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3 Enzymes in Food Analysis 51
formed the basis for a broad range of techniques for pesticide
analysis (see reviews by Mulchandani et al. 2001, Amine et al.
2006). These have used:
- amperometric transducers to measure thiocholine and p-
aminophenol produced by acetylcholinesterase hydrolysis
of butyrylthiocholine, and p-aminophenyl acetate or hydro-
gen peroxide produced by oxidation of choline following
acetylcholine hydrolysis in presence of choline oxidase; - potentiometric transducers for measuring pH changes as a
result of acetic acid production from enzyme activity; - fiber optics for monitoring pH changes using a fluorescein-
labeled enzyme.
These acetylcholinesterase inhibitory methods, however, are
nondiscriminating between the organophoshates, carbamates,
and other pesticides. In their review, Mulchandani et al. (2001)
have discussed the use of organophosphorus hydrolase inte-
grated with electrochemical and optical transducers to specif-
ically measure organophosphate pesticides. The enzyme hy-
drolyzes organophosphates to produce acids or alcohols, which
generally tend to be either electroactive or chromophores that
can be monitored.
Bioelectronic tongues have also been proposed for the de-
tection of pesticides such as organophosphates, carbamates, as
well as phenols using an amperometric bioelectronic tongue in-
volving cholinesterase, tyrosinase, peroxidases, and cellobiose
dehydrogenase. The corresponding substrates for the enzymes
were acetylcholine, phenols, hydrogen peroxide, and cellobiose,
respectively, with detection limits in the nanomolar and micro-
molar range (Solna et al. 2005). A similar bioelectronic tongue
with acetylcholinesterase as biosensor has been used to resolve
pesticide mixtures of dichlorvos and methylparaoxon (Valdez-
Ramirez et al. 2009).
Botrytis, a fungal disease responsible for significant losses of
agricultural produce particularly in vineyards (e.g., wine grapes,
strawberries), is controlled by preharvest or postharvest treat-
ment with the fungicide fenhexamid, which inhibits growth of
the germ tube and mycelia of the fungus. Detection and analysis
of fenhexamid levels in must and wines have been accomplished
by direct competitive ELISA containing horseradish peroxidase
(Mercader and Abad-Fuentes 2009). Atrazine, a herbicide with
widespread contamination of waterways and drinking water sup-
plies and implicated in certain human health defects (e.g., birth
defects, menstrual problems, low birth weight), is tightly regu-
lated in the European Union with a limit of 0.1 g/L in potable
water and fruit juices. Conductimetric and amperometric biosen-
sors based on tyrosinase inhibition have been developed for the
analysis of atrazine and its metabolites (Hipolito-Moreno et al.
1998, Vedrine et al. 2003, Anh et al. 2004). The method is
based on deactivation of tyrosinase under aqueous conditions.
The enzyme normally catalyzes oxidation of monophenols to
o-diphenols, which are eventually dehydrogenated to the cor-
responding o-quinones. In aqueous environments, these form
polymers that inhibit the enzyme (Hipolito-Moreno et al. 1998).
Therefore, atrazine inhibition of the enzyme is conducted in
nonaqueous media, where the quinone effect is eliminated, thus
ensuring the inhibition is primarily due to the herbicide. Some
recent reviews have also captured the application of enzymatic
biosensors for determination of fertilizers (nitrates, nitrites, and
phosphates), as well as several pesticides and heavy metals that
are potentially toxic to humans when they enter the food chain
(Amine et al. 2006, Cock et al. 2009). Some of the enzymes
applied to these analyses include (i) urease, glucose oxidase,
acetylcholinesterase, alkaline phosphatase, ascorbate oxidase,
alcohol oxidase, glycerol-3-phosphate oxidase, invertase, and
peroxidase for heavy metals and (ii) nitrate reductase, polyphe-
nol oxidase, phosphorylase, glucose-6-phosphaste dehydroge-
nase, and phosphoglucomutase for nitrates, nitrites, and phos-
phates.
ENZYMATIC ANALYSIS OF FOOD
QUALITY
While enzymes generally exert positive influence on food qual-
ity, the activities of some enzymes need to be controlled posthar-
vest or postprocessing to avoid compromising the quality of
food products. For instance, protease activity is desirable for
meat tenderization during ageing, but left uncontrolled, meats
become mushy and overtenderized. Similarly, excessive lipase
and oxidase activities in foods during storage, especially under
temperature-abuse conditions, may cause rancidity and impact
quality attributes such as color, flavor, and texture. Therefore, a
number of preservation techniques developed through the years
have been aimed at controlling such undesirable postharvest en-
zymatic as well as microbial activities to ensure extended shelf
life of food products.
The activities of a number of enzymes as well as the content
of certain food components and secondary metabolites resulting
from postharvest or postprocessing biochemical and microbial
activities have been recognized as quality indices for various
foods and consequently monitored. An example is the milk en-
dogenous alkaline phosphatase, which is inactivated following
heat treatment at 60◦C for 5 seconds. With milk generally sub-
jected to HTST/flash pasteurization (i.e., 71.5–74◦C/160–165◦F
for 15–30 seconds) or ultra-high-temperature treatment (i.e.,
135 ◦C/275◦F for 1–2 seconds), residual alkaline phosphatase
activity following such treatment provides a good indication
of efficacy of the treatment. The phosphatase test is based on
hydrolysis of disodium phenyl phosphate to liberate phenol,
which reacts with dichloroquinonechloroimide to form a blue
indophenol that is measured colorimetrically at 650 nm (Mur-
phy et al. 1992). An alternative fluorimetric method for alkaline
phosphatase analysis has also been developed and commercial-
ized (Rocco 1990). The adequacy of blanching, an essential
step in vegetable processing, is also established by measuring
residual lipoxygenase or peroxidase activities (Powers 1998).
Amylase activity in malt, also referred to as its diastatic power,
is a very essential quality parameter that influences dextrinizing
time and, therefore, closely monitored (Powers 1998). In most
seafoods, freshness is rapidly compromised postharvest, par-
ticularly when handled under temperature-abuse conditions that
promote endogenous enzyme activity. The sequence of reactions
(Figure 3.8) involved in ATP breakdown in postmortem fish