesterol, sterols, glycerides, phospho-
lipids, etc. The most simplistic definition of a lipid is based
on its solubility, that is, it is soluble in organic solvents (e.g.,
alcohol) but insoluble in water. Lipid molecules are hydropho-
bic, but this generality is sometimes not totally correct as some
lipids are amphiphilic, that is, partially soluble in water and