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52 Part 1: Principles/Food AnalysisAT P ⇒ADP⇒AMP⇒IMP⇒HxR⇒Hx⇒X⇒UFigure 3.8.Sequence of reactions involved in postmortem ATP
breakdown. ADP, adenosine diphosphate; AMP,
adenosine-5′-phosphate; IMP, inosine-5-phosphate; HxR, inosine;
Hx, hypoxanthine; X, xanthine; U, uric acid.muscle has been very well investigated, and the relative amounts
of the different intermediates are generally accepted as fish fresh-
ness indicator.
These key freshness indicators such as ornithine, amines, and
hypoxanthine, which rapidly build up in fresh seafoods, have
been analyzed by various enzymes. These include ornithine
carbamoyl transferase, nucleoside phosphorylase, xanthine oxi-
dase, and diamine oxidase for ornithine and amines; and xanthine
oxidase for hypoxanthine (Cock et al. 2009). In meats, lactic acid
levels tend to increase postmortem, and thus their levels have
been monitored as freshness indicators using xanthine oxidase,
diamine oxidase, and polyamide oxidase.
In addition to the indirect indicators of food quality or safety
described, there are other enzymatic methods for direct detec-
tion of contaminating microflora. The latter has gained even
greater significance for controlling not only food-borne diseases
but also the potential for bioterrorism. The USDA Pathogen Re-
duction Performance Standards has set limits for the presence of
Salmonellafor all slaughter facilities and raw ground meat prod-
ucts (Alocilja and Radke 2003). Other well-known pathogens
recognized as being responsible for food-borne diseases areEs-
cherichia coli0157:H7,Campylobacter jejuni,Listeria monocy-
togenes, Bacillus cereus, Staphylococcus aureus, Streptococci,
etc. While there are a number of conventional methods for de-
tection of such contaminating microflora or pathogens, they tend
to be labor intensive and results are usually not available until
after a couple of days. A number of ELISA methods with high
sensitivity have since been developed that address this drawback
(Croci et al. 2001, Delibato et al. 2006, Salam and Tothill 2009).
Some of these ELISA kits (e.g., LOCATE SALMONELLA pro-
duced by R-Biopharm) are also commercially available. In their
sandwich ELISA method forSalmonelladetection in precooked
chicken, Salam and Tothill (2009) immobilized the capture an-
tibody onto a gold electrode surface, and a second antibody
conjugated to horseradish peroxidase was used as the detection
system for recognition of captured microbial cells. Detection or
binding of the enzyme label is then conducted by an electro-
chemical system using tetramethylbenzidine dihydrochloride as
electron transfer mediator and hydrogen peroxide as substrate.CONCLUDING REMARKS
It is evident from the foregoing that enzymes play a tremen-
dous role in food analysis and their application for this purpose
will likely increase in importance, taking into consideration the
developments in the food industry. With consumers becoming
increasingly aware of the health implications of foods and thegrowing demand for functional foods and nutraceuticals, new
ingredients are being pervasively introduced into the food sup-
ply at a rapid pace. Analysis of these novel ingredients will
invariably require novel analytical tools with the sensitivities
and specificities that are inherent in enzymes. The convergence
of these unique characteristics of enzymes with developments
in biotechnology and biosensors certainly make the application
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