Antonie van Leeuwenhoek 76: 207–215, 1999.

© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

207

Bioactive peptides encrypted in milk proteins: proteolytic activation and

thropho-functional properties

Hans Meisel1,∗ & Wilhelm Bockelmann2

Federal Dairy Research Centre,
1 Institute for Chemistry and Physics,
2 Institute for Microbiology, P.O. Box 6069, D-24121 Kiel, Germany. (∗ Author for correspondence)

Key words: bioactive peptides, functional foods, lactic acid bacteria, milk proteins

Abstract
The bioactivities of peptides encrypted in major milk proteins are latent until released and activated by enzymatic
proteolysis, e.g. during gastrointestinal digestion or food processing. The proteolytic system of lactic acid bacteria
can contribute to the liberation of bioactive peptides. In vitro, the purified cell wall proteinase of Lactococcus
lactis was shown to liberate oligopeptides from β- and α-caseins which contain amino acid sequences present in
casomorphins, casokinines, and immunopeptides. The further degradation of these peptides by endopeptidases and
exopeptidases of lactic acid bacteria could lead to the liberation of bioactive peptides in fermented milk products.
However, the sequences of practically all known biologically active peptides can also be cleaved by peptidases from
lactic acid bacteria. Activated peptides are potential modulators of various regulatory processes in the body: Opioid
peptides are opioid receptor ligands which can modulate absorption processes in the intestinal tract, angiotensin-
I-converting enzyme (ACE)-inhibitory peptides are hemodynamic regulators and exert an antihypertensive effect,
immunomodulating casein peptides stimulate the activities of cells of the immune system, antimicrobial peptides
kill sensitive microorganisms, antithrombotic peptides inhibit aggregation of platelets and caseinophosphopeptides
may function as carriers for different minerals, especially calcium. Bioactive peptides can interact with target sites
at the luminal side of the intestinal tract. Furthermore, they can be absorbed and then reach peripheral organs.
Food-derived bioactive peptides are claimed to be health enhancing components which can be used for functional
food and pharmaceutical preparations.

Introduction for example by the action proteinases and peptidases

In the last two decades a number of studies have been
performed on bioactive peptides that are present in
the amino acid sequence of milk proteins. Although
other animal as well as plant proteins contain potential
bioactive sequences, milk proteins are currently the
main source of a range of biologically active peptides
(Table 1). The structures of biologically active se-
quences were obtained from in vitro enzymatic and/or
by in vivo gastrointestinal digests of the appropriate
precursor proteins; chemical synthesis has been car-
ried out to confirm the sequence of potential bioactive
peptides.
Milk-protein derived bioactive peptides are inact-
ive within the sequence of the parent protein and can
be released and activated by enzymatic proteolysis
from lactic acid bacteria. Lactic acid bacteria are nu-
tritionally fastidious and need amino acids for nitrogen
nutrition. In milk, caseins are the major source for
amino acids (Juillard et al. 1995). The proteolytic sys-
tem of lactic acid bacteria consists of a cell wall bound
proteinase and several intracellular peptidases (Pelis-
sier 1984; Monnet et al. 1993; Bockelmann 1995;
Poolman et al. 1995; Law & Haandrikman 1997).
Transport systems specific for amino acids, di- and
tripeptides and oligopeptides (up to 18 amino acids in
length) are present in lactic acid bacteria for nitrogen
uptake (Konings et al. 1997). Longer oligopeptides,
not transported into the cells can be a source for
the liberation of bioactive peptides in fermented milk
products when further degraded, for example by in-
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Table 1. Bioactive peptides derived from milk proteins

Bioactive peptide Protein precursor Bioactivity

Casomorphins α-, β-Casein Opioid agonist

α-Lactorphin α-Lactalbumin Opioid agonist
β-Lactorphin β-Lactoglobulin Opioid agonist
Lactoferroxins Lactoferrin Opioid antagonist
Casoxins κ-Casein Opioid antagonist
Casokinins α- β-Casein ACE-inhibitory
Lactokinins α-Lactalbumin, ACE-inhibitory
β-Lactoglobulin, Serum albumin
Immunopeptides α- β-Casein Immunomodulatory
Lactoferricin Lactoferrin Antimicrobial
Casocidin α S2 -Casein Antimicrobial
Isracidin α S1 -Casein Antimicrobial
Casoplatelins κ-Casein Antithrombotic
Phosphopeptides α-, β-Casein Mineral binding

tracellular peptidases of lactic acid bacteria after cell
lysis (Law & Haandrikman 1997). In the human
gastrointestinal tract, digestive enzymes will contrib-
ute to the further breakdown of long casein-derived
oligopeptides which may also lead to release of bio-
active peptides. Once they are liberated in the body,
bioactive peptides may act as regulatory compounds
with hormone-like activity. The following overview is
based on recent reviews on the specific biochemical
properties and possible dietary and pharmaceutical
applications of peptides derived from milk proteins
(Meisel & Schlimme 1996; Meisel 1997a, b; Meisel
1998).
Phenomenon of multifunctional bioactive peptides
Many milk-derived peptides reveal multifunctional
properties: i.e. specific peptide sequences having two
or more different biological activities have been repor-
ted (Table 2; peptides are indicated in bold numbers as
listed in the table). Some regions in the primary struc-
ture of caseins contain overlapping peptide sequences
that exert different biological effects. These regions
have been considered as ‘strategic zones’ which are
partially protected from proteolytic breakdown (Fiat
& Jollès 1989; Schanbacher et al. 1997; Meisel 1998).
Three strategies have generally been used in the iden-
tification and characterisation of such peptides, i.e.
isolation of inhibitory peptides from in vitro enzymatic
or from in vivo gastrointestinal digests of precursor
proteins and chemical synthesis of peptides or peptide
fragments having similar structures to those known to
be bioactive.
Opioid peptides, i.e. opioid receptor (µ-, δ- or κ-
type) ligands with agonistic activity, originate from
different milk proteins and exert naloxone-inhibitable
opioid activities in both receptor studies and during
bioassays (Brantl et al. 1981; Chiba and Yoshikawa
1986; Loukas et al. 1993). The atypical opioid pep-
tides 1-7 derived from milk proteins have N-terminal
sequences different from that of the ‘typical’ endogen-
ous opioid peptides (Teschemacher et al. 1997). The
major exogenous opioid peptides, β-casomorphins,
are fragments of the β-casein sequence 1 (Meisel
1986) and have been characterized as µ-type lig-
ands (Teschemacher & Brantl 1994). β-caso-morphins
were also found in analogous positions in sheep, wa-
ter buffalo and human β-casein (for review: Fiat and
Jollès 1989). Five opioid peptides were isolated from
wheat gluten (Fukudome & Yoshikawa 1994). These
gluten exorphins were found at 15 sites in the primary
structure of glutenin and were highly selective for
δ-receptors.
Opioid antagonists have been found in bovine and
human κ-casein (casoxins 8, 9) and in human αS1 -
casein (Chiba et al. 1989; Yoshikawa et al. 1994). Fur-
thermore the opioid antagonist lactoferroxin has been
found in human lactoferrin (Yoshikawa et al. 1988).
Various casoxins which are opioid receptor ligands of
the µ-type were isolated as C-terminally methoxylated
peptides, e.g. the derivatives corresponding to the κ-
casein sequences 8 (Chiba & Yoshikawa 1986). The
Table 2. Examples for multifunctional bioactive peptides encrypted in bovine milk proteins1

Biological activity
No. Peptide sequence 2 Fragment Name Preparation Opioid ACE- Immunomodulatory5 Ca2+ binding
IC350 inhibitory Kapp (1·mol−1 )6
IC450

1 YPFPGPIPNSL β-CN(f60-70) β-Casomorphin-11 Intest. Chyme 10

2 YPFPGPI β-CN(f60-66) β-Casomorphin-7 Trypsin 14 500 −21/+26
3 YPFPG β-CN(f60-64) β-Casomorphin-5 Trypsin 1.1 0
4 RYLGYLE α S1 -CN(f90-96) α-Casein Exorphin Pepsin 1.2
5 YGLF α-LA(f50-53) α-Lactorphin Synthesis 300 733
6 YLLF β-LG(f102-105) β-Lactorphin Synthesis 160 172
7 YGFQNA SA(f399-404) Serorphin Pepsin 85
8 SRYPSY·OCH3 κ-CN(f33-38) Casoxin 6 Pepsin 15 (↓)
9 YIPIQYVLSR κ-CN(f25-34) Casoxin C Trypsin 50 (↓)
10 YL β-LG(f102-103) Lactokinin Synthesis 122
11 ALPMHIR β-LG(f142-148) Lactokinin Trypsin 43
12 VPP β-CN (f84-86) β-Casokinin Sour Milk 9
13 IPP β-CN (f74-76) β-Casokinin Sour Milk 5
14 AVPYPQR β-CN(f177-183) β-Casokinin Trypsin 15
15 FFVAPFPEVFGK α S1 -CN(f23-34) α S1 -Casokinin Trypsin 77
16 FFVAP α S1 -CN(f23-27) α S1 -Casokinin Tryp.+Peptidase 6
17 VAP α S1 -CN(f25-27) α S1 -Casokinin Synthesis 2
18 YQQPVLGPVR β-CN(f193-202) β-Casokinin-10 Synthesis 300 −28/+14
19 TTMPLW α S1 -CN(f194-199) α S1 -Immunocasokinin Trypsin 16 +162
20 PGPIPN β-CN(f63-68) Immunopeptide Synthesis +139
21 LLY β-CN(f191-193) Immunopeptide Synthesis +148
22 YG α-LA(f50-51)(f18-19), κ-CN(f38-39) Synthesis >1000 +101
23 YGG α-LA(f18-20) Immunopeptide Synthesis +35
24 RELEELNVPGEIVES∗LS∗ S∗ S∗ EESITR β-CN(f1-25)4P Caseinophosphopeptide Trypsin 629
25 DIGS∗ ES∗ TEDQAMEDIM α S1 -CN(f43-58)2P Caseinophosphopeptide Trypsin 328
26 QMEAES∗ IS∗ S∗ S∗ EEIVPNS∗ VEQK α S1 -CN(f59-79)5P Caseinophosphopeptide Trypsin 841
27 FKCRRWQWRMKKLGAPSITCVRRAF LF(f17-41) Lactoferricin Pepsin antimicrobial: disruption of bacterial cell membranes
28 MAIPPKKNQDK κ-CN(f106-116) Casoplatelin Trypsin antithrombotic: inhibition of platelet aggregation

1 Details are available within the references used to generate this Table (adapted from Meisel 1997): Bellamy et al. 1992; Brantl et al. 1981; Chiba & Yoshikawa 1986; Chiba et al. 1998;
FitzGerald & Meisel 1999; Jollès et al. 1986; Kayser & Meisel 1996; Loukas et al. 1983; Meisel 1986; Meisel & Schlimme 1994; Migliore-samoure et al. 1989; Mullally et al. 1996,
1997; Nakamura et al. 1995; Teschemacher et al. 1997.
2 The one-letter amino acid codes were used; Phosphoserin=S∗ .
3 IC values (µmol/l) are given for peptide concentrations inhibiting [3 H]-ligand binding by 50%; (↓) indicates antagonistic activity.
50
4 IC values (µmol/l) are given for peptide concentrations inhibiting the activity of angiotensin-converting-enzyme (ACE) by 50%.
50
5 Figures indicate the maximum% stimulation (+) and/or inhibition (−), respectively, in relation to control (=100).
6 Apparent calcium binding constants (K ) were determined with samples obtained from tryptic fractions containing the indicated peptide as the main component.
app
209
210
chemically modified casoxins were more active than
the non-methoxylated fragments.
Casein derived immunopeptides including frag-
ments of α S1 -casein 19 and β-casein 18, 20, 21
stimulate phagocytosis of sheep red blood cells by
murine peritoneal macrophages and exert a protective
effect against Klebsiella pneumoniae infection in mice
after intravenous administration of peptides (Migliore-
Samour et al. 1989). The C-terminal β-casein se-
quence 193-209 containing β-casokinin-10 18 in-
duced a significant proliferative response in rat lymph-
ocytes (Coste et al. 1992). Depending on peptide
concentration β-casokinin-10 18 and β-casomorphin-
7 2 showed a suppression as well as a stimulation of
lymphocyte proliferation (Table 2). β-Casomorphin-7
2 inhibits the proliferation of human colonic lamina
propria lymphocytes where the anti-proliferative ef-
fect was reversed by the opiate receptor antagonist
naloxone (Elitsur & Luk, 1991). Recently, Kayser and
Meisel (1996) reported that the peptides Tyr-Gly 22
and Tyr-Gly-Gly 23 significantly enhanced the pro-
liferation of human peripheral blood lymphocytes in
vitro.
Antimicrobial peptides have been derived from the
whey protein lactoferrin (Tomita et al. 1991). Lacto-
ferrin is an iron-binding glycoprotein present in most
biological fluids of mammals including milk. The ex-
istence of an antimicrobial sequence named lactofer-
ricin 27 near the N-terminus of lactoferrin in a region
distinct from its iron-binding sites has been reported
(Bellamy et al. 1992). The antimicrobial activity of
lactoferricin and synthetic analogs seems to be correl-
ated with the net positive charge of the peptides. These
cationic peptides kill sensitive microorganisms by in-
creasing cell membrane permeability (Bellamy et al.
1993). Dionysius & Milne (1998) reported on the an-
tibacterial activity of lactoferricins towards enterotox-
igenic Escherichia coli and Listeria monocytogenes.
The α S2 -casein fragment casocidin-I (position 165-
203) containing a high proportion (10 of 39) of basic
amino acid residues was found to inhibit the growth of
Esherichia coli and Staphylococcus carnosus (Zucht
et al. 1995, 1996). Another antibacterial α S1 -casein
fragment (position 1-23) was significantly effective
against lethal infection of mice by Staphylococcus
aureus (Lahov & Regelson 1996).
Antithrombotic peptides, casoplatelins, are derived
from the C-terminal part (caseinoglycomacropeptide)
of bovine κ-casein. The main antithrombotic peptides
of κ-casein are the sequence 28 and smaller fragments
thereof (Bouhallab et al. 1992).
Several food protein sources including fish gelatin
and maize protein contain peptides which are inhibit-
ors of the Angiotensin-Converting-Enzyme and thus
have an antihypertensive effect (for review: Ariy-
oshi 1993; Meisel 1993; Yamamoto 1997). ACE-
inhibitors derived from milk protein represent differ-
ent fragments of caseins 12-18 (casokinins; Meisel &
Schlimme 1994) or whey proteins 10, 11 (lactokinins;
FitzGerald & Meisel 1999).
Mineral binding caseinophosphopeptides corres-
pond to different phosphorylated regions of α S1 -, α S2 -
and β-casein 24, 25, 26 (for review: FitzGerald 1998).
The negatively charged side chains of phosphoseryl
residues represent the anionic binding sites for min-
erals. Binding and solubilisation of calcium in the
presence of phosphate has many potential beneficial
consequences, e.g. inhibition of the precipitation of
insoluble calcium phosphate.
Proteolytic activation of encrypted bioactive
peptides
Relatively high amounts of bioactive peptides could
potentially be produced during proteolysis of 1 g
of each of the major casein and whey protein com-
ponents (Meisel 1998). For chemical characterization
and identification, most of the yet known bioact-
ive peptides were proteolytically released from their
parent milk protein by hydrolysis with pancreatic pro-
teinases preferably with trypsin (Table 2). However,
other enzyme activities and combinations of endopro-
teinases including chymotrypsin, pepsin, thermolysin
and proline-specific endopeptidase were also used to
generate bioactive peptides (for review: Meisel &
Schlimme 1996, and cited ref.). Proteinase prepar-
ations of bacterial, fungal, plant and animal origin
have recently been used to produce caseinophos-
phopeptides (McDonagh & FitzGerald 1998).
The release of bioactive peptides from their im-
mediate precursor sequence is a prerequisite for any
functional role in the living system. In addition to the
possible liberation of bioactive peptides during intest-
inal proteolysis such peptides may already be gener-
ated during manufacture of several milk products and
thus be ingested as food components. For example,
partially hydrolyzed milk proteins for hypoallergenic
infant formulae and for clinical applications in enteral
nutrition consist exclusively of peptides.
A number of bacterial species used in food pro-
duction or which occur in the human intestine may

produce bioactive peptides. Cheese contains phos-
phopeptides as natural constituents and secondary
proteolysis during cheese ripening leads to forma-
tion of various ACE-inhibitory peptides (Meisel et al.
1997). Data obtained from ‘non-starter’ lactic acid
bacteria (Lactobacillus ssp.) present in fermented milk
products and in the human intestine indicate that their
proteolytic system is comparable to Lactococcus lac-
tis. In Lactococcus lactis, cell wall-bound proteinases
catalyze the first steps of casein degradation (Kunji et
al. 1996). Two types of proteinases (PI, PIII) were
detected which are distinguished by their specificity,
PI with a preference for β-casein, PIII cleaving α- and
β-casein at the same rate (Law & Haandrikman 1997).
The specificities of these proteinases towards caseins
have been determined by several authors. Early pa-
pers indicated that only small parts of caseins were
degraded, and that relatively large fragments (chain
lengths >10 amino acids) were formed (Visser et
al. 1988; Monnet et al. 1989; Reid et al. 1991;
Pritchard & Coolbear 1993). In a more recent study,
Juillard et al. (1995) analysed 95% of the peptides
formed by a PI-type proteinase by HPLC and mass
spectrometry. The authors found that hydrolysis of
β-casein proceeded much further than reported pre-
viously, more than 40% of the peptide bonds were
cleaved resulting in the formation of more than 100
different oligopeptides. Many of the peptides had
chain lengths of less than 10 amino acids and thus
were substrates for the oligopeptide transport system.
A further intracellular breakdown is catalyzed by a
number of peptidases (Mierau et al. 1997). In spite
of the presence of a di-/tripeptide transport system
in Lactococcus the liberation of di- and tripeptides
was not detected. Casein-derived peptides with chain
lengths of more than 10 amino acids are abundant
and contain various amino acid sequences represent-
ing bioactive peptides within a longer peptide (Juillard
et al. 1995). Two degradation products of β-casein
with biological activity were detected, β-CN(f60-68)
and β-CN(f190-193) which are part of the sequence
of β-casomorphin-11 1 and immunopeptide 21, re-
spectively. The presence of the tetrapeptide can be
concluded indirectly from the presence of two flank-
ing peptides β-CN(f183-189) and β-CN(f194-209) in
proteolytic digests of β-casein (Juillard et al. 1995).
Several casokinins derived from β-casein were
liberated by a cell-wall-associated serine-type pro-
teinase isolated from Lactobacillus helveticus CP790
(Yamamoto et al. 1994) and fermentation of milk
with starter cultures containing Lactobacillus helve-
211
ticus CP790 and Saccharomyces cerevisiae produced
the β-casokinins 12 and 13. An antihypertensive pep-
tide, β-CN(f169-175), was identified from casein
hydrolysate produced by the purified extracellular pro-
teinase from Lactobacillus helveticus (Maeno et al.
1994). This peptide did not have a strong ACE-
inhibitory activity (IC50 >1000 µmol/l). However, the
corresponding hexapeptide, Lys-Val-Leu-Pro-Val-Pro,
which was obtained after liberation of the C-terminal
Gln residue by pancreatic digestion in vitro, had a
strong ACE-inhibitory activity (IC50 =5 µmol/l) as
well as a remarkable antihypertensive effect in vivo
(Maeno et al. 1994). Liberation of α S1 -casokinins 15,
16 and 19 by the lactococcal proteinase seems unlikely
since no appropriate cleavage sites were found (Reid
et al. 1991). Cleavage sites in α S1 -casein were often
within the sequences of bioactive peptides (α S1 -CN
f23/24, 33/34, 37/38, 74/75). Studies on the degrada-
tion of κ-casein showed that proteolytic cleavage sites
were within the sequences of bioactive peptides (κ-CN
f32/33, 106/107). One peptide, κ-CN(f33-41), was
isolated that contained casoxin-6 8 (Reid et al. 1994).
The formation of bioactive peptides by lactic acid
bacteria in fermented milk products seems to be a
rare event. Various long oligopeptides are liberated
by degradation of caseins which could be precursors
of peptides with biological activity when cleaved by
other enzymes. In fermented milk products intracellu-
lar peptidases of lactic acid bacteria will most likely
contribute to further degradation after cell lysis (Pool-
man et al. 1995; Kunji et al. 1996; Mierau et al. 1997).
The specificities of the known peptidases suggest that
practically all peptide bonds in caseins can be cleaved
(Law & Haandrikman 1997). The formation of caso-
morphins in fermented milk products is particularly
unlikely since all lactic acid bacteria (Lactococcus
ssp. and Lactobacillus ssp.) are equipped with a X-
prolyl-dipeptidyl-aminopeptidase (Law & Haandrik-
man 1997). The alternating X-Pro sequence of the
casomorphins is the ideal substrate for this peptidase
(Bockelmann et al. 1991). It was found that en-
zymatic degradation of β-casomorphins with bacterial
cell lysate of Lactococcus lactis subsp. cremoris was
influenced by the combination of pH and salt concen-
trations and proceeded over 15 weeks at cheese storage
temperatures (Muehlenkamp & Warthesen 1998); β-
casomorphins were more resistant to proteolysis at
lower pH and higher salt concentrations (e.g. pH
5.0, 5% salt). In bovine milk samples incubated with
caseolytic bacterial species such as Pseudomonas aer-
uginosa and Bacillus cereus, varying amounts of β-
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casomorphin immunoreactive materials were detected
(Hamel et al. 1985). With regard to casokinins and
lactokinins, Pihlanto-Leppälä et al. (1998) have been
shown that commercial lactic acid starters were not
able to produce ACE-inhibitory activity from casein
or whey in vitro, but further proteolysis with pep-
sin and trypsin was needed to liberate ACE-inhibitory
peptides. No data are available on the action of the
proteolytic enzymes from lactic acid bacteria in the
human gut after ingestion of fermented milk products.
Tropho-functional properties
Milk-protein derived bioactive peptides may function
as exogenous regulatory substances which may act
on different intestinal and peripheral target sites of
the mammalian organism. Orally ingested peptides or
intestinal proteolytic products may produce local ef-
fects at the luminal side of the gastrointestinal tract.
Moreover, regulatory peptides may be absorbed via
carrier-mediated transport or via paracellular transport
and then reach target tissues. The paracellular per-
meability is regulated by intestinal tight junctions, and
modulation of tight-junction structure by other food
substances facilitates paracellular transport (Shimizu
1999).
Bioactive peptides that have been produced by
limited proteolysis during processing and/or intestinal
digestion of milk proteins could be further diges-
ted by intestinal proteinases or brush-border pepti-
dases. However, evidence for the liberation of β-
casomorphins (Svedberg et al. 1985; Meisel 1986;
Meisel & Frister 1989) and casein phosphopeptides
(Meisel & Frister 1988; Kasai et al. 1992) from ca-
sein into the intestinal lumen of mammals after intake
of milk or a casein-containing diet, i.e. under in vivo
conditions, has already been obtained. Furthermore,
evidence for in vivo production of antibacterial pep-
tides has been provided by Tomita et al. (1994) who
isolated lactoferricin from gastrointestinal contents of
rats fed a diet containing bovine lactoferrin.
Opioid peptides derived from milk proteins appear
to have physiological significance in the female or-
ganism (liberation of casomorphin in the mammary
gland) and in neonates, and they appear to parti-
cipate in the control of gastrointestinal functions in
adults (Teschemacher et al. 1997). Opioid recept-
ors are located in the nervous endocrine and immune
systems as well as in the intestinal tract of the mam-
malian organism and can interact with their endo-
genous ligands as well as with exogenous opioids
and opioid antagonists (Teschemacher & Brantl 1994).
Orally given opioid peptides derived from milk pro-
teins are able to modulate absorption processes in
the gut. The enhancement of net water and electro-
lyte absorption by β-casomorphins in the small and
large intestine is a major component of their anti-
diarrhoeal action (Daniel et al. 1990a, b). This effect
could be mediated via subepithelial opioid receptors
or specific luminal binding sites at the brush border
membrane (Tomé et al. 1987; Brandsch et al. 1994).
β-Casomorphins 1, 2, 3 are claimed to be rapidly de-
graded once they enter the blood stream. However, the
presence of β-casomorphin-7 immunoreactive mater-
ial has been demonstrated in the plasma of newborn
calves after their first milk intake (Umbach et al.
1985). This material revealed a greater molar mass
than β-casomorphin-7 1 and thus has been considered
as a β-casomorphin precursor. Such pre-casomorphins
could reach any potential site of action in the organism
to elicit effects after liberation of the protected active
sequence from the precursor molecule. Opioid casein
fragments have not been detected in the plasma of
adult mammals (Umbach et al. 1985; Teschemacher
et al. 1986). Thus, only the neonatal intestine appears
to be permeable to (pre-)casomorphins. A variety of
opioid activities in further test systems have been de-
scribed which may be of pharmacological but not of
physiological significance (for review: Teschemacher
et al. 1997).
The immunopotentiatory peptides Tyr-Gly 22 and
Tyr-Gly-Gly 23 were found to be the active com-
ponents in a dialysed leukocyte extract from normal
donors that was recently used in a large multicenter
trial to inhibit the development of infections in pa-
tients with pre-AIDS (Hadden 1991). It was suggested
that casein peptides are implicated in the stimulation
of the newborn’s immune system (Migliore-Samour et
al. 1989). The immunostimulating activities may also
have a direct effect on the resistance to bacterial and
viral infections of adult humans.
Bactericidal peptides may assist in protecting
against a microbial challenge, especially in the
neonatal intestinal tract, and thus support the non-
immune defence of the gut (Schanbacher et al. 1997).
It is not yet clear how antibacterial effects of food-
derived peptides are of physiological importance.
Casoplatelins are inhibitors of the aggregation of
ADP-activated platelets as well as binding of hu-
man fibrinogen γ -chain to a specific receptor site on
the platelet surface (Jollès et al. 1986). Data on the

physiological significance of food-derived peptides
with antithrombotic activity are not yet available.
Inhibition of the multifunctional enzyme ACE
which is located in different tissues (e.g. plasma, lung,
kidney, heart, skeletal muscle, pancreas, brain, mam-
mary arteries, testes, uterus, intestine) may influence
different regulatory systems of the organism (Ondetti
& Cushman 1982; Bruneval et al. 1986; Johnston
1992; Meisel 1993). ACE has been classically associ-
ated with the renin-angiotensin system regulating peri-
pheral blood pressure where ACE-inhibition results
in an antihypertensive effect. When ACE-inhibitory
peptides were orally given to rats blood pressure was
reduced in a dose-dependent manner (Suetsuna &
Osajima 1989). It is known that small peptides, such
as di- and tripeptides, are easily absorbed in the intest-
ine without being decomposed by digestive enzymes.
Accordingly, small ACE-inhibitory peptides can dir-
ectly pass across the intestine to reach peripheral target
sites (for review: Yamamoto 1997). In addition, ACE-
inhibitory peptides having Pro residues, especially at
the C-terminal end, are highly resistant to the de-
gradation by digestive enzymes. Recently a placebo-
controlled study showed that the daily intake of 95 ml
of Calpis sour milk results in a significant decrease
of blood pressure in hypertensive subjects (Hata et
al. 1996). The sour milk drink, which was prepared
by fermenting reconstituted skim milk powder with
a starter culture of Lactobacillus helveticus CP790
and Saccharomyces cerevisiae, contains two ACE-
inhibitory tripeptides Val-Pro-Pro 12 and Ile-Pro-Pro
13 (Nakamura et al. 1995) that were not present in the
placebo drink. It is worth noting that the ingested dose
of these peptides was in the range of only 1.2 to 1.6
mg.
The formation of soluble complexes with min-
erals and the proteolytic resistance of caseinophos-
phopeptides (CPPs) in the intestinal lumen is a pre-
requisite for their function as mineral carriers (for
review: FitzGerald 1998). Under normal physiological
conditions the paracellular transport of calcium plays
a major role independent of vitamin D in calcium ab-
sorption from the distal small intestine (for review:
Kitts & Yuan 1992). Hence CPPs might exert an in-
fluence on the absorption of calcium or other minerals
and trace elements in the intestine. A recent human
feeding trial reported an increase in calcium and zinc
absorption following CPP incorporation into a rice-
based infant food (Hansen 1997). Furthermore, it has
been shown that calcium binding CPPs can have an
anticariogenic effect in that they inhibit caries lesions
213
through recalcification of the dental enamel (Reynolds
1987).
Potential uses in functional foods and
pharmaceuticals
Although a number of studies indicate the regulatory
potential of food-derived biologically active peptides,
their role as food hormones or ‘formones’ needs fur-
ther clarification. Even if some bioactive peptides are
not released under physiological conditions in vivo,
they could be produced commercially and used as in-
gredients in functional foods. A physiologically func-
tional food can be defined as a food derived from
naturally occurring substances that can and should
be consumed as part of the daily diet, and thus
provides health benefits in reducing the risk of de-
veloping a disease. Functional foods should not be
confused with medical food products that are de-
signed to supply missing nutrients or to be used for
prevention and treatment of disease where pharmaco-
logically active compounds (drugs) are needed. The
question of what kinds of bioactive peptides are be-
neficial and desirable as food constituents or as drugs
should be always carefully examined. However, the
possibilities for the design of new dietary products and
drugs to help reduce or control diet-related chronic
diseases look promising. According to the present
state of knowledge ACE-inhibitory peptides, caseino-
phosphopeptides and immunomodulating peptides are
the most favourite bioactive peptides for application
to foodstuffs formulated to provide specific health
benefits.
Casein-derived peptides, which can be manufac-
tured on industrial scale, have already been considered
for interesting applications both as dietary supple-
ments and as pharmaceutical preparations (for review:
Meisel 1998). The production of bioactive peptides
during food processing, for example by use of specific
bacterial enzymes or genetically transformed microor-
ganisms, is of interest for future research work.
Acknowledgements
Prof. E. Schlimme is gratefully acknowledged for
the generous and stimulating support of the work on
bioactive peptides and for helpful discussions.
214
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