Food Biochemistry and Food Processing (2 edition)

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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:

1. 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;


2. potentiometric transducers for measuring pH changes as a
   result of acetic acid production from enzyme activity;


3. 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