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3 Enzymes in Food Analysis 49studied of the mycotoxins. It has since been shown to occur
mainly in the tropics in such food ingredients as peanuts, pista-
chios, Brazil nuts, walnuts, cottonseed, maize, and other cereal
grains such as rice and wheat. Aflatoxins are considered to in-
duce hepatotoxicity, mutagenicity, teratogenicity, immunosup-
pressant, and carcinogenic effects, and their presence in foods
is highly regulated at a level of 0.05–2μg/kg in the European
Union (EC Report 2006). With such low limits, the analyti-
cal methods need to be of high sensitivity and specificity, and
ELISA has developed to be one of the main methods for afla-
toxin analysis. Some of the enzymes that have been used include
horseradish peroxidase, which oxidizes tetramethylbenzidene to
a product that can be measured by its electrical properties, and
alkaline phosphatase for dephosphorylation of naphthyl phos-
phate to naphthol that is measured by pulse voltametry (Am-
mida et al. 2004, Parker and Tothill 2009). Detection limits for
these methods were in the range of 20–30 pg/mL. Other ELISA
methods for aflatoxin analysis have been reported (Lee et al.
2004, Liu et al. 2006, Jin et al. 2009, Piermarini et al. 2009,
Tan et al. 2009). In their method, Liu et al. (2006) developed a
biosensor by immobilizing horseradish peroxidase and aflatoxin
antibodies onto microelectrodes. The presence of aflatoxin in the
food resulted in immunocomplex formation with the antibody,
which blocked electron transfer between the enzyme and the
electrode, thereby modifying conductivity in proportion to afla-
toxin concentration. Similar ELISA methods involving alkaline
phosphatase and horseradish peroxidase has been used for anal-
ysis of ochratoxin A in wine (Prieto-Simon and Campas 2009).
A fast and sensitive spectrophotometric method has also been
reported based on acetylcholinesterase inhibition by aflatoxin
(Arduini et al. 2007).
Unlike aflatoxins, which are mainly associated with foods
from warm tropical regions, trichothecene mycotoxins are com-
monly found in cereals like wheat, barley, maize, oats, and
rye, particularly in cold climates. Like the aflatoxins, these
have also been found to cause immunosuppressive and cyto-
toxic effects as well as other disorders due to their inhibition of
protein, DNA, and RNA synthesis. AOAC-approved methods
(www.aoac.org/testkits/testedmethods.html) based on ELISA
kits are commercially available for analysis of trichothecenes.
These include the RIDASCREEN FAST and AgraQuant man-
ufactured by Biopharm GmbH and Romer Labs, respectively,
using horseradish peroxidase (Lattanzio et al. 2009).
Linamarin and lotaustralin are cyanogenic glycosides consid-
ered toxic and found in food crops like lima beans and cas-
sava. The endogenous linamarase released on tissue damage hy-
drolyzes the glucosides to produce hydrogen cyanide, a highly
toxic compound (Cooke 1978). In the analysis of these toxins,
the endogenous linamarase is first inactivated, followed by in-
troduction of the exogenous enzyme. The cyanide produced as a
result of the enzyme activity is then measured by a spectropho-
tometer as index of toxin content. The linamarase-catalyzed re-
action normally exists in equilibrium but is made to proceed in
the forward direction by alkali treatment (Cooke 1978).
Biogenic amines are organic bases found in a broad range
of foods such as meat, fish, wine, beer, chocolate, nuts, fruits,
dairy products, sauerkraut, and some fermented foods, but arepotentially toxic when consumed. These amines, most of which
are produced by microbial decarboxylation of the correspond-
ing amino acids, include putrescine, cadaverine, tyramine, sper-
mine, spermidine, and agmatine. That is, putrescine, histamine,
tryptamine, tyramine, agmatine, and cadaverine are formed from
ornithine, histidine, tryptophan, tyrosine, arginine, and lysine,
respectively (Teti et al. 2002). Histamine is the most regulated of
these amines. In Germany, the maximum limit in fish products is
set at 200 mg/kg, whereas in Canada, Finland, and Switzerland,
it is set at 100 mg/kg. Various methods have been developed for
analysis of these compounds (Onal 2007). For histamine deter-
mination, one of the methods has been based on the sequential
activities of diamine oxidase and horseradish peroxidase (Lan-
dete et al. 2004). The diamine oxidase catalyzes breakdown of
histamine, releasing imidazole acetaldehyde, ammonia, and hy-
drogen peroxide. The hydrogen peroxide produced is oxidized
by the peroxidase, resulting in color change of a chromogen,
which is measured by a colorimeter. Muresan et al. (2008) re-
cently developed an amperometric biosensor involving use of
amine oxidase and horseradish peroxidase for amine detection.
In this method, the amines were first separated on a weak acid
cation exchange column followed by enzymatic reactions that
produce a potential difference, which is measured by a bio-
electrochemical detector. An enzyme sensor array for simul-
taneous detection of putrescine, cadaverine, and histamine has
also been reported by Lange and Wittman (2002). The principle
underlying the method is illustrated in Figure 3.7. Changes in
the levels of the various biogenic amines during ageing of salted
anchovies were determined using immobilized diamine oxidase
coupled to electrochemical detection of the hydrogen peroxide
produced by the enzyme activity (Draisci et al. 1998).Process-Induced ToxinsAcrylamide is a process-induced toxin that has gained signifi-
cant global attention in the past decade following a publication
by the Swedish National Food Administration (2002), which in-
dicated its widespread presence in numerous food products and
the risk posed to humans from consumption of such foods. Foods
considered to have high levels of acrylamide include baked and
fried products like potato chips (or crisps), biscuits, crackers,
breakfast cereals, and French fries. The mechanism of acry-
lamide formation in these foods is attributed to the high lev-
els of asparagine in the raw materials (especially wheat and
potatoes) used in their production. Initiation of the Maillard re-
action in presence of reducing sugars with asparagine as amino
group donor channels the complex series of reactions through
a pathway that results in the production of acrylamide (Mot-
tram et al. 2002). While methods involving gas chromatography,
liquid chromatography, and mass spectrometry still dominate
analysis of this compound, a recent report has demonstrated
the potential application of ELISA for analyzing acrylamide
content in foods (Preston et al. 2008). To overcome the prob-
lem with the small size of acrylamide, which has limited the
use of immunoassay for its analysis, these authors derivatized
acrylamide with 3-mercaptobenzoic acid (3-MBA) and conju-
gated that to a carrier protein (bovine thyroglobulin) to form an