3 Recent Advances 53propriate tissues, conferring better antifreeze prop-
erties in farm fish.
BIOENGINEERED
MICROORGANISMS
For over 5000 years, mankind has, knowingly and
unknowingly, made use of spontaneous fermenta-
tion of a variety of food items, which include bread,
alcoholic beverages, dairy products, vegetable prod-
ucts, and meat products. But it was more recently,
just in the last century, that scientists realized that
the process of fermentation was effected by the ac-
tion of microorganisms and that each microorgan-
ism responsible for a specific food fermentation
could be isolated and identified. Now, with advanced
bioengineering techniques, it is possible to charac-
terize with high precision important food strains,
isolate and improve genes involved in the process of
fermentation, and transfer desirable traits between
strains or even between different organisms.
ELIMINATION OFCARCINOGENICCOMPOUNDS
Brewer’s yeast (Saccharomyces cerevisiae)is one of
the most important and widely used microorganisms
in the food industry. This microorganism is cultured
not only for the end products it synthesizes during
fermentation, but also for the cells and the cell com-
ponents (Aldhous 1990). Today, yeast is mainly
used in the fermentation of bread and of alcoholic
beverages. Recombinant DNA technologies have
made it possible to introduce new properties into
yeast, as well as eliminate undesirable by-products.
One of the undesirable by-products formed during
yeast fermentation of foods and beverages is ethyl-
carbamate, or urethane, which is a potential carcino-
genic substance (Ough 1976). For this reason, the
alcoholic beverage industry has dedicated a large
amount of its resources to funding research oriented
to the reduction of ethylcarbamate in its products
(Dequin 2001). Ethylcarbamate is synthesized by
the spontaneous reaction between ethanol and urea,
which is produced from the degradation of arginine,
found in large amounts in grapes. Yeasts, used in
wine fermentation, possess the enzyme arginase that
catalyzes degradation of arginine. If this enzyme can
be blocked, arginine will no longer be degraded into
urea, which in turn will not react with ethanol to
form ethylcarbamate. In industrial yeast, the gene
CAR1encodes the enzyme arginase (EC 3.5.3.1)
(Dequin 2001). To reduce the formation of urea in
sake, Kitamoto et al. (1991) developed a transgenic
yeast strain in which theCAR1gene is inactivated.
The researchers constructed the mutant yeast strain
by introducing an ineffectiveCAR1gene, flanked by
a DNA sequence homologous to regions of the
arginase gene. Through homologous recombination,
the ineffective gene was integrated into the active
CAR1gene in the yeast chromosome, interrupting its
function (Fig. 3.12). As a result, urea was eliminated
and ethylcarbamate was no long formed during sake
fermentation. This same procedure can be used to
eliminate ethylcarbamate from other alcoholic bev-
erages, including wine (Kitamoto et al. 1991).INHIBITION OFPATHOGENICBACTERIATo increase safety, hygiene, and efficiency in the
production of fermented foods, the use of starter and
protective bacterial cultures is a common practice in
the food industry today (Gardner et al. 2001). Starter
culture is a liquid consisting of a blend of selected
microorganisms, used to start a commercial fermen-
tation. The difference between starter and protective
cultures is that starter cultures give the food a de-
sired aroma or texture, while protective cultures
inhibit the growth of undesirable pathogenic micro-
organisms, but do not change the food property
(Geisen and Holzapfel 1996). For the purpose of
practicality during food processing, the same micro-
organism should be used for both starter and protec-
tive cultures, but unfortunately this is not always
possible. Genetic engineering methods help improve
available strains of microorganisms used in starter
and protective cultures, so that new characteristics
can be added and undesirable properties eliminated
(Hansen 2002).
Genetic engineering research aimed at optimizing
starter cultures is focused on three main goals: (1) to
enhance process stability, (2) to increase efficiency,
and (3) to improve product safety (Geisen and Hol-
zapfel 1996). During the production of some fer-
mented food, such as mold-ripened cheese, pH level
rises in the culture due to lactic acid degradation by
fungal activity. This alkaline media offers an ideal
condition for the proliferation of foodborne patho-
genic microorganisms such as Listeria monocyto-
genes(Lewus et al. 1991). The safety of food prod-
ucts could be greatly improved by the use of starter