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25 Biochemistry of Milk Processing 479to acid or rennet casein, and may be more suitable for use in
applications such as cheese manufacture, as it closely resembles
the micellar casein in milk.
Whole casein may be resolved intoβ-andαs/κ-casein-rich
fractions by processes that exploit the dissociation ofβ-casein
at a low temperature (reviewed by Mulvihill and Ennis 2003), or
low-temperature dissociation after renneting of milk (Huppertz
et al. 2006). Chromatographic or precipitation processes can be
used to purify the other caseins, although such processes are
generally not easy to scale up for industrial-level production
(Coolbear et al. 1996, Farise and Cayot 1998).EXOGENOUS ENZYMES IN DAIRY
PROCESSINGThe dairy industry represents one of the largest markets for
commercial enzyme preparations. The use of some enzymes in
the manufacture of dairy products, for example rennet for the
coagulation of milk, is in fact probably the oldest commercial
application of enzyme biotechnology.ProteinasesThe use of rennets to coagulate milk and the ripening of cheese,
were discussed earlier. A further application of proteolytic en-
zymes in dairy products, that is the production of dairy protein
hydrolysates, is discussed later in this chapter. The applications
of other enzymes of significance for dairy products are discussed
in this Section.β-GalactosidaseHydrolysis of lactose (4-O-β-d-galactopyranosyl-d-
glucopyranose), either at a low pH or enzymatically (by
β-galactosidases), yields two monosaccharides,d-glucose and
d-galactose (Mahoney 1997, 2002). A large proportion of the
world’s population suffers from lactose intolerance, leading to
varying degrees of gastrointestinal distress if lactose-containing
dairy products are consumed. However, glucose and galactose
are readily metabolised; they are also sweeter than lactose.
Glucose/galactose syrups are one of the simplest and most
common commercial products of lactose hydrolysis.
β-Galactosidases is available from several sources, includ-
ingE. coli, A. niger, Aspergillus oryzae and K. lactis(Whitaker
1994; Mahoney 2002, Husain 2010).β-Galactosidase for use in
dairy products must be isolated from a safe source and be ac-
ceptable to regulatory authorities; for this reason, genes for some
microbialβ-galactosidases have been cloned into safe hosts for
expression and recovery. Of particular interest are enzymes that
are active either at a low or a very high temperature, to avoid
the microbiological problems associated with enzymatic treat-
ment at temperatures that encourage the growth of mesophilic
microorganisms (Vasiljevic and Jelen 2001). Microorganisms
capable of producing thermophilicβ-galactosidases include
Lb. bulgaricussubsp.bulgaricus(Vasiljevic and Jelen 2001)
andThermus thermophilus(Maciunska et al. 1998), while cold-activeβ-galactosidase has been purified from psychrophilic bac-
teria (Nakagawa et al. 2003).
Enzymatic hydrolysis of lactose rarely leads to complete
conversion to monosaccharides, due to feedback inhibition of
the reaction by galactose, as well as concurrent side-reactions
(due to transferase activity) that produce isomers of lactose and
oligosaccharides (Chen et al. 2002). While initially considered
to be undesirable by-products of lactose hydrolysis, galacto-
oligosaccharides are now recognised to be bifidogenic factors,
which enhance the growth of desirable probiotic bacteria in
the intestine of consumers and suppress the growth of harmful
anaerobic colonic bacteria (Shin et al. 2000).
Lactose has a number of other properties that cause diffi-
culty in the processing of dairy products, such as its tendency
to form large crystals on cooling of concentrated solutions of
the sugar; lactose-hydrolysed concentrates are not susceptible
to such problems. For example, hydrolysis of lactose in whey
concentrates can preserve these products through increased os-
motic pressure, while maintaining physical stability (Mahoney
1997, 2002).
For the hydrolysis of lactose in dairy products,
β-galactosidase may be added in free solution, allowed suffi-
cient time to react at a suitable temperature and inactivated by
heating the product. To control the reaction more precisely and
avoid uneconomical single use of the enzyme, immobilised en-
zyme technology (e.g., where the enzyme is immobilised on an
inert support, such as glass beads) or systems where the en-
zyme is recovered by UF of the product 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 permeabilised bacterial or yeast cells (e.g.,K. lactis)
withβ-galactosidase activity. In such processes, the cells are
treated with agents, for example ethanol, to damage their cell
membrane and allow diffusion of substrate and reaction prod-
ucts across the damaged membrane; the cell itself becomes the
immobilisation matrix and the enzyme is active in its natural cy-
toplasmic environment (Fontes et al. 2001, Becerra et al. 2001).
This provides a crude but convenient and inexpensive enzyme-
utilisation strategy. Overall, however, few immobilised systems
for lactose hydrolysis are used commercially, due to technolog-
ical limitations and high cost of such processes (Zadow 1993);
one strategy of potential interest involves the recovery of sol-
uble enzyme by UF, allowing separation from the product and
substrate, and recovery and reuse of the enzyme.
One of the more common applications of lactose hydrol-
ysis 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
β-galactosidase to packaged UHT milk, or the consumer may
addβ-galactosidase to milk during domestic refrigerated storage
(Modler et al. 1993). In the case of ice cream, lactose hydrolysis
reduces the incidence of sandiness (due to lactose crystallisation)
during storage and, due to the enhanced sweetness, permits the
reduction of sugar content. Lactose hydrolysis may also be ap-
plied in yoghurt manufacture, to make reduced-calorie products.
Overall, despite considerable interest, industrial use of lactose
hydrolysis byβ-galactosidases has not been widely adopted,