BLBS102-c02 BLBS102-Simpson March 21, 2012 11:54 Trim: 276mm X 219mm Printer Name: Yet to Come
2 Analytical Techniques in Food Biochemistry 27For the past several decades, protein analysis from food prod-
ucts has been performed by determining the nitrogen content
after complete acid hydrolysis and digestion by the Kjeldahl
method followed by an analytical step in which the resulting
ammonium ion is quantified by titrimetry, colorimetry, or by the
use of an ion-specific electrode. The result is then multiplied
by a pre-established protein conversion factor that determines
the final protein content of the sample (Chang 1998, Dierckx
and Huyghebaert 2000). While this “wet” analytical technique
is still the gold standard in protein analysis, it is time-consuming
and involves the use of many dangerous chemicals both to the
analyst and to the environment. Its main advantage is that the
food sample used in this analytical procedure is considered large
enough to be a genuine representative of the entire product. On
the other hand, Dumas combustion is a more recent and faster
“dry” analytical instrumental method of determining the protein
content in foods and is based on the combustion of a very small
sample at 900◦C in the presence of oxygen. The resulting liber-
ated nitrogen gas is analyzed in three minutes by the equipment
through built-in programmed processes with the resultant value
also multiplied by pre-determined conversion factors, requiring
no further analysis or the use of dangerous chemicals. Both of
these methods assume that all the nitrogenous compounds in
the sample are proteins, but other organic molecules such as
nucleotides, nucleic acids, some vitamins, and pigments (e.g.,
chlorophyll) also contains nitrogen, overestimating the actual
protein content of the sample. Both techniques measure crude
protein content, not actual protein content. While the Kjeldahl
method is the internationally accepted method of protein de-
termination for legal purposes, Dumas combustion is slowly
becoming more acceptable as its accuracy and repeatability will
soon be superior to that of the Kjeldahl method (Schmitter and
Rihs 1989, Simonne et al. 1997).
As far back as the turn of the century, colorimetric meth-
ods for protein determination became available with procedures
such as the Biuret, Lowry (original and modified), bicinchoninic
acid (BCA), Bradford, and ultraviolet (UV) absorption at
280 nm (Bradford 1976). These colorimetric methods exploit
the properties of specific proteins, the presence of specific amino
acid functional groups, or the presence of peptide bonds. All re-
quire the extraction, isolation, and sometimes purification of the
protein molecule of interest to attain an accurate absorbance
reading. Considering that the nutrients in foods exist in complex
matrices, these colorimetric methods are not practical for food
analysis. Additionally, only a few dye-binding methods (official
methods 967–12 and 975–17) have been approved for the direct
determination of protein in milk (AOAC 1995).
The Biuret procedure measures the development of a purplish
color produced when cupric salts in the reagent complex react
with two or more peptide bonds in a protein molecule under
alkaline conditions. The resultant color absorbance is measured
spectroscopically at 540 nm with the color intensity (absorbance)
being proportional to the protein content (Chang 1998) and with
a sensitivity of 1–10 mg protein/mL. This method “measures”
the peptide bonds that are common to cellular proteins, not just
the presence of specific side-groups. While it is less sensitive
compared to other UV methods, it is considered to be a goodgeneral protein assay for which yield is not an important issue.
Likewise, the presence of interfering agents during absorbance
measurement is not an issue as these substances usually absorb
at lower wavelengths. While the color is stable, it should be mea-
sured within 10 minutes for best results. The main disadvantage
of this method is that it consumes more material as well as it
requires a 20-minute incubation period.
Over the years, further modifications to the colorimetric
measurement of protein content have been made with the
development of the Lowry method (Lowry et al. 1951, Pe-
terson 1979), which combines both the Biuret reaction with
the reduction of the Folin-Ciocalteu (F-C) phenol reagent
(phosphomolybdic–phosphotungstic acid). The divalent cupric
cations form a complex with the peptide bonds in the pro-
tein molecules, which cause them to be reduced to monovalent
cations. The radical side groups of tyrosine, tryptophan, and cys-
teine then react with the Folin reagent, producing an unstable
molybdenum/tungsten blue color when reduced under alkaline
conditions. The resulting bluish color is read at both 500 nm and
750 nm wavelengths, which are highly sensitive to both high and
low protein concentrations with a sensitivity of 20–100 ug, re-
spectively. The modified Lowry method requires the absorbance
measurement within 10 minutes, whereas the original Lowry
method needs precise timing due to color instability. Addition-
ally, the modified Lowry method is more sensitive to protein
than the original method but less sensitive to interfering agents.
The BCA protein assay is used to determine the total protein
content in a solution being similar to the Biuret, Lowry, and
Bradford colorimetric protein assays. The peptide bonds in the
protein molecules reduce the cupric cations in the BCA in the
reagent solution to cuprous cations, a reaction that is dependent
upon temperature. Afterwards, two molecules of BCA chelate
the curprous ions, changing the solution color from green to
purple, which strongly absorbs at 562 nm. The amount of cupric
ions reduced is dependent upon the amount of protein present,
which can be measured by comparing the results with protein
solutions with known concentrations. Incubating the BCA as-
say at temperatures of 37–60◦C and for longer time periods
increases the assay’s sensitivity as the cuprous cations complex
with the cysteine, cystine, tyrosine, and tryptophan side-chains
in the amino acid residues, while minimizing the variances
caused by unequal amino acid composition (Olsen and Markwell
2007).
Other methods exploit the tendency of proteins to absorb
strongly in the UV spectrum, that is, 280 nm primarily due
to the presence of tryptophan and tyrosine amino acid residues.
Since tryptophan and tyrosine content in proteins are generally
constant, the absorbance at 280 nm has been used to estimate
the concentration of proteins using Beer’s law. As each protein
has a unique aromatic amino acid composition, the extinction
coefficient (E 280 ) must be determined for each individual protein
for protein content estimation.
Although these methods are appropriate for quantifying the
actual amounts of protein, they do have the ability to differen-
tiate and quantify the actual types of proteins within a mixture.
The most currently used methods to detect and/or quantify spe-
cific protein components belong to the field of spectrometry,