Food Biochemistry and Food Processing (2 edition)

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