BLBS102-c03 BLBS102-Simpson March 21, 2012 11:56 Trim: 276mm X 219mm Printer Name: Yet to Come
40 Part 1: Principles/Food Analysisproteins, carbohydrates, and lipids) and others in extremely low
amounts (e.g., flavor components, micronutrients such as vita-
mins, antioxidants, etc.). Analysis of these different components
requires techniques that are not only simple and affordable but
also, more importantly, very specific and sensitive to ensure that
the “needle in the haystack” can be picked up (identified) and its
size (amount) determined. These challenges have been largely
met by technological advancements in chromatography, elec-
trophoresis, mass spectrometry, etc. The specificity and relative
sensitivity of enzymes make them ideally suited and versatile
candidates for addressing these challenges as well. The protein
nature of enzymes, however, means their efficacy for such ap-
plications is influenced by factors such as pH, temperature, and
ionic strength of the analytes and the catalytic milieu. These
factors tend to limit the scope of enzyme applications for this
purpose as they may destabilize or denature the enzyme, com-
promise specificity, or reduce affinity of the enzyme for the
substrate or analyte of interest. Some of these limitations have
been addressed using immobilization techniques. Furthermore,
the significant developments in biosensor technology provide
an even greater opportunity for use of enzymes for this purpose
by providing the necessary robustness, versatility, and afford-
ability. Basically, biosensors are analytical tools consisting of
two main components: (i) a bioreceptor for selective analyte
or food ingredient detection and (ii) a transducer that transmits
the resulting effect of the bioreceptor–analyte interaction to a
measurable signal (Viswanathan et al. 2009). These signals may
be amperometric, piezoelectric, potentiometric, fluorometric, or
colorimetric. Furthermore, developments in biotechnology have
made possible the commercial production, and thus availability,
of a broad range of enzymes with the desired characteristics for
use in such applications. This chapter is aimed at discussing the
application of enzymes in analysis of foods, with emphasis on
the principles underlying their role in these analyses. Obviously,
any attempt at reviewing the analytical role of enzymes for all
food components will be a massive endeavor that cannot be cap-
tured in one chapter, and there have been a number of reviews
on the subject in the past, several of which have been appro-
priately cited. Therefore, the emphasis of this chapter is on the
enzymatic methods for analysis of a few selected topics related
to food components, including the major food components (i.e.,
proteins, carbohydrates, and lipids), toxins, contaminants, and
food quality.ANALYSIS OF THE MAJOR FOOD
COMPONENTSProteins and Amino AcidsProteins are major components of foods, and while the primary
interest in proteins is due to their nutritional and functional
impact on foods, the allergic reactions induced in sensitized in-
dividuals following consumption of certain proteinaceous foods
have been a key driver for the development of highly selective
and sensitive tools for protein detection and analysis to ensure
that food products conform to regulatory standards. For exam-
ple, lysozyme (muramidase) is an enzyme derived from henegg white and is approved for use as an antimicrobial agent for
preservation of the quality of ripened cheese. However, it is a
potential allergen and the joint Food and Agricultural Organiza-
tion/World Health Organization Codex Alimentarius Commis-
sion require its presence in foods to be declared on the label
as an egg product (Codex Standard I-1985). Enzyme-linked im-
munosorbent assay (ELISA) has been a major technique for
analysis of these and other proteins with limits of detection
ranging from 0.05 to 10 mg/kg (Schubert-Ulrich et al. 2009).
The widespread application of ELISA for these analyses has
been attributed to its specificity, sensitivity, simple sample han-
dling, and high potential for standardization and automation.
Schneider et al. (2010) recently reported an ELISA for anal-
ysis of lysozyme in different kinds of cheese. Their method
involved use of a peroxidase-labeled antibody that undergoes
a redox reaction with tetramethylbenzidine dihydrochloride to
form a product that can be measured by the absorbance change
at 450 nm. A number of ELISA methods have been used for
analysis of lysozyme in hen egg white, wines, cheese, and other
foods (Rauch et al. 1990, Yoshida et al. 1991, Rauch et al. 1995,
Vidal et al. 2005, Kerkaert and De Meulenaer 2007, Weber et al.
2007). Other proteins that have been analyzed by ELISA in-
clude those found in hazelnuts (Holzhauser and Vieths 1999),
peanuts (Pomes et al. 2003), walnuts (Doi et al. 2008), soy (You
et al. 2008), sesame (Husain et al. 2010), etc. In soybeans, as
many as 16 allergenic proteins have been detected, of which
β-conglycinin is considered to be the major culprit (Maruyama
et al. 2001, Xiang et al. 2002). In the method of You et al. (2008)
forβ-conglycinin analysis, they had used an epitope correspond-
ing to amino acid residues 78–84 of the protein to synthesize
an immunogen by linking it (the epitope) to chicken ovalbu-
min and bovine serum albumin as carrier proteins. Horseradish
peroxidase was used as the enzyme for detection by measuring
absorbance at 492 nm. Table 3.1 shows a list of ELISA-based
methods commercially available for analysis of a number of
food proteins.
ELISA is a major enzyme-based analytical tool for a broad
range of food components and other ingredients, and therefore,
an explanation of the underlying principles is appropriate here.
Figures 3.1 and 3.2 illustrate the general principles underlying
the use of sandwich and competitive ELISA (both of which may
be direct or indirect) for protein analysis. In the direct sand-
wich ELISA, a capture antibody specific for the protein being
analyzed is first immobilized onto a solid phase. When analytes
containing the protein of interest are introduced or present in
the system or food material, they bind specifically to the im-
mobilized antibody. Detection of such binding is achieved by
introducing a second enzyme-labeled analyte-specific antibody
into the system, thereby forming a sandwich. In the indirect
sandwich ELISA, a second analyte-specific antibody binds to
a secondary site on the protein of interest (analyte) to form
the sandwich before the enzyme-labeled antibody binding. The
requirement for a secondary binding site or more than one epi-
tope for specific binding makes this type of ELISA primar-
ily applicable to large molecules like proteins. In both types,
the binding reaction may be linked to a chromophore, with the
color intensity being proportional to the protein concentration.