20 Biochemistry of Milk Processing 471linking reactions involve the caseins; heat-induced
denaturation renders the whey proteins susceptible
to cross-linking, both to each other and to casein
molecules.
TGase treatment has several significant effects on
the properties of milk and dairy products. For exam-
ple, TGase treatment of fresh raw milk increases its
heat stability; however, if milk is preheated under
conditions that denature the whey proteins prior to
TGase treatment, the increase in heat stability is
even more marked (O’Sullivan et al. 2002a). This is
probably due to the formation of cross-links be-
tween the caseins and the denatured whey proteins,
brought into close proximity by the formation
of disulphide bridges between -lactoglobulin and
micellar -casein. There has also been considerable
interest in the effects of TGase treatment on the
cheese-making properties of milk, in part due to the
potential for increasing cheese yield. It has been
suggested that TGase treatment of milk before ren-
neting can achieve this effect. However, a number
of recently published studies (Lorenzen 2000,
O’Sullivan et al. 2002a) have indicated that the ren-
net coagulation properties of milk and the synere-
tic properties of TGase-cross-linked, renneted milk
gels, as well as the proteolytic digestibility of ca-
sein, are impaired by cross-linking the proteins,
which may alter cheese manufacture and ripening.
Further studies are required to evaluate whether
limited, targeted cross-linking may give desirable
effects.
Other potential benefits of TGase treatment of
dairy proteins include physical stablilization and
structural modification of products such as yogurt,
cream, and liquid milk products, particularly in for-
mulations with a reduced fat content; and the pro-
duction of protein products, such as caseinates or
whey protein products, with modified or tailor-made
functional properties (Dickinson and Yamamoto
1996, Lorenzen and Schlimme 1998, Færgamand
and Qvist 1998, Færgamand et al. 1998).
Overall, it is likely that TGase will have commer-
cial applications in modifying the functional charac-
teristics of milk and dairy products. However, some
important issues remain to be clarified. For example,
knowledge of the heat inactivation kinetics of the
enzyme is needed to facilitate control of the reaction
through inactivation at the desired extent of cross-
linking. There is also a dearth of information on the
effects of processing variables (e.g., temperature,
pH) on the nature and rate of cross-linking reactions.
Finally, a clear commercial advantage for using
TGase over other methods of manipulating the
structure and texture of dairy products (such as addi-
tion of proteins or hydrocolloids) must be estab-
lished.LIPASESLipases, that is, enzymes that produce free fatty
acids (FFAs) and flavor precursors from triglyc-
erides, diglycerides, and monoglycerides, are pro-
duced by a wide range of plants, animals, and
microorganisms (Kilara 2002). For example, calves
produce a salivary enzyme called pregastric esterase
(PGE), which is present in high levels in the stom-
achs of calves, lambs, or kids slaughtered immedi-
ately after suckling (Kilcawley et al. 1998). Micro-
bial lipases have been isolated from Aspergillus
niger, Aspergillus oryzae, Pseudomonas fluores-
cens,and Penicillium roqueforti. Exogenous lipases
may be used for a range of applications (Kilara
2002) including (1) modification of milk fat to
improve physical properties and digestibility, reduce
calorific value, and enhance flavor (Balcão and
Malcata 1998); (2) production of enzyme-modified
cheese flavors (e.g., “buttery,” “blue cheese,” or “yo-
gurt”), and (3) production of lipolyzed creams for
bakery applications.
The correct choice of lipase for particular applica-
tions is very important, as the FFA profile differs
with the type of enzyme used, and different lipases
vary in their pH and temperature optima, heat stabil-
ity, and other characteristics. Calf PGE, for exam-
ple, generates a buttery and slightly peppery flavor,
while kid PGE generates a sharp peppery flavor
(Birsbach 1992).
The intense flavor of blue cheese is derived from
lipolysis and thus catabolism of free fatty acids;
most other cheese varieties undergo very little lipol-
ysis during ripening. A method for the production
of blue cheese flavor concentrates was described by
Tomasini et al. (1995). It has been reported that the
use of exogenous lipases (e.g., PGE) can enhance
the flavor of Cheddar cheese and accelerate the
development of perceived maturity (for review see
Kilcawley et al. 1998). Enzyme-modified cheese pre-
parations (e.g., slurries or powders) can also be pro-
duced by incubation of cheese emulsified in water
with commercial enzymes (such as those produced