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3 Enzymes in Food Analysis 43this using gold-nanoparticle–carbon-nanotube hybrid linked to
horseradish peroxidase as a biosensor for measuring the amount
of protein in serum (Cui et al. 2008). In this method, the
hybrid-bound peroxidase catalyzes oxidation of o-
phenylenediamine in the presence of hydrogen peroxide
to produce diaminobenzene, which, being electroactive, gen-
erates an amperometric response. Comparison of this method
with ELISA showed no significant differences between the two
methods. Table 3.1 shows a list of some commercially available
ELISA kits for analysis of food proteins.
The free amino acid content as well as amino acid composition
of food proteins constitutes very important food components due
to their impact on general protein metabolism and a number of
food qualities. For example, the presence of glutamate is gener-
ally associated with umami or savory flavor, while high content
of hydrophobic amino acids tend to cause bitterness in protein
hydrolysates. Similarly, high content of free amino acids provide
free amino groups that promote Maillard reactions particularly
when these foods are subjected to high-temperature treatments.
The products or intermediates of the Maillard reaction may un-
dergo other reactions that eventually influence flavor and color
of the food product. In the age of protein-fortified sports drinks,
free amino acid and peptide content of foods also provides the
athlete the “boost” needed for recovery from intense muscle
activity during fitness training. A number of analytical meth-
ods for determination of amino acid content in foods involve
application of enzymes. For instance, one of the early meth-
ods for amino acid analysis involved the use of amino acid de-
carboxylases to decarboxylate amino acids. The stoichiometric
release of carbon dioxide was then analyzed using glass elec-
trodes containing sodium bicarbonate (Wiseman 1981). In the
analysis of tryptophan, tryptophanase was used to catalyze hy-
drolysis of the amino acid to produce pyruvate. Coupling of the
pyruvate production to lactate dehydrogenase converted NADH
to NAD+and the corresponding change in absorbance at 340
nm was recorded as the index of tryptophan content (Wiseman
1981). The principle underlying the method is shown in Figure
3.3. A similar method involving coupling to NADH has been
exploited in the aspartate aminotransferase analysis of aspartic
acid by conversion of aspartic acid to oxaloacetate in the pres-
ence of malate dehydrogenase (Wiseman 1981). A number ofL-Tryptophan + H 2 O Pyruvate + Indole + NH 3TryptophanaseNADHNAD+Lactate
dehydrogenaseMeasure change in absorbance at 340 nmLactateFigure 3.3.Principle underlying tryptophan analysis in foods using
the coupled tryptophanase and lactate dehydrogenase (LDH)
reaction.other enzyme-based methods have been developed for analysis
of amino acids, with a significant number of these also using
coupling to NADH–NAD+conversion (see Table 3.2).SugarsGlucose oxidase is by far the most predominant and widely used
of enzymes for detection of glucose in foods. The underlying
principle is based on hydrolysis of glucose to form gluconic
acid and hydrogen peroxide. The latter is then coupled to per-
oxidase catalysis, which oxidizes a colored precursor, resulting
in absorbance change that is proportional to the glucose content
(Wiseman 1981). Other methods using enzyme complexes or
combinations for analysis of glucose are available. One such
method was based on the glucose-6-phosphate transferase cat-
alyzed transfer of an acyl phosphate from a donor molecule
to form glucose 6-phosphate. Coupling this reaction to NADP
conversion to NADPH resulted in absorbance change that could
be measured at 340 nm. Glucose oxidase has also been applied
to the determination of sucrose (common sugar). The analysis
involves invertase-catalyzed conversion of sucrose tod-fructose
andα-d-glucose. This is followed by mutarotase-catalyzed iso-
merization of theα-d-glucose toβ-d-glucose, which in turn
is oxidized by glucose oxidase tod-glucono-δ-lactone and hy-
drogen peroxide (Morkyavichene et al. 1984, Matsumoto et al.
1988). As discussed earlier, the amount of peroxide released
by the coupled reaction with peroxidase is proportional to
the sucrose content. Fructose is another monosaccharide with
widespread use in several foods and thus of significant interest
in the food industry. The level of fructose in foods provides
an indication of the stage of ripening, adulteration of products
such as honey, and the sweetness of various foods. There have
been several analytical methods based on the highly specific
pyrroloquinoline quinone (PQQ) enzyme,d-fructose dehydro-
genase (Paredes et al. 1997). The enzyme catalyzes oxidation
ofd-fructose to 5-keto-d-fructose with corresponding reduction
of the covalently bound cofactor PQQ to PQQH 2. Coupling re-
oxidation of the reduced cofactor to electrodes produces electric
current in direct proportion to the amount of fructose. Trivedi
et al. (2009) recently reported another method involving use of
d-fructose dehydrogenase with ferricyanide as electron acceptor.
Figure 3.4 is a schematic representation of the principle underly-
ing this method. The enzyme oxidizes fructose to keto-fructose
with the reduction of ferricyanide. The reduced ferricyanide is
subsequently re-oxidized, producing electrical current propor-
tional to fructose concentration.
In a recent review, Zeravik et al. (2009) discussed the use of
biosensors referred to as bioelectronic tongues for detection and
analysis of sugars and other food components or contaminants.
Glucose and ascorbic acid content of fruit juices have been an-
alyzed using one of these “tongues” involving glucose oxidase
and metal catalysts (Pt, Pd, and Au–Pd). The metal catalysts
improved the sensitivity of the tongue by reducing the oxidation
potential of the hydrogen peroxide released as a result of the glu-
cose oxidase activity (Gutes et al. 2006). Similar simultaneous
detection of glucose, fructose, and sucrose has been achieved
by amperometric flow injection analysis using an immobilized