Food Biochemistry and Food Processing (2 edition)

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BLBS102-c03 BLBS102-Simpson March 21, 2012 11:56 Trim: 276mm X 219mm Printer Name: Yet to Come


50 Part 1: Principles/Food Analysis

R-CH 2 -NH 2 + O 2 + 2 O R-CHO ++NH 3 H 2 O 2

Amine oxidase

Enzymatic reaction:

+HReduced dye (colorless) 2 O + Oxidized dye (colored)

Peroxidase

Color reaction:

Electrochemical reaction:

H 2 O 2

H 2 O 2

O 2 + 2H+ 2e–

H

Figure 3.7.Principle underlying amine detection by coupling amine oxidase and peroxidase reactions.

immunogen. Antisera raised against the immunogen and shown
to have high affinity for the 3-MBA-derivatized acrylamide was
used in the development of a peroxidase-based ELISA detec-
tion system with about 66μg/kg limit of detection. A com-
mercial ELISA kit has been recently introduced by Abraxis
(www.abraxis.com) for acrylamide analysis.

Environmental Toxins

Certain toxin-producing phytoplanktons present in the aquatic
environment have generally been recognized as the source of
marine toxins in the human food chain following consumption
of contaminated fish and shellfish. Safety concerns have led to
regulatory limits being set for the presence of these toxins in
seafoods.
Okadaic acid and its derivatives, which are considered respon-
sible for diarrheic shellfish poisoning, are very potent inhibitors
of protein phosphatase, and a number of methods have been de-
veloped around inhibition of the enzyme for its analysis (Tubaro
et al. 1996, Della Loggia et al. 1999, Campas and Marty 2007,
Volpe et al. 2009). Analysis of this toxin has also been accom-
plished by ELISA methods (Kreuzer et al. 1999, Campas et al.
2008). The toxic effects of yessotoxins remain to be elucidated
even though in vitro studies indicate cardiotoxicity. Analytical
methods based on ELISA (Garthwaite et al. 2001, Briggs et al.
2004) and yessotoxin interaction with pectinesterase enzymes
have been developed (Alfonso et al. 2004, Pazos et al. 2005,
Fonfria et al. 2008).
Another group of marine toxins of relevance to food are
the brevetoxins responsible for neurotoxic shellfish poisoning.
The inhibition by brevetoxins of the desulfo-yessotoxin inter-
action with phosphodiesterase provided the basis for an analyt-
ical method (Mouri et al. 2009), and also reported are various
ELISA methods (Baden et al. 1995, Garthwaite et al. 2001, Naar
et al. 2002). Saxitoxins, responsible for paralytic shellfish poi-
soning, have been analyzed in foods by ELISA (Chu and Fan
1985, Garthwaite et al. 2001). The paralytic effect is attributed to
their high-affinity binding to specific sites on sodium channels,
thereby blocking sodium flux across excitable cells. Like the

saxitoxin group of toxins, amnesic shellfish poisoning is associ-
ated with consumption of foods contaminated with the domoic
acid group of water-soluble neurotoxins produced by the gen-
eraPseudonitzschia,Nitzschia,andChondria armata(Vilarino
et al. 2009). Ciguatoxin poisoning is characterized by symptoms
of nausea, diarrhea, abdominal cramps, memory loss, dizziness,
headaches, and death in extreme cases. An ELISA method ap-
proved by AOAC has been adopted in many countries (Kleivdahl
et al. 2007). Tsai et al. (2009) have reported the identification
of moray eel species responsible for ciguatera poisoning based
on analysis of the cyt b gene in mitochondrial DNA using the
PCR and restriction enzymes. In a recent study, Garet et al.
(2010) compared relative sensitivities of ELISA and conven-
tional methods (HPLC-UV and mouse bioassay) for detection
of amnesic shellfish poisoning and paralytic shellfish poison-
ing toxins in naturally contaminated fresh, frozen, boiled, and
canned fish and shellfish. Their findings showed lower limits
of detection (50μg saxitoxin and 60μg per kg shellfish meat
for saxitoxin and domoic acid, respectively) compared to 350
μg saxitoxin and 1.6 mg domoic acid per kg shellfish meat,
respectively, when analyzed by conventional methods.
Detection of marine palytoxins and tetrodotoxins using
ELISA techniques have also been reported (Bignami et al. 1992,
Lau et al. 1995, Rivera et al. 1995, Kawatsu et al. 1997, Kreuzer
et al. 2002, Neagu et al. 2006). Palytoxins and their analogues
elicit their toxic effects by binding to the Na+/K+ATPase
pumps, thereby altering the selectivity of ion flux and membrane
potential. Much like the saxitoxins, the tetrodotoxins elicit their
neurotoxicity by inhibiting voltage-gated sodium channels. The
method of Kreuzer et al. (2002) for analysis of tetrodotoxins
involves an alkaline phosphatase-labeled antibody, the activity
of which produces p-aminophenol, which can be detected by
the amount of current generated. Alkaline phosphatase activity
is competitively inhibited by tetrodotoxin, thereby reducing the
current produced.
The presence of pesticides in foods and their impact on health
is an issue of critical concern to the food industry, and therefore,
regulatory measures are in place establishing maximum resid-
ual limits. Inhibition of acetylcholinesterase by pesticides has
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