BLBS102-c03 BLBS102-Simpson March 21, 2012 11:56 Trim: 276mm X 219mm Printer Name: Yet to Come
3 Enzymes in Food Analysis 47as incorporation of antioxidants in various food product for-
mulations. Though generally added for their ability to maintain
food quality during processing and storage, antioxidants are also
considered to protect consumers against some of the potential
health risks associated with free radicals. An enzymatic assay
recently developed for antioxidant analysis involves oxidation
of the yellowish compound syringaldazine by laccase to pro-
duce the purple-colored tetramethoxy azobismethylene quinone
(TMAMQ) with maximum absorbance at 530 nm. In the pres-
ence of antioxidants, the free-radical intermediates are unavail-
able or converted, thereby proportionately blocking formation
of the purplish TMAMQ (Prasetyo et al. 2010). This method
has been used to measure the level of antioxidant activity in a
wide range of fruits and vegetables (e.g., apples, carrots, gar-
lic, kiwi, lettuce, spinach, tomatoes), beverages (e.g., green tea,
coffee), and oils. While in most cases, enzymes have been used
for specific identification and analysis of these ingredients, they
have also been used in extraction of some compounds for anal-
ysis. Analysis of flavanoids, which are often present in foods
as the glycosylated or sulfated derivatives, generally involves
enzymatic hydrolysis to the corresponding aglycones and desul-
fated forms during their extraction. Typically used in the extrac-
tion process are theβ-glucuronidase orβ-glycosidases and aryl
sulfatase, as demonstrated in a recent report for determination
of hesperitin and naringenin in foods (Shinkaruk et al. 2010).
The extracted products were then subjected to peroxidase-linked
ELISA.Organic AcidsThe presence and content of organic acids has long been known
to exert significant influence on quality attributes of a wide
variety of food products, including wines, milk, vinegar, soy
sauce, and fermented products. Therefore, a number of enzy-
matic methods have been developed to detect and quantify these
acids such as lactic, acetic, citric, malic, ascorbic, and succinic
acids in a number of food products (Wiseman 1981). Also see
Table 3.2.
The most widely studied of these acids is lactic acid due to
its impact on a broad range of food products. In yogurt pro-
duction and cheese maturation, lactic acid is a major interme-
diate ofLactobacillusfermentation and determines the flavor
of the final product. In wine production, organic acids impact
flavor, protect against bacterial diseases, and may slow down
ripening. Excessive lactic acid in wines has a negative effect
on wine taste due to formation of acetate, diacetyl, and other
intermediates. These irreversible transformations designated by
the French term “piqure lactique” have been attributed to the ac-
tivities of some heterolactic bacteria during malo-lactic fermen-
tation, which compromises wine race (Avramescu et al. 2001,
Shkotova et al. 2008). Lima et al. (1998) developed a method
for the simultaneous measurement of both lactate and malate
levels in wines. This involved injection of enzymes (malate de-
hydrogenase and lactate dehydrogenase) into a buffer carrier
stream flowing to a dialysis unit, where they react with the wine
donor containing the two acids. Detection is based on absorbance
change at 340 nm due to reduction of NAD+to NADH. Simi-lar NAD+-dependent lactate dehydrogenase application for lac-
tate analysis has been developed using amperometric biosensors
(Avramescu et al. 2001, 2002). Another enzymatic method for
lactate determination in wines is the immobilized FAD-bound
lactate oxidase. The enzyme catalyzes conversion of lactate to
pyruvate and peroxide. Decomposition of the hydrogen perox-
ide results in generation of electrons that are recorded by an
amperometric transducer. Table 3.2 lists a number of enzymatic
methods and commercial test kits used for analysis of various
acids in foods.ANALYSIS OF FOOD CONTAMINANTS
PharmaceuticalsThe growing use of antibiotics and other drugs for treatment of
animals meant for food (e.g., cattle, chicken, pigs) is of great
concern to regulatory authorities due to the potential carry-over
effect on consumers as these drugs enter the food supply. This
has led to strict regulations on the prophylactic and therapeutic
use of these drugs being introduced in many countries. There-
fore, a number of methods have been developed for analysis
of these drugs to ensure food safety. For instance, the high inci-
dence of aplastic anemia in some countries has been attributed to
the use of chloramphenicol in the treatment of food-producing
animals, particularly aquaculture (Bogusz et al. 2004). Clen-
buterol, aβ-adrenergic agonist widely used in the treatment of
some pulmonary diseases, also promotes muscle growth, and
therefore, it is sometimes abused in animal feed to boost the
lean meat-to-fat ratio and as a doping agent by athletes (Clarkson
and Thompson 1997). Prolonged use of clenbuterol is known to
cause adverse physiological effects, and incidents of poisoning
have been reported in many countries following consumption of
foods containing high concentrations of the drug (Pulce et al.
1991). ELISA methods based on monoclonal and polyclonal
antibodies are the most widely used of the enzymatic methods
for analysis (Petruzzelli et al. 1996, Matsumoto et al. 2000).
He et al. (2009) recently reported development of a polyclonal
indirect ELISA with high sensitivity for clenbuterol analysis in
milk, animal feed, and liver samples. Another set of drugs sub-
ject to European Union Maximum Residual Limits are the nons-
teroidal anti-inflammatory drugs (NSAIDs) used for treatment of
some food-producing animals (porcine and bovine). Consump-
tion of meat products containing these drugs has been shown
to cause gastrointestinal problems such as diarrhea, nausea, and
vomiting by irritation of the gastric mucosa. A simple enzy-
matic method involving cyclooxygenase has been developed
for analysis of NSAIDs in milk and cheese (Campanella et al.
2009). In absence of NSAIDs, cyclooxygenase catalyzes oxida-
tive conversion of arachidonic acid to prostaglandins. However,
the presence of NSAIDs results in competitive inhibition of this
activity, thereby reducing prostaglandin synthesis. The NSAID
concentration is detected by coupling the catalytic reaction with
an amperometric electrode for oxygen. Chloramphenicol, a po-
tent broad-spectrum antibiotic banned in Europe and the United
States due to potential toxic effects (aplastic anemia), is gen-
erally analyzed using various conventional methods such as