144 2 Enzymes
nitude due to amplification which is millionfold
after 20 cycles and billionfold after 30 cycles.
Heated food can be analyzed because DNA is
considerably more stable than proteins. It is also
possible to detect GMOs which do not contain al-
tered or added proteins identifiable by chemical
methods. Acidic foods can cause problems when
they are strongly heated, e. g., tomato products. In
this case, the DNA is hydrolyzed to such an ex-
tent that the characteristic sequences are lost. The
exceptional sensitivity of this method can also
give incorrect positive results. For this reason, it
is important that the PCR is quantitatively evalu-
ated, especially when controlling limiting values.
A known amount of a synthetic DNA is added to
the sample and amplified competitively with the
analyte. For calibration, mixtures of the target and
competing DNA are subjected to PCR analysis.
2.6.4.2 Examples
2.6.4.2.1 Addition of Soybean
The addition of soybean protein to meat and
other foods can be detected with the help of
the primers GMO3 (5′-GCCCTCTACTCCA-
CCCCCATCC-3′) and GMO4 (5′-GCCCATC-
TGCAAGCCTTTTTGTG-3′). They label a small
but still sufficiently specific sequence of 118 base
pairs (bp) of the gene for a lectin occurring in
soybean. A small amplicon is of advantage since
the DNA gets partially fragmented when meat
preparations are heated.
2.6.4.2.2 GeneticallyModifiedSoybeans............................
Genetically modified soybeans are resistant to
the herbicide glyphosphate (cf. 9.4.3), which
inhibits the key enzyme, 5-enolpyruvylshiki-
mi-3-phosphate synthase (EPSPS), in the
metabolism of aromatic amino acids in plants.
However, glyphosphate is inactive against the
EPSPS of bacteria. Hence transgenic soybeans
contain a genetic segment which codes for an
EPSPS fromAgrobacterium sp.and a peptide
for the transport of this enzyme. To detect this
segment and, consequently, genetically modified
soybeans, primers are used which induce the
amplification of a segment of 172 bp in the PCR.
2.6.4.2.3 GeneticallyModifiedTomatoes............................
During ripening and storage, tomatoes soften due
to the activity of an endogenous enzyme poly-
galacturonase (PG). The expression of the gene
for PG is specifically inhibited in a particular
tomato, resulting in extended storage life and bet-
ter aroma. PCR methods have been developed to
detect these transgenic tomatoes. However, this
detection can fail if the DNA is too strongly hy-
drolyzed on heating the tomato products.2.6.4.2.4 SpeciesDifferentiation...................................
If specific primers fail, a PCR with universal
primers can be applied in certain cases, followed
by an RFLP analysis (restriction fragment length
polymorphism). The DNA of a meat sample is
first determined with a primer pair which exhibits
a high degree of correspondence in its binding
sites to the DNA of many animal species. In the
case of various animal species, the PCR yields
equally long products which should be relatively
large (ca. 300–500 bp). The amplicon is cleaved
in the subsequent RFLP analysis with different
restriction endonucleases. After electrophoretic
separation, the pattern of the resulting DNA
fragments can be assigned to individual animal
species. This method is suitable for samples of
one type of meat. Preparations containing meat
of several animal species or DNA which is more
strongly fragmented on heating can be reliably
analyzed only with animal species-specific
primers.2.7 Enzyme Utilization in the Food Industry
Enzyme-catalyzed reactions in food processing
have been used unintentionally since ancient
times. The enzymes are either an integral part
of the food or are obtained from microorgan-
isms. Addition of enriched or purified enzyme
preparations of animal, plant or, especially, mi-
crobial origin is a recent practice. Most of these
enzymes come from microorganisms, which
have been genetically modified in view of their
economic production. Such intentionally used