470 Part IV: Milk
well as to concurrent side reactions (due to trans-
ferase activity) that produce isomers of lactose and
oligosaccharides (Chen et al. 2002). While initially
considered to be undesirable by-products of lactose
hydrolysis, galactooligosaccharides are now recog-
nized to be bifidogenic factors, which enhance the
growth of desirable probiotic bacteria in the intes-
tine of consumers and suppress the growth of harm-
ful anaerobic colonic bacteria (Shin et al. 2000).
Lactose has a number of other properties that
cause difficulty in the processing of dairy products,
such as its tendency to form large crystals on cool-
ing of concentrated solutions of the sugar; lactose-
hydrolyzed concentrates are not susceptible to
such problems. For example, hydrolysis of lactose
in whey concentrates can preserve these products
through increasing osmotic pressure, while main-
taining physical stability (Mahoney 1997, 2002).
For the hydrolysis of lactose in dairy products, -
galactosidase may be added in free solution, allowed
sufficient time to react at a suitable temperature, and
inactivated by heating the product. To control the
reaction more precisely and avoid the uneconomical
single use of the enzyme, immobilized enzyme tech-
nology (e.g., where the enzyme is immobilized on
an inert support, such as glass beads) or systems
where the enzyme is recovered by UF of the prod-
uct after hydrolysis and reused, have been studied
widely (Obon et al. 2000). A further technique with
potential for application in lactose hydrolysis is the
use of permeabilized bacterial or yeast cells (e.g.,
Kluyveromyces lactis) with -galactosidase activity.
In such processes, the cells are treated with agents
(e.g., ethanol) that damage their cell membrane and
allow diffusion of substrate and reaction products
across the damaged membrane; the cell itself be-
comes the immobilization matrix, and the enzyme is
active in its natural cytoplasmic environment (Fontes
et al. 2001, Becerra et al. 2001). This provides a
crude but convenient and inexpensive enzyme uti-
lization strategy. Overall, however, few immobilized
systems for lactose hydrolysis are used commercial-
ly, due to the technological limitations and high cost
of such processes (Zadow 1993).
One of the more common applications of lactose
hydrolysis is the production of low-lactose liquid
milk, suitable for consumption by lactose-intolerant
consumers; this may be achieved in a number of
ways, including adding a low level of-galactosidase
to packaged UHT milk; alternatively, the consumer
may add -galactosidase to milk during domestic
refrigerated storage (Modler et al. 1993). In the case
of ice cream, lactose hydrolysis reduces the inci-
dence of sandiness (due to lactose crystallization)
during storage and, due to the enhanced sweetness,
permits the reduction of sugar content. Lactose
hydrolysis may also be applied in yogurt manufac-
ture, to make reduced-calorie products.
Overall, despite considerable interest, industrial
use of lactose hydrolysis by -galactosidases has
not been widely adopted, although such processes
have found certain niche applications (Zadow 1993).TRANSGLUTAMINASETransglutaminases (TGase; protein-glutamine:amine
-glutamyl transferase) are enzymes that create
inter- or intramolecular cross-links in proteins by cat-
alyzing an acyl-group transfer reaction between the
-carboxyamide group of peptide-bound glutamine
residues and the primary amino group of lysine res-
idues in proteins. TGase treatment can modify the
properties of many proteins through the formation
of new cross-links, incorporation of amines, or de-
mamidation of glutamine residues (Motoki and
Seguro 1998).
TGases are widespread in nature; for example,
blood factor XIIIa, or fibrinoligase, is a TGase-type
enzyme, and TGase-mediated cross-linking is in-
volved in cellular and physiological phenomena
such as cell growth and differentiation, as well as
blood clotting and wound healing. TGases have been
identified in animals, plants, and microbes (e.g.,
Streptoverticillium mobaraense, which is the source
of much of the TGase used in food studies). TGase
may be either calcium-independent (most microbial
enzymes) or calcium-dependent (typical for mam-
malian enzymes).
TGase-catalyzed cross-linking can alter the solu-
bility, hydration, gelation, rheological and emulsify-
ing properties, rennetability, and heat stability of a
variety of food proteins. The structure of individual
proteins determines whether cross-linking by TGase
is possible. Caseins are good substrates for TGase
due to their open structure, but the whey proteins,
due to their globular structure, require modifica-
tion (e.g., heat-induced denaturation) to allow cross-
linking (Ikura et al. 1980; Sharma et al. 2001;
O’Sullivan et al. 2002a,b). When the whey proteins
in milk are in the native state, the principal cross-