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International Dairy Journal 16 (2006) 533–545
www.elsevier.com/locate/idairyj
Review
Physiological role of indigenous milk enzymes:
An overview of an evolving picture
Nissim Silanikovea,, Uzi Merinb, Gabriel Leitnerc
a
b
Department of Ruminant Physiology, Institute of Animal Science, A.R.O., The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel
Department of Food Science, Institute of Technology and Storage of Agricultural, Products, A.R.O., The Volcani Center, P.O. Box 6, Bet Dagan 50250,
Israel
c
National Mastitis Reference Center, Kimron Veterinary Institute, P.O.B. 12, Bet Dagan 50250, Israel
Received 29 May 2005; accepted 26 August 2005
Abstract
Over 60 indigenous enzymes have been identiﬁed so far in the milk of various mammalian species. The vast majority of research in this
area has focused on their use as indicators of processing (mainly pasteurization), contribution to dairy product quality and investigating
the factors that affect their level in milk. The aim of this article is to provide an overview of data accumulated during the last 5 years,
mostly for bovine and human milk, which shows that milk indigenous enzymes play a key role in regulating lactogenesis, e.g., inducing
active involution, and that they are essential components of antioxidation and the innate immune system of milk.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Milk; Colostrum; Indigenous enzymes; Review; Physiological role
Contents
1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Physical nature of milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3. Distribution of milk enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.4. Factors governing the level of enzymes in milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Physiological roles of milk enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Role of milk enzymes as pro-digestive factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Role of the plasmin system in the regulation of mammary secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Role of the plasmin system in induction of involution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. The plasminogen activator–plasminogen–plasmin-based negative feedback mechanism as a working hypothesis
2.5. Milk enzymes as part of the innate immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
1.1. Overview
Corresponding author. Tel.: +972 8 9484436; fax: +972 8 9475075.
E-mail address: [email protected] (N. Silanikove).
0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2005.08.015
Milk synthesis starts in the epithelial cells of the
mammary gland at the end of pregnancy to support the
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nutrition and promote the health of the off-spring. Milk
contain vital nutrients such as proteins, carbohydrates,
lipids, minerals and vitamins, together with bioactive
substances including immunoglobulins, peptides, antimicrobial factors, hormones and growth factors (Clare &
Swaisgood, 2000; Grosvenor, Picciano, & Baumrucker,
1993). Over 60 indigenous enzymes have been identiﬁed so
far in the milk of various mammalian species (Fox, 2003).
Because of its complex composition, lactogenesis requires
the concerted action of many transport processes and the
presence of many milk constituents may have biological
effects on both the mother (Silanikove, Shamay, Shinder,
& Moran, 2000), and the survival of her off-spring
(Koldovsky, 1998). The purpose of the present review is
to provide an overview of data, mostly on bovine and
human milk, showing that indigenous milk enzymes play a
key role in regulating lactogenesis, including inducing
active involution, and that they are essential components of
antioxidation and the innate immune system of milk.
A brief review of literature regarding the physical nature of
milk, the distribution of enzymes between milk compartments and factors governing their level in milk is presented
initially as a background.
1.2. Physical nature of milk
Though its structure appears to be continual and
homogeneous, milk is composed of at least ﬁve physically
and functionally discrete phases (Fig. 1):
Whey: Milk serum, commonly known as whey, is the
medium in which all compartments are homogeneously
dispersed. Whey is composed of water in which minerals,
various organic molecules, proteins and peptides are
dissolved.
Fat globules: The fat is dispersed in milk as small
droplets that are enveloped by a plasma membrane rich in
phospholipids and commonly known as the milk fat
globule membrane (MFGM). In bovine milk, these
Fig. 1. Schematic representation of physical phases of milk. The area
between the milk particles represents the milk serum (whey), the phase in
which all other phases are homogenously dispersed.
globules range in size from 1 to 8 mm and average 3–4 mm
in diameter (Heid & Keenan, 2005).
Casein micelles: The main protein in milk is casein, of
which there are 4 types in bovine milk, arranged as large
colloidal particles commonly known as micelles. Highresolution ﬁeld-emission scanning electron microscope
micrographs of casein micelles revealed spherical particles
in the range of 200 nm; the surface of the micelle is not
smooth but contains gaps between the tubular substructures (Dalgleish, Spagnuolo, & Goff, 2004).
Membrane vesicles: The secretion of various membrane
vesicles into the extracellular space is a frequent phenomenon described in normal and tumor cells (Hugel, Carmen
Martinez, Kunzelmann, & Freyssinet, 2005). Two types of
vesicles are secreted by cells.
Exosomes, typically 40–100 nm in diameter, originate
from endocytic multivesicular bodies, and are released in
an exocytic manner. Although the functions of exosomes
remain largely unresolved, they are thought to play
immunoregulatory and antitumoral roles (Fevrier &
Raposo, 2004).
Microvesicles, with a diameter in the range 100–1000 nm,
originate from the cell surface membrane and are shed
directly into the extracellular space, a process that seems to
be important for membrane turnover, tumor ganglioside
metabolism and vascular regulation (Hugel et al., 2005).
The processes of exosome secretion and membrane
shedding are poorly understood.
In bovine milk, 40–60% of the membranous phospholipids are in the skim milk, the remainder being associated
with the MFGM (Huang & Kuksis, 1967; Morton, 1954;
Plantz & Patton, 1973). The skim milk membrane vesicles
comprise approximately 1% of the total milk lipids (Huang
& Kuksis, 1967) and 5%, w/w, of the total milk proteins
(Morton, 1954). Morton (1954) was the ﬁrst to show that
normal cows’ milk contains enzymatically active lipoprotein particles, which he called ‘milk microsomes’. Based on
their size (30–200 nm), enzymatic nature and chemical
composition, it was concluded that milk microsomes
originate directly from the microsomes of secretory
epithelial cells (Bailie & Morton, 1958a, b). Thus, milk
microsomes most likely reﬂect the secretion of endosomes
(i.e., vesicles that originate deep in the cell) from mammary
epithelial cells. The possibility that microvesicles are also
released into the milk by shedding fragments of the apical
membrane of the mammary epithelial cells should be taken
into consideration and is supported by some histological
data, which show budding of microvilli (Wellings, Deome,
& Pitelka, 1960), and the presence of detached microvilli in
the ‘ﬂuff’ (membranous) fraction of skim milk (Plantz &
Patton, 1973). The secretion of microvesicles into milk may
also function in regulating homeostasis of the apical
membrane of the mammary epithelial cells (Kanno,
1990). Intracellular vesicles are constantly fused into the
apical membrane during exocytotic secretion of milk
proteins, lactose, calcium and other components of the
aqueous phase of milk (McManaman & Neville, 2003),
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while cytoplasmic lipid droplets that move to the apical
membrane are enveloped by apical membrane and secreted
as membrane-bound milk fat globules (Wu, Howell,
Neville, Yates, & MacManaman, 2000).
In general, the composition of phospholipids derived
from milk vesicles and the MFGM are very similar (Huang
& Kuksis, 1967), which points to a common origin of the
two types of membrane (Patton, Mccarthy, & Durdan,
1966). Shennan (1992) found that K+ and Cl ions crossmembrane vesicles by conductance pathways. The similarity between ion transport by skim milk membrane vesicles
and by the apical side of the intact mammary epithelium
suggests that the former may be a good model in which to
study solute transport by the apical membrane of
mammary secretory cells. Silanikove et al. (2000) have
identiﬁed a casein-derived peptide that blocks K+ uptake
into skim milk membrane vesicles and demonstrated that
this peptide down-regulates milk secretion in vivo. Thus,
skim milk membrane vesicles appear to maintain their
integrity and functionality during storage in the mammary
gland.
Milk cells: The milk of various mammals contains a
heterogeneous population of cells, commonly referred to as
somatic cells (SC). In most species, the predominant cells
are leukocytes, composed of lymphocytes, polymorphonuclear neutrophils (PMNs) and macrophages, which serve
as important components in the mammary defense against
potential pathogens, mostly bacteria (Paape, Bannerman,
Zhao, & Lee 2003). In bacteria-free cows’ milk, macrophages (Concha, Holmberg, & Astrom, 1986) and epithelial cells (Leitner, Shoshani, Krifucks, Chaffer, & Saran,
2000) are the predominant cell type (35–79%). Following
detection of an invasion of pathogens into the mammary
gland, macrophages release chemo-attractants, which
trigger the migration of PMNs from the blood toward
the infection in the gland, increasing their proportion from
a basal level of 5–25% to approximately 90% of the total
cell population. PMNs phagocytose the invaders and then
destroy them by using reactive oxygen species and a range
of proteolytic enzymes (Paape, Wergin, Guidry, & Pearson,
1979).
1.3. Distribution of milk enzymes
Milk is not a homogeneous solution of enzymes; rather,
given enzymes are speciﬁcally associated with one or more
of the above-described ﬁve distinct phases (see Table 1 in
Shahani, Harper, Jensen, Parry, & Zittle, 1973). The
distribution of enzymes in milk most likely reﬂects the
way in which they were secreted into the milk and their
tendency to associate with particular milk constituents or
phases.
Little is known about the mechanisms by which
indigenous enzymes enter milk, but knowledge of these
processes is important for understanding their physiological role. The term ‘spilling over’ from epithelial mammary
cells or serum during milk secretion was used in earlier
535
(Kitchen, Taylor, & White, 1970) and later (Farkye, 2003)
studies as an explanation for their presence in milk.
However, it is doubtful if the term ‘spill over’ is justiﬁed
in explaining the origin of enzymes in milk. Milk
components are secreted through organized pathways
(McManaman & Neville, 2003) without evidence for
spillage or waste of cytoplasm during the process, except
for caprine milk, which contain a lot of cytoplasmic
particles (Neveu, Riaublanc, Miranda, Chich, & Martin,
2002). In some cases, there is an association of crescent
material with 1% of the MFGM in bovine milk (Huston
& Patton, 1990). A variable but low level of crescent
material was also found in the milk of other mammals,
such as goats, rats, pigs, sheep, rabbits and humans
(Janssen & Walstra, 1982). According to Wooding
(1977), these crescents contain trapped cytoplasma, and
therefore may contain cellular enzymes. However, Huston
and Patton (1990) suggested that an abnormality of the
inner protein coat of the MFGM may be responsible for
crescent formation and, in that case, the crescent would be
expected to be a poor source of enzymes. In any event,
there is no reason to assume that the secretion of crescents
is an unregulated phenomenon. In the mammary gland, the
tight junctions of the alveolar epithelial cells are impermeable even to small atoms or molecules such as Na+ and K+
(Nguyen & Neville, 1998). It should also be borne in mind
that tight junctions separate milk and extracellular ﬂuid, in
which the concentration of proteins is much lower than in
the blood serum. Nevertheless, it is commonly believed that
the source of albumin in milk is blood serum and
abbreviations such as human serum albumin (HSA) and
bovine serum albumin (BSA) are commonly used to
describe the albumin in milk. Shamay et al. (2005) have
shown recently that the mammary gland itself is a source of
milk albumin. The increase in the level of albumin in milk
was previously taken as evidence for disruption of the tight
junctions (Nguyen & Neville, 1998). However, Shamay
et al. (2005) showed that, as part of the innate immune
non-speciﬁc defense system the synthesis and secretion of
albumin increased under conditions that typically cause
disruption of tight junctions. Thus, if blood serum is a
source of a given milk enzyme, it is probably through
dedicated transcellular transport systems, such as that
responsible for the transfer of immunoglobulins (McManaman
& Neville, 2003)
The largest group of indigenous milk enzymes is that
associated with the MFGM and vesicle membranes
(Shahani et al., 1973). This phenomenon probably reﬂects
the cellular sources of these membranes, i.e., Golgi
membranes (Powell, Jarlfors, & Brew, 1977), the rough
endoplasmic reticulum (RER) (Jarasch, Bruder, Keenan, &
Franke, 1977) and plasma membranes (Dowben, Brunner,
& Philpott, 1967). A prominent marker of Golgi membranes from bovine mammary gland epithelial cells is
galactosyltransferase (Keenan, Morre, & Huang, 1972),
which also has high speciﬁc activity in the MFGM (Powell
et al., 1977). A b-type cytochrome which is speciﬁcally
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associated with the RER in cows has also been identiﬁed in
MFGM and milk microsomes (Jarasch et al., 1977). The
‘sidedness’ in the membrane is also important in considering the physiological role and function of membraneassociated enzymes. Enzymes located within the inner side
of MFGM or milk vesicles are intact and therefore their
activity would be latent in respect to the outer membrane
environment. Patton and Trams (1971) showed that
nucleotide pyrophosphatase is conﬁned largely to the inner
membrane surface, 50 -nucleotidase is located on the outer
surface and Mg2+-activated ATPases are in both surfaces.
In uninfected glands, sporadic invasion of bacteria into
the gland is counteracted by macrophages, which phagocytose and digest them intracellulary (Paape et al., 2003).
Thus, in the uninfected udder, leukocyte enzymes most
likely do not interact with milk proteins. Intramammary
infection causes an elevation of the somatic cell count
(SCC), mostly through increased number of PMNs,
associated with increased proteolytic activity in milk
(Leitner, Chaffer et al., 2004; Leitner, Merin, & Silanikove,
2004). There is evidence that the oxidative stress associated
with clinical mastitis induces massive liberation of proteolytic enzymes from PMNs, which in turn affect proteolysis
and cause damage to the mammary tissue (Le Roux,
Laurent, & Moussaoui, 2003). Milk from mastitic cows is
not used by the dairy industry. However, udders of
30–50% of cows in herds in modern dairy systems may
be subclinically infected and this milk is collected routinely
by the industry. An important question which arises is
whether leukocyte enzymes, under subclinical conditions,
affect milk components. PMNs contain a number of
lysosymal proteolytic enzymes, including neutral and acidic
proteases, elastase and cathepsin B and D (Considine,
Healy, Kelly, & McSweeney, 2004). During inﬂammation,
there is also increased secretion of lysosomal enzymes, such
as N-acetyl-b-D-glucosaminidase (NAGase) from epithelial
cells (Leitner, Chaffer et al., 2004; Leitner, Merin,
Silanikove, Ezra et al., 2004). Therefore, it would be
difﬁcult to distinguish between leukocyte-derived and
epithelial cell-derived enzymes during subclinical mastitis.
1.4. Factors governing the level of enzymes in milk
The composition of milk is adapted to support the
demands of the off-spring during the pre-weaning period,
which is reﬂected in great interspecies variability in the
gross composition of milk. Limited information, particularly comparison between human and cows, suggests that
signiﬁcant inter-species differences exist also in the level of
enzymes in milk (Table 1 in Shahani, Kwan, & Friend,
1980). In general, the level of enzymes in human milk
is much greater than in bovine milk, e.g., adenosine
triphosphatase ( 23), alanine aminotransferase ( 400),
a-amylase ( 40) lysozyme ( 100) and NAGase ( 3000).
However, in certain cases, the level of particular enzymes
are much higher in bovine than in human milk, e.g.,
lactoperoxidase ( 100), alkaline phosphatase ( 40) and
xanthine oxidase ( 10). Peroxidase activity in llama milk
is less than 10% that in bovine or ovine milk but NAGase
activity is over 20-fold higher; the activity in llama milk of
both enzymes resemble more closely their activities in
human milk than bovine milk (Morin, Rowan, & Hurley,
1995).
In general, the enzyme content of human or ruminant
colostrum is higher from that in corresponding mature
milk (Shahani et al., 1973) or in ruminant milk (Shahani
et al., 1980). Some milk enzymes, such as lactic dehydrogenase and malic dehydrogenase, may reﬂect the activity of
epithelial cells. Kjellber and Karlsson (1967) found that the
zymogram pattern of lactic and malic dehydrogenase in
milk is different from that in blood serum, strongly
suggesting that they are synthesized in the mammary
gland. These authors found also that the activity of these
two enzymes is inversely related to body weight, e.g., in
mouse, human and cow, in agreement with the higher
metabolic rate in cells from small animals than large ones.
Grigor and Hartmann (1985), who compared the activity
of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, malic enzyme, lactate dehydrogenase and malate dehydrogenase in
the milk of sows, rats and rabbits, concluded that the
activity of these enzymes accurately reﬂects enzyme activity
in the epithelial cells of the mammary gland. The activity of
lactic dehydrogenase in milk increased by a factor of 2–3
between parturition and peak milk production, whereas
other enzymes, in particular glucose 6-phosphate dehydrogenase, may increase by up to 20-fold between these
times (Gul & Dils, 1969; Richards & Hilf, 1972, in rat;
Gumaa, Greenbaum, & McLean, 1973, in sheep). Thus, it
may be concluded that some milk enzymes are constitutive
components of milk, while others are induced at particular
periods of the lactation cycle. In cows, the marked increase
in secretion of NAGase in response to inﬂammation,
provides a sensitive marker for the detection of mastitis
(Pyorala, 2003).
Shahani et al. (1980) reviewed some data showing that
increased dietary fat intake in lactating women increased
the levels of lipase, esterase and alkaline phosphatase,
enzymes that play a role in digestion, assimilation and
metabolism of fat; whereas protein supplementation of
malnourished women increased alkaline phosphatase and
xanthine oxidase activity in their milk. Neville, Waxman,
Jensen, and Eckel (1991) presented data suggesting that
lipoprotein lipase in human milk is regulated by plasma
insulin, similarly to adipose tissue. Expression of plasma
membrane Ca2+-ATPase type 4b in MFGM was lower in
cows that developed milk fever than in control cows
(Prapong, Reinhardt, Goff, & Horst, 2005).
It may be concluded that the levels of enzymes in milk
are species-speciﬁc, and are affected by the metabolic
activity of cells, stage of lactation, whether the enzyme is
secreted in constitutive or inductive manner and the
hormonal, nutritional and metabolic status of the producing animal.
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2. Physiological roles of milk enzymes
2.1. Role of milk enzymes as pro-digestive factors
Human milk provides digestive enzymes (amylase and
lipase) that compensate the newborn for immature
pancreatic functions (Hamosh, 1998). In breast-fed infants,
digestion of milk triglycerides, the major source of energy
and long-chain polyunsaturated fatty acids, is catalyzed by
the concerted action of gastric lipase, colipase-dependent
pancreatic lipase and bile salt-stimulated lipase (BSSL).
The major proportion of BSSL is present in the milk and a
lesser part originates from the infant’s exocrine pancreas
(Hernell & Blackberg, 1994). Human milk BSSL hydrolyses ceramides and may thus have a role in sphingomyelin
digestion, but only after initial hydrolysis to ceramide and
phosphorylcholine. Thus, both sphingomyelinase and
BSSL may be important for optimal use of human milk
sphingolipids (Nyberg et al., 1998). BSSL is also present in
milk from dog and cat (Freed, York, Hamosh, Sturman, &
Hamosh, 1986) and ferret (Sbarra et al., 1996), suggesting
that it may be important also in non-primate species.
It is well known that colostrum is an important source of
immunoglobulins for the neonate. Studies with cows show
that colostrum intake inﬂuences plasma enzyme activities,
either because colostral enzymes are absorbed, or as a
consequence of endogenous production, which for some
enzymes appears to be modiﬁed by the time of ﬁrst
colostrum intake (Zanker, Hammon, & Blum, 2001). While
the absorption of colostral g-glutamyltransferase has been
conﬁrmed amply (Zanker et al., 2001), it is not fully clear
whether this is also the case for alkaline phosphatase and
aspartate-aminotransferase. The physiological importance,
if any, of the absorbed enzymes in the neonatal calf is not
clear at present.
2.2. Role of the plasmin system in the regulation of
mammary secretion
Milk secretion and mammary function are regulated by
local mechanisms sensitive to the frequency and efﬁciency
of milking (Daly, Owens, & Hartmann, 1993; Wilde &
Peaker, 1990). Acute local control of milk secretion occurs
through autocrine feedback inhibition by milk-borne
factors. Sustained changes in the frequency of milking
and milk secretion are associated with longer-term
adaptation in the degree of differentiation and, ultimately,
the number of mammary epithelial cells. Differentiation of
cultured mammary cells is suppressed by a milk fraction
containing the inhibitor, suggesting that intra-mammary
regulation of differentiation in vivo is elicited by the same
autocrine regulator subsequent to its acute effect on milk
secretion. This autocrine factor may affect mammary cell
differentiation by modulating the number of cell surface
hormone receptors for prolactin, thereby changing their
sensitivity to circulating hormones (Wilde & Peaker, 1990).
In addition to the regulation by milking, milk secretion
537
also depends on external factors, such as emotional stress,
as well as harsh physical conditions such as heat stress and
water deprivation (Silanikove, 2000). The fast modulation
of milk secretion in response to these challenges may point
to a similar mechanism of both phenomena (Silanikove
et al., 2000).
Wilde, Addey, Boddy, and Peaker (1995) presented
evidence suggesting that local regulation of milk secretion
by milk removal is through autocrine feedback inhibition
by a single goats’ whey protein of M(r) 7600, which they
termed FIL (Feedback Inhibitor of Lactation). However,
despite the fact that a decade has passed since this work
was published, there is no information regarding the
complete amino acid sequence of FIL or identiﬁcation of
the gene coding it. This information is critical for
veriﬁcation of the concept.
The involvement of the plasminogen activator (PA)–
plasminogen–plasmin system in many biological phenomena reﬂects the ubiquitous presence of plasminogen in
biological ﬂuids and the ability of numerous cell types to
synthesize, in a highly regulated manner, PA, and
inhibitors of plasmin and PA. This system is particularly
intensively studied in respect to its role in the lysis of blood
clots (Sidelmann, Gram, Jespersen, & Kluft, 2000) and
regulation of cell activity and cell attachment in various
tissues (Saksela & Riﬁrin, 1988). Plasmin, the principal
proteolytic enzyme in milk, is found mainly in its inactive
or zymogen form, plasminogen. The conversion of
plasminogen to plasmin is modulated by PA (Politis,
1996), two types of which exist in mammals: urokinasetype PA (u-PA) and tissue-type PA (t-PA) (Heegaard,
Rasmussen, & Andreasen, 1994; Politis, 1996). Plasmin,
plasminogen, and t-PA are closely associated with the
casein micelles (Politis, 1996), whereas u-PA is associated
with neutrophils (Politis, Voudouri, Bizelis, & Zervas,
2003; Politis, Zavizion, Cheli, & Baldi, 2002) and inhibitors
of PA and plasmin are in the milk serum (Precetti, Oria, &
Nielsen, 1997; Politis, 1996). The close proximity of
plasmin to its substrate ensures that hydrolysis is an
efﬁcient process (Korycka-Dahl, Dumas, Chene, & Martal,
1983) and it is the primary agent of proteolysis in goodquality milk (Kelly & McSweeney, 2003). The association
of t-PA with the casein micelle suggests that it plays a
pivotal role in regulating plasmin activity under nonpathological conditions. However, during bacterial infection and the increase in neutrophil numbers in the
mammary gland, u-PA activity increases (Leitner, Chaffer
et al., 2004; Zachos, Politis, Gorewit, & Barbano, 1992).
Plasmin preferentially cleaves polypeptide chains after a
lysine or, to a lesser extent, an arginine residue (Ueshima,
Okada, & Matsuo, 1996). b-CN is the preferred substrate
for plasmin and its hydrolysis results in the production
of g-caseins and proteose–peptones (Andrews, 1983).
aS1-Casein (McSweeney, Olson, Fox, Healy, & Højrup,
1993) and aS2-casein (Le Bars & Gripon, 1989) are also
susceptible to proteolysis by plasmin and the l-caseins are
products of hydrolysis of aS1-casein (Aimutis & Eigel,
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538
1982). However, k-casein is resistant to proteolysis by
plasmin (Diaz, Gouldsworthy, & Leaver, 1996).
The insulin-like growth factor binding proteins
(IGFBPs) have been shown to interact with several
proteins present in milk including as2-casein, lactoferrin
and transferrin. These interactions implicate the IGFBPs in
the regulation of plasminogen activation since plasminogen
and t-PA also bind to as2-casein (Flint et al., 2001) (Fig. 2).
There is evidence for the existence of a close association
between the PA–plasminogen–plasmin system and gradual
involution (the decline phase of lactation). Increased
plasmin and PA activity in bovine milk are correlated with
gradual involution (Politis, 1996). Treatment with bovine
somatotrophin prevents the increase in plasmin during
gradual involution, indicating that bovine somatotrophin
interferes with the conversion of plasminogen to plasmin
(Politis, 1996). Brown, Law, and Knight (1995) found that
relative amounts of g-caseins were highly negatively
correlated with milk yield in the declining phase of
lactation, reﬂecting the gradual involution of the gland at
this time. The counter-part of g-casein is the N-terminal
fraction b-CN f (1–28) (Andrews, 1983), and therefore this
peptide should also be highly negatively correlated with
milk yield in the declining phase of lactation. Thus, the
data of Brown et al. (1995) support the concept that b-CN f
(1–28) plays a role in the regulation of milk secretion
(Silanikove et al., 2000).
Milk volume is determined by osmotic-coupled water
ﬂow. In bovines, the secretion of K+, Na+ and Cl
determines approximately 40% of the driving force, with
the rest being determined by lactose (Shennan & Peaker,
2000). The currently held view is that lactose and
monovalent ions are secreted into the lumen of the
mammary gland mainly via vesicles (Shennan & Peaker,
2000). However, a direct contact between monovalent
ions inside the epithelial cells and ﬂuid stored in the lumen
of the gland is possible, since the apical membrane of the
epithelial cells contains K+, Na+ and Cl channels
(Shennan & Peaker, 2000). In the pancreas, regulation of
cellular secretion of insulin could result from changes in the
ionic permeability, which modulates membrane potential
(Newgard & McGarry, 1995). This perspective led Silanikove et al. (2000) to test the possibility that ion channels
expressed in the apical regions of mammary gland
epithelium are involved in the regulation of milk secretion.
Indeed, Silanikove et al. (2000) showed that a distinct
plasmin-induced b-CN peptide f (1–28) is a potent blocker
of K+ channels in the apical membrane of mammary
epithelial cells. The production of b-CN f (1–28) by
plasmin during the storage of milk in the udder represents
between 8% and 12% of the total proteose–peptone
fraction in whey, and matches the formation of g1-casein
(the C-terminal residues 29–209) from b-CN (Andrews,
1978). b-CN f (1–28) is resistant to further degradation by
plasmin (Andrews, 1978, 1983). As far as we are aware, no
genetic substitutions in this part of b-CN have been found,
so that all genetic variants of b-CN will result in the same
fragment. All these characteristics make b-CN f (1–28) an
Anti-Lactogenic
hormones:
e.g.
Cortisol, Estrogen
Lactogenic
hormones:
e.g.
GH, Prolactin
Blood side
PA =plasminogen
activator
PAI = PA inhibitor
PLG = plasminogen
PL = plasmin
PLI = PL inhibitor
CN = casein
β-CN f (1-28) =fraction
1-28 of β-CN
IGFBP = IGF binding
protein
PAI
IGFBP
β-CN f (1-28)
PA
β-CN
Gland lumen
PLG
PL
PLI
Alveoli, the
basic milk
secreting unit
Milk stasis, or
bacterial invasion
Frequent milking
or suckling
External effects:
milking, suckling,
bacterial invasion
Fig. 2. Overview of the PA–plasminogen–plasmin negative feedback mechanism that down-regulate milk secretion. The contribution of most elements is
described in the text. Bold arrows indicate ﬂow signal along the feedback loop, dotted arrows positive effects and dashed arrows suppressive effects.
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N. Silanikove et al. / International Dairy Journal 16 (2006) 533–545
ideal candidate for negative feedback control of milk
secretion. Infusion of a solution composed of a casein
digest enriched with b-CN f (1–28) into the cistern of cows,
or infusion of pure b-CN f (1–28) into the cistern of goats,
led to a transient reduction in milk secretion in the treated
gland (Silanikove et al., 2000).
Stress and stress-related hormones such as glucocorticoids inhibit lactation in cows (Shamay, Shapiro, Barash,
Bruckental, & Silanikove, 2000). Silanikove et al. (2000)
proposed a novel mechanism connecting stress with the
PA–plasminogen–plasmin system. They showed that stress
activates the PA–plasminogen–plasmin system leading to
an increase in plasmin activity and to the formation of
b-CN f (1–28). The reduction in milk production due to
dehydration stress or glucocorticoid (dexamethsone) was
correlated with the activity of plasmin and channelblocking activity in the milk of the tested cows (Silanikove
et al., 2000). Complexes of both types of PA with PAinhibitor-1 have been detected in the culture medium of
bovine mammary epithelial cells (Heegaard, White, Zavizion,
Turner, & Politis, 1994).
So far, a better understanding of the role of the
hypothalamus–pituitary–adrenocortical axis and PA–plasminogen–plasmin system in milk has been hampered by a
lack of basic knowledge of the physiological function of
these two systems in milk synthesis and secretion. The
results and concept presented in Fig. 2 provides an
explanation for the already-known correlation between
the activity of the above systems and reduced milk
secretion. Accordingly, activation of the hypothalamus–
pituitary–adrenocortical axis by external stress liberates
cortisol into blood plasma, which in turn induces the
liberation of PA from the mammary epithelial cells into the
mammary cistern, where it activates the plasmin system
and enhances the release of b-CN f (1–28) from b-CN.
Inhibition of ion channels by b-CN f (1–28) triggers an as
yet unknown process which reduces the secretion of lactose
and monovalent ions into the lumen of the gland, leading
to the decrease in milk volume. This rapid modulation of
milk secretion increases the potential for survival in
response to stress.
Based on the evidence described above, this system may
explain also the effects of frequency and efﬁciency of udder
emptying and, as will be described below, the response in
levels of milk secretion to the presence of bacterial infection
of the udder. A direct association between decreased
milking frequency and an increase in the activity of the
PA–plasminogen–plasmin system has been found (Kelly,
Reid, Joyce, Meaney, & Foley, 1998; Stelwagen, Politis
et al., 1994).
2.3. Role of the plasmin system in induction of involution
Mammary gland involution proceeds through several
distinct stages that involve cessation of milking, apoptosis
of epithelial cells and tissue remodelling. Unilateral
cessation of milking in goats (Quarrie, Addey, & Wilde,
539
1994) and teat sealing in mice (Li et al., 1997; Marti, Feng,
Altermatt, & Jaggi, 1997; Quarrie, Addey, & Wilde, 1998)
induced involution in the treated gland only. This
speciﬁcity suggests that mammary involution is triggered
by local stimuli. Reinitiating milk removal can reverse the
ﬁrst stage of involution, but the second stage of involution
is irreversible and is characterized by activation of
proteases that destroy the lobular–alveolar structure of
the gland by degrading the extracellular matrix and
basement membrane, and cause massive loss of alveolar
cells (Capuco & Akers, 1999; Hurley, 1989).
In cows, involution is complete by 21–30 days after
drying-off, and during this period the mammary secretion
becomes scant, watery, turbid (serum-like) and rich in
leukocytes (41 106mL1) (Capuco & Akers, 1999). The
compositional changes include a dramatic decrease in the
concentrations of lactose and fat, and parallel increases in
the concentrations of lactoferrin and immunoglobulins,
which are part of the innate immune system (Capuco &
Akers, 1999; Shamay, Shapiro, Leitner, & Silanikove,
2003). It takes dairy cows considerably more time than
rodents to reach involution (Capuco & Akers, 1999).
In cows reaching involution while still producing a lot of
milk (X20–30 L day1), following cessation of milking,
plasmin activity in mammary secretion increased gradually
and became substantially higher within 13 days (Shamay
et al., 2003). In mice, plasmin activity rose sharply
immediately following the induction of drying-off
(Ossowski, Biggel, & Reich, 1979), which may explain the
differences between the species in their rate of involution.
In support of this hypothesis, Shamay, Shapiro, Mabjeesh,
and Silanikove (2002) for goats and Shamay et al. (2003)
for cows, have shown that the infusion of casein hydrolyzates (CNHs) which contain products of plasmin activity
dramatically accelerated the rate of involution, to the
extent that it was complete within 3 days. The infusion of
CNH was followed by rapid (within 3 days) drying-off of
mammary secretion and was associated with earlier
increases in the concentrations of components of the
innate immune system: lactoferrin (an antimicrobial
protein), immunoglobulin type G (Shamay et al., 2003)
and the formation of free radicals with bactericidal activity
by milk enzymes (Silanikove, Shapiro, Shamay, & Leitner,
2005). In conventional drying-off, induced by thee abrupt
cessation of milking, ﬂuid volume declined precipitously
only after 3–7 days (Hurley, 1989; Noble & Hurley, 1999;
Shamay et al., 2003). It was concluded that CNH treatment
induced involution in the treated gland that imitates the
natural involution process, albeit at a rate which accelerates and synchronizes the involution process among the
treated cows in comparison to involution induced by the
cessation of milking (Shamay et al., 2002, 2003).
Milk stasis also induces the disruption of tight junctions
between epithelial cells due to the accumulation of local
negative feedback signals (Nguyen & Neville, 1998). In
CNH-treated glands, disruption of the tight junctions
occurred within 8 h after the ﬁrst treatment. In comparison,
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it took 18 h in dairy cows to ﬁnd the ﬁrst signs of tight
junction leakiness (Stelwagen, Farr, McFadden, Prosser, &
Davis, 1997) and 3 days to complete the process (Shamay
et al., 2003). On the resumption of milking, the gland
rapidly resumes tight junction integrity (Stelwagen et al.,
1997). Thus, milking frequency affects the leakiness of the
tight junctions, and the increased leakiness, induces
reduced milk yield, increased leukocytes content in the
gland and increased proteolysis of casein (Stelwagen,
Politis et al., 1994; Stelwagen, Davis, Farr, Eichler, &
Politis, 1994), and hence the deterioration of milk quality
for the dairy industry (Kelly et al., 1998). These responses
are accentuated in late lactation when there is natural
increase in the activity of the PA–plasminogen–plasmin
system (Kelly et al., 1998; Lacy-Hulbert, Woolford,
Nicholas, Prosser, & Stelwagen, 1999). Sørensen, Muir,
and Knight (2001) concluded that thrice-daily milking, in
comparison to twice-daily milking, helps to prevent the
usual increase in casein degradation associated with late
lactation. Part of this effect is due simply to reduced
exposure to plasmin as a result of the reduced storage time
in the udder, but it is partly due to a better maintenance of
epithelial tight junction integrity as lactation advances.
Thus, the results of Sørensen et al. (2001) are in agreement
with the ﬁnding that casein degradation products induce
the disruption of tight junction integrity (Shamay et al.,
2002, 2003).
The data of Shamay et al. (2002, 2003) indicate that the
ﬁrst step in the induction of involution in the mammary
gland is disruption of the tight junctions between epithelial
cells. Maintaining the tight junctions open for a critical
time (about 48 h) initiates the second phase of involution,
which is irreversible. Wilde, Blatchford, Knight, and
Peaker (1989) presented data indicating that incomplete
milking over a long period caused partial secretory
cellular involution via a local chemical feedback mechanism in the gland. Thus, collectively, the data of Kelly et al.
(1998), Lacy-Hulbert et al. (1999) and Sørensen et al.
(2001) strongly suggest that the mammary gland may
undergo partial involution while still in the lactogenesis
phase.
2.4. The plasminogen activator–plasminogen–plasmin-based
negative feedback mechanism as a working hypothesis
The PA–plasminogen–plasmin-based negative feedback
mechanism which down-regulates milk secretion was
presented in Section 2.2 and, in Section 2.3, the PA–plasminogen–plasmin-based negative feedback mechanism
which induces the disruption of tight junctions and
involution was described. It was also noted that partial
involution of the mammary gland may be induced even
during lactogenesis. We are aware that formal approval of
these two concepts should wait for further elucidation of
the interaction between the casein derived peptide/s
(ligand/s) and respective receptor/s or ion channel/s on
the apical membrane of the secretory cells. Nevertheless,
the utility of a concept can also be judged by its ability to
explain current data and to provide tools that predict
experimental results. In this section, we will demonstrate
how these two concepts explain some apparent contradictions in respect to changes in milk composition
associated with the effects of milking frequency and
subclinical mastitis. Firstly, it should be emphasized that
the down-regulation of milk secretion is associated with
mild activation of plasmin (10–40% increase in activity).
Under such conditions, the response induced by b-CN f
(1–28) affects speciﬁcally ﬂuid secretion (water, lactose and
ions), without affecting lactose concentration or fat and
protein secretion and without affecting the integrity of tight
junctions and the leukocyte level in the gland. Consequently, mild activation of the plasmin system results in
reduced milk yield but with higher concentrations of fat
and proteins, as seen in the response to injecting cows with
dexamethasone (Shamay et al., 2000). On the other hand,
massive activation of the plasmin activity (X150%) is
associated with extensive degradation of casein, disruption
of tight junctions, an inﬂammatory response (increase in
leukocytes and NAGase activity), and a reduction in
lactose concentration. As a result, ﬂuid, fat and protein
secretion are negatively affected, as seen in the response to
treatment with CNH of goats (Shamay et al., 2002) and
cows (Shamay et al., 2003).
Remond, Pomies, Dupont, and Chilliard (2004) found
that once-daily milking, in comparison with twice-daily
milking, reduced milk yield by 30%. The lactose concentration and SCC were similar, but fat and protein content
were higher for once-daily milking. A response of a similar
nature with respect to gross milk composition was reported
by Smith, Ely, Graves, and Gilson (2002), who compared
the effect of thrice-daily milkings with twice-daily milking
in cows and Bar-Peled et al. (1995) who compared six daily
milkings and 3 daily milkings plus 3 daily sucklings with
thrice-daily milked cows. Similar results were also observed
in twice-daily milked dairy goats in comparison with oncedaily milking (Salama et al., 2003). It seems that increased
milking frequency dilute the content of b-CN f (1–28),
which in turn reduces the inhibition on ﬂuid secretion.
However, because b-CN f (1–28) affects fat and protein
secretion less than ﬂuid secretion, it results in increasing fat
and protein concentration in the milk. In contrast, when
plasmin activity is already high (Kelly et al., 1998;
Stelwagen, Politis et al., 1994), or when milk stasis extends
much more than 12 h (Stelwagen et al., 1997), caseinderived active components disrupt the epithelial tight
junctions, induces inﬂammatory response and affects ﬂuid,
fat and protein secretion.
Our group recently compared the effect of subclinical
infection in one gland in comparison to an uninfected
gland in the same animal on milk composition and yield in
sheep (Leitner, Chaffer et al., 2004), goats (Leitner et al.,
2004) and on milk composition in cows (Leitner, Krifucks,
Merin, Lavi, & Silanikove, 2005). Milk yield was reduced
in the infected glands in comparison with the uninfected
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ones, and these changes were associated with increases in
plasmin activity, indices of inﬂammation (SCC, NAGase
activity), proteolysis of casein (increased proteose–peptone
content and lower casein number) and a decrease in lactose
concentration. However, the results for fat, total protein
and casein concentration were variable in the case of sheep,
a signiﬁcant decrease in fat, total protein and casein
concentration and a signiﬁcant increase in whey protein
concentration were found. In the case of goats, subclinical
mastitis was associated with no change in fat concentration
and a signiﬁcant increase in total protein, but no change in
casein level and with a signiﬁcant increase in whey protein
concentration. In the case of cows, infection was not
associated with changes in fat, total protein or casein, but
was associated with increase of total whey protein
concentration. In view of the evidence for extensive
degradation of casein in the infected glands, the lack of
response in total casein concentration in goats and cows is
striking. This contradiction may be explained as an
outcome of the reduction in casein secretion and casein
content due to enhanced hydrolysis and the increased
casein concentration as a result of a greater reduction in
ﬂuid secretion. The more severe response in sheep,
in a comparison to goats and cows, is consistent with a
higher basal level of plasmin activity, a higher level of
plasmin activity in the infected glands, a higher basal level
of casein, and hence a higher level of casein degradation
products (i.e., proteose–peptones) in the infected glands.
These inter-species differences are also associated with a
greater reduction in lactose concentration in sheep than in
goats and cows, in line with the primary osmotic role of
lactose in milk and its effect on ﬂuid secretion (Shamay
et al., 2000).
The fact that all glands treated with CNH (so far, 4than
100 cases in dairy cows) resumed normal lactation after
parturition supports the conclusion that the treatment
imitated natural phenomena rather than inducing a
necrotic response which would irreversibly damage the
secretory function of the udder (Shamay et al., 2003). In
several aspects, such as induction of inﬂammation and
changes in milk composition, CNH treatment resembles
studies in which the mammary gland was challenged with
endotoxin or colchicines (Olivier & Smith, 1983; Persson,
Carlsson, Hambleton, & Guidry, 1992). However, this
treatment cannot induce complete drying-off of mammary
secretion, and the mammary gland becomes partially
refractory to frequent treatment with endotoxin (Shuster
& Harmon, 1991). These qualities of CNH treatment have
led us to examine its use for drying-off secretion in infected
mammary quarters, and evaluate its ability to cure
microbiological infection. CNH treatment appears to be
an effective tool for improving milk hygiene (i.e., reducing
SCC) in cows exposed to sub-clinical or chronic
mastitis and for eliminating bacterial infection (Silanikove,
Iscovich, & Leitner, 2005). The latter effects of CNH
treatment also suggest that it may be use as an effective
non-antibiotic dry cow treatment.
541
2.5. Milk enzymes as part of the innate immune system
Milk and colostrum contain several antimicrobial
factors, which exert both speciﬁc and non-speciﬁc bacteriostatic and bactericidal activity. These factors may be used
to protect the mammary gland itself or transferred from the
mother to the neonate and contribute to the protection of
the off-spring against infectious diseases. For many species,
the milk-derived immunoglobulins are crucial for survival
of the newborn. Human milk contains high level of
lysozyme, which has been found to be effective at
signiﬁcantly slowing the growth of, or killing various types
of bacteria (e.g., Maga, Anderson, Cullor, Smith, &
Murray, 1998; Reddy, Bhaskaram, Raghuramulu, &
Jagadeesan, 1977). NAGase activity, which is constitutively
present in human milk at a high concentration, and at a
high concentration in cows’ milk during inﬂammation, also
confers antimicrobial activity (Pompei, Ingianni, Cagetti,
Rrizzo, & Cotti, 1993).
Xanthine oxidoreductase and lactoperoxidase are two
ubiquitously expressed enzymes in bovine milk which are
capable of forming free radicals. The former uses xanthine,
hypoxanthine, or reduced nicotinamide adenine nucleotide
as electron-donating substrates and catalyzes the last two
steps in the formation of urate. The enzyme is synthesized
as xanthine dehydrogenase, but can be readily converted to
xanthine oxidase by oxidation of sulfhydryl residues or by
proteolysis (Harrison, 2002). The weak microbicidal
activity of xanthine oxidase relates to its ability to form
superoxide and hydrogen peroxide (Bjorck & Claesson,
1979). However, recent studies have shown that the
microbicidal activity of XO in milk may also be related
to the formation of NO from nitrite (Hancock et al., 2002;
Li, Samouilov, Liu & Zweier, 2003; Stevens et al., 2000);
XO converts nitrate into nitrite, and so may substantially
increase the substrate for NO generation (Li et al., 2003).
Lactoperoxidase, a heme-containing peroxidase, is likely
to contribute to oxidative mechanisms in milk (Østdal,
Bjerrum, Pedersen, & Andersen, 2000). Thiocyanate,
considered to be the physiological substrate of LPO, is
oxidized by the enzyme to hypothiocyanite, with H2O2
serving as the electron acceptor (Wolfson & Sumner, 1993).
However, the lactoperoxidase/SCN/H2O2 system in milk is
inactive (Althaus, Molina, Rodriguez, & Fernandez, 2001;
Silanikove, Shapiro et al., 2005), and its activation requires
the addition of exogenous thiocyanate and H2O2 to levels
exceeding their physiological concentration in milk (Wolfson & Sumner, 1993). On the other hand, nitrite reacts with
mammalian peroxidases such as lactoperoxidase to produce the potent radical, nitric dioxide (NO2 ) (Van der
Vliet, Eiserich, Ilalliwell, & Cross, 1997) and this system
has been shown to be active in milk (Silanikove, Shapiro
et al., 2005).
Silanikove, Shapiro et al. (2005) have shown that NO
and H2O2 were constantly produced in the mammary gland
secretions. Nitrite formed either by autooxidation of NO
or by conversion of nitrate to nitrite by xanthine oxidase
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was converted into the powerful nitric dioxide radical by
lactoperoxidase and H2O2 which is derived from the
metabolism of xanthine by xanthine oxidase. Nitric dioxide
is most likely responsible for the formation of nitrosothiols
on thiol-bearing groups, which allow prolonged presence of
NO in mammary secretion. Nitrite is effectively converted
to nitrate, which accumulated in the range of 25–1000 mM
from the start of involution (i.e., in normal milk) to the
complete involution of glands. The conversion of active
nitrite into less active nitrate is mediated by catalase.
Catalase, therefore, appears to play a central role in redox
control in milk (Silanikove, Shapiro et al., 2005). The
secretion of all mammary glands was bactericidal and
bacteriostatic during established involution, and this
appeared sooner and more acutely in glands treated with
CNH, within 8–24 h. It was concluded that xanthine
oxidase, lactoperoxidase and NO are components of the
mammary innate immune system that has bactericidal and
bacteriostatic activity in mammary secretions. The innate
immune system plays a major role in preventing intramammary infection during milk stasis and its activation
may increase its effectiveness.
In other species, other enzymes that produce radicals
may be important for gland defense; for example, in mouse
milk, H2O2 produced using endogenous amino acids by
L-amino acid oxidase kills bacteria in the mammary gland
(Sun et al., 2002).
3. Conclusion
Milk enzymes have an important biological role and so
far have been found to be involved in the control of milk
secretion, developmental stage (involution), the gland
innate immune system and preventing oxidative damage
to its essential nutrients. Many more secrets are probably
waiting to be revealed. In executing their function, milk
contains many enzymes which constantly consume metabolites, produce free-radicals and modify its composition,
if necessary. Milk enzymes, along with other components
(e.g., cytokines, enzyme inhibitors), in many cases form
complex metabolic pathways.
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