BLBS102-c25 BLBS102-Simpson March 21, 2012 13:23 Trim: 276mm X 219mm Printer Name: Yet to Come
480 Part 4: Milkalthough such processes have found certain niche applications
(Zadow 1993).TransglutaminaseTransglutaminases (TGase; protein-glutamine: amine γ-
glutamyl-transferase) are enzymes that create inter- or intra-
molecular cross-links in proteins by catalysing an acyl-group
transfer reaction between theγ-carboxyamide group of peptide-
bound glutamine residues and the primary amino group of lysine
residues in proteins. TGase treatment can modify the properties
of many proteins through the formation of new cross-links, in-
corporation of amines or deamidation of glutamine residues.
TGases are widespread in nature; for example blood factor
XIIIa, or fibrinoligase, is a TGase-type enzyme, and TGase-
mediated cross-linking is involved 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 mo-
baraense, which is the source of much of the TGase used in food
studies). TGase may be either calcium-independent (most mi-
crobial enzymes) or calcium-dependent (typical for mammalian
enzymes).
TGase-catalysed cross-linking can alter the solubility, hy-
dration, gelation, rheological and emulsifying properties, ren-
netability and heat stability of a variety of food proteins (Motoki
and Seguro 1998). The structure of individual proteins deter-
mines 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 modifi-
cation, for example 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-linking 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 proper-
ties of milk and dairy products. For example, TGase treatment
of fresh raw milk increases its heat stability; if milk is pre-
heated 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 for-
mation of cross-links between the caseins and denatured whey
proteins, brought into close proximity by the formation of disul-
phide bridges betweenβ-lg and micellarκ-casein. There has
also been considerable interest in the effects of TGase treatment
on the cheesemaking properties of milk, in part due to the po-
tential for increasing cheese yield. It has been suggested that
TGase treatment of milk before renneting can achieve this ef-
fect. However, a number of studies (Lorenzen 2000, O’Sullivan
et al. 2002a) have indicated that the rennet coagulation proper-
ties of milk and the syneretic properties of TGase-cross-linked
renneted milk gels, as well as the proteolytic digestibility of
casein, are impaired by cross-linking the proteins, which may
alter cheese manufacture and ripening. Further studies are re-
quired to evaluate whether limited, targeted cross-linking may
give desirable effects.Other potential benefits of TGase treatment of dairy pro-
teins include physical stablilisation and structural modification
of products such as yoghurt, cream and liquid milk products,
particularly in formulations with a reduced fat content, and the
production of protein products, such as caseinates or whey pro-
tein 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 commercial appli-
cations in modifying the functional characteristics of milk and
dairy products. However, some important issues remain to be
clarified; for example the heat inactivation kinetics of the en-
zyme, to facilitate control of the reaction through inactivation at
the desired extent of cross-linking. There is also a dearth of infor-
mation on the effects of processing variables (e.g., temperature,
pH) on the nature and rate of cross-linking reactions. Finally, a
clear commercial advantage of using TGase over other methods
for manipulating the structure and texture of dairy products (such
as addition of proteins or hydrocolloids) must be established.LipasesLipases, that is enzymes that produce free fatty acids (FFAs) and
flavour precursors from triglycerides, diglycerides and mono-
glycerides, are produced by a wide range of plants, animals and
microorganisms (Kilara 2003). For example, calves produce a
salivary enzyme called PGE, which is present at high levels in the
stomachs of calves, lambs or kids slaughtered immediately after
suckling (Kilcawley et al. 1998). Microbial lipases have been
isolated fromA. niger,A. oryzae,Pseudomonas fluorescensand
P. roqueforti. Exogenous lipases may be used for a range of
applications (Kilara 2011), including:
modification of milk fat to improve its physical properties
and digestibility, reduce calorific value and enhance flavour
(Balc ̃ao & Malcata 1998);
production of EMC flavours (e.g., ‘buttery’, ‘blue cheese’
or ‘yoghurt’); and
production of lipolysed creams for bakery applications.The correct choice of lipase for particular applications 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 stability and other characteristics. Calf PGE, for
example generates a buttery and slightly peppery flavour, while
kid PGE generates a sharp peppery flavour (Birsbach 1992).
The intense flavour of blue cheese results from lipolysis and
catabolism of FFAs; most other cheese varieties undergo very
little lipolysis during ripening. A method for the production
of blue cheese flavour concentrates was described by Tomasini
et al. (1995). It has been reported that the use of exogenous li-
pases (e.g., PGE) can enhance the flavour of Cheddar cheese and
accelerate the development of perceived maturity (for review see
Kilcawley et al. 1998). EMC preparations (e.g., slurries or pow-
ders) can also be produced by incubation of cheese emulsified
in water with commercial enzymes (such as those produced by
Novo Nordisk) that have 5–20 times the flavour intensity of mild
Cheddar cheese.