ARTICLE IN PRESS

International Dairy Journal 16 (2006) 1277–1293

www.elsevier.com/locate/idairyj

Review

Physiological, chemical and technological aspects of

milk-protein-derived peptides with antihypertensive and
ACE-inhibitory activity
R. López-Fandiñoa,, J. Otteb, J. van Campc
a
Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
b
Department of Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
c
Department of Food Safety and Food Quality, Faculty of Bio-Science Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
Received 15 September 2005; accepted 12 May 2006

Abstract
Among the bioactive peptides derived from milk proteins, those with blood pressure-lowering effects are receiving special attention due
to the prevalence and importance of hypertension in the Western population. A few antihypertensive products based on milk-protein-
derived peptides with clinically proven health benefits already exist. This paper reviews the current literature on milk-derived peptides
with antihypertensive effects. The structure-activity characteristics of angiotensin converting enzyme (ACE) inhibitory peptides are
discussed, as well as their bioavailability, potential physiological affects and the existence of mechanisms of action other than ACE
inhibition. The paper also focuses on the technological aspects of the production of bioactive dairy products with antihypertensive
peptides, either by fermentation with selected microorganisms or by in vitro-hydrolysis and enrichment. Finally, the stability of the
peptides during production and processing is addressed, including the potential interactions with other food components and their
influence on peptide bioactivity and bioavailability.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Milk proteins; Bioactive peptides; Angiotensin-converting enzyme; Antihypertensive; Bioavailability; Fermentation; Proteolysis; Enrichment;
Stability

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1278
2. Milk-protein-derived peptides with antihypertensive effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1278
2.1. ACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1278
2.2. Assays for ACE-inhibitory and antihypertensive activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1278
2.3. Structure-activity relationship for ACE-inhibitory peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1279
2.4. Bioavailability and physiological relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1280
2.5. Other possible mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1280
2.6. Milk-derived products with antihypertensive effects in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1281
3. Technologies for the production of milk-protein-derived antihypertensive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1282
3.1. Manufacture of fermented dairy products with ACE-inhibitory peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1283
3.2. Production of antihypertensive milk protein hydrolysates in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1284
3.3. Enrichment of hydrolysates with ACE-inhibitory peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1285
3.4. Other strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1286

Corresponding author. Tel.: +34 91 5622900; fax. +34 91 5644853.

E-mail address: rosina@ifi.csic.es (R. López-Fandiño).

0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2006.06.004
 ARTICLE IN PRESS
1278 R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293

4. Chemistry and stability of milk-protein-derived antihypertensive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1286

4.1. Influence of heat processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1287
4.2. Influence of oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288
4.3. Potential role of functional properties of milk-derived bioactive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1289
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1289

1. Introduction side of various oligopeptides. As part of the renin–angio-

Hypertension, which is estimated to affect one third of
the Western population, is a risk factor for cardiovascular
disease and stroke. In view of the role of the diet in the
prevention and treatment of the disease, efforts are being
put into the production of foods with antihypertensive
activity. Despite the higher doses needed in comparison
with antihypertensive drugs, the consumption of food
products containing antihypertensive peptides has shown
to significantly reduce the blood pressure of moderately
hypertensive subjects. The purpose of this paper is to
review the current literature on dairy products with
antihypertensive effects. Special attention was paid to
update the information covered in recent reviews on the
subject, in particular FitzGerald, Murray, and Walsh
(2004) and Vermeirssen, Van Camp, and Verstraete
(2004), with respect to the structure-activity characteristics
of angiotensin-converting enzyme (ACE) inhibitory pep-
tides, and to the existence of mechanisms of action other
than ACE inhibition. The paper also focuses on the
technological aspects of the production of bioactive dairy
products with antihypertensive peptides and on the
stability of the peptides during production and processing.
2. Milk-protein-derived peptides with antihypertensive
effects
Hypertension, i.e. high blood pressure, is a key factor in
the development of cardiovascular diseases such as myocar-
dial infarction, stroke and heart failure. In view of its
prevalence and importance, changes in life-style (reduction of
overweight, cessation of smoking and physical activity),
dietary approaches and pharmacological treatments are
broadly applied to treat hypertension. In clinical practice,
vasodilators, diuretics, calcium channel blockers, angiotensin
II receptor blockers and ACE inhibitors are normally used.
These substances interfere with the different interacting
biochemical pathways that control blood pressure, fluid and
electrolyte balance, namely, the renin–angiotensin system,
the kinin–kallikrein system, the neutral endopeptidase system
and the endothelin-converting enzyme system. The metabolic
pathways associated with the control of blood pressure have
been reviewed recently by FitzGerald et al. (2004).
2.1. ACE
ACE (peptidyldipeptide hydrolase, EC 3.4.15.1) is an
exopeptidase that cleaves dipeptides from the C-terminal
tensin system, ACE hydrolyses an inactive decapeptide,
angiotensin I, to the potent vasoconstrictor angiotensin II.
ACE also takes part of the kinin–kallikrein system as it
hydrolyses bradykinin, which has a vasodilator action.
ACE is widely distributed in many tissues, in some of
which other components of the rennin–angiotensin or the
kinin–kallikrein system are not present: this reinforces
the idea that ACE has probably other roles in addition to
the production of angiotensin II and the inactivation of
bradykinin. ACE inhibitors were first discovered in snake
venom. Since then, synthetic ACE inhibitors such as
captopril, enalapril, alecepril and lisinopril are used
extensively in the treatment of essential hypertension
despite their undesirable side effects, such as hypotension,
cough, increased potassium levels, reduced renal function,
angioedema, etc. (FitzGerald et al., 2004).
2.2. Assays for ACE-inhibitory and antihypertensive activity
The search for ACE inhibitory activity is the most
common strategy followed in the selection of antihyper-
tensive hydrolysates and/or peptides derived from milk
proteins, as well as from other food sources. The classical
approaches involve the in vitro determination of the ACE
inhibitory activity of milk protein hydrolysates, obtained
by enzymatic digestion or microbial fermentation, followed
by the identification of peptide structures and the chemical
synthesis of potentially active peptides, or their analogues,
in order to confirm their activity.
In order to facilitate the characterisation of ACE inhibitory
peptides, the establishment of a simple, sensitive and reliable
in vitro ACE inhibition assay is desirable. There are spectro-
photometric, fluorimetric, radiochemical, HPLC and capillary
electrophoresis methods to measure ACE activity. These can
also be used to obtain information on the inhibitory potency
of different substances (Li, Liu, Shi, & Le, 2005). This is
usually expressed as the IC50, or concentration needed to
inhibit 50% of the enzyme activity. The spectrophotometric
method of Cushman and Cheung (1971) is most commonly
utilized. It is based on the hydrolysis of hippuryl-His-Leu
(HHL) by ACE to hippuric acid (HA) and HL. The extent of
HA release from HHL is measured after it is extracted with
ethyl acetate, which is tedious and may overestimate ACE
activity if unhydrolyzed HHL is also extracted. Direct,
extraction-free methods have been published recently (Li
et al., 2005; Shalaby, Zakora, & Otte, 2006). Another broadly
used spectrophotometric method is based on the hydrolysis of
a furanocryloyl tripeptide (FA–Phe–Gly–Gly, FAPGG) to
 ARTICLE IN PRESS
R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
FAP and the dipeptide GG (Vermeirssen, Van Camp, &
Verstraete, 2002). However, the observation that the ACE
inhibitory activity differed with the method employed creates
a need to standardize the methodologies to evaluate ACE
inhibitory activity in vitro (Vercruysse, Smagghe, Herregods,
& Van Camp, 2005). In practice, differences may arise among
the results of different assays due to the use of different
substrates or, within the same assay, due to the use of
different test conditions or ACE from different origins. In
particular, ACE activity levels need to be carefully controlled
to obtain comparable and reproducible values (Murray,
Walsh, & FitzGerald, 2004).
The in vivo effects are tested in spontaneously hyperten-
sive rats (SHR), which constitute an accepted model for
human essential hypertension. In addition, in many in vivo
studies it is also checked that antihypertensive peptides
from milk proteins do not modify the arterial blood
pressure of Wistar-Kyoto (WKY) rats, that are the
normotensive control of the SHR.
The hypotensive effects caused by the short-term
administration to SHR of milk protein hydrolysates,
fermented products and isolated milk-derived peptides
have been summarized by FitzGerald et al. (2004). In
general terms, the results of those tests have highlighted an
important lack of correlation between the in vitro ACE
inhibitory activity and the in vivo action. This poses doubts
on the use of the in vitro ACE inhibitory activity as the
exclusive selection criterium for potential antihypertensive
substances, as it does not take into consideration the
physiological transformations that determine the bioavail-
ability of the peptides and because there might be other
mechanisms of action than ACE inhibition.
The influence of the long-term intake of milk products
on blood pressure of SHR also has been addressed. It was
demonstrated that there was a dose dependent attenuation
of the development of hypertension in SHR during 14
weeks of treatment with milk containing the potent ACE
inhibitory peptides IPP and VPP (IC50 5 and 9 mM,
respectively; Nakamura, Yamamoto, Sakai, Okubo, Ya-
mazaki, & Takano, 1995; Sipola, Finckenberg, Korpela,
Vapaatalo, & Nurminen, 2002). The long-term feeding of
the fermented milk to SHR was more effective than the
pure tripeptides, probably because calcium, potassium and
magnesium have an independent effect on blood pressure
and they intensify the antihypertensive effects of IPP and
VPP (Jauhiainen et al., 2005). Similarly, feeding SHR for
17 weeks a fermented milk devoid of IPP and VPP, but
containing other in vitro ACE inhibitory peptides, such as
LHLPLP (b-casein f133-138), exerted antihypertensive
properties that increased with the calcium content of the
fermented product (Miguel et al., 2005; Quirós et al., 2006).
2.3. Structure-activity relationship for ACE-inhibitory
peptides
Several review papers are now available that give an
overview of amino acid sequences from milk peptides with
1279
ACE-inhibitory activity (FitzGerald & Meisel, 2000;
FitzGerald et al., 2004; Gobbetti, Stepaniak, De Angelis,
Corsetti, & Cagno, 2002; Meisel, 1997a; Meisel, 2004;
Pihlanto-Leppälä, 2001).
The structure-activity relationship of ACE inhibitory
peptides from food proteins is not well studied. However,
some general features have been found (FitzGerald et al.,
2004; Meisel, 1997a, b). ACE-inhibitory peptides usually
contain 2–12 amino acids, although active peptides with up
to 27 amino acids have been identified (Robert, Razaname,
Mutter, & Juillerat, 2004; Saito, Nakamura, Kitazawa,
Kawai, & Itoh, 2000; Yamamoto, Akino, & Takano, 1994).
The binding to ACE is strongly influenced by the
C-terminal sequence, whereby hydrophobic amino acids,
e.g., Pro, are more active if present at each of the three
C-terminal positions. In addition, the presence of the
positive charge of Lys (e-amino group) and Arg (guanidino
group) as the C-terminal residue may contribute to the
inhibitory potency. Pripp, Isaksson, Stepaniak, and Sor-
haug (2004) established quantitative structure–activity
relationships (QSAR) for ACE-inhibitory peptides derived
from milk proteins. For peptides up to six amino acids, a
relationship was found between the ACE-inhibitory
activity and some of the peptide characteristics (hydro-
phobicity and a positively charged amino acid at the
C-terminal position). No relationship was found between
the N-terminal structure and the ACE-inhibitory activity.
The fact that the catalytic sites of ACE have different
conformational requirements may indicate that there is a
need for developing a complex mixture of peptides, with
slightly different conformational features, in order to
inhibit ACE activity more completely (Gobbetti et al.,
2002). Furthermore, it has been postulated that the
mechanism of ACE inhibition may involve the interaction
of the inhibitor with subsites not normally occupied by
substrates or with an anionic inhibitor binding site that is
different from the catalytic site of the enzyme (Meisel,
1997a).
Peptides can adopt different configurations depending
on the environmental conditions, which determine their
bioactivity. e.g., bradykinin, as an extended or random coil
structure, is open and sensitive for cleavage by ACE. A
b-turn at the C-terminal end of bradykinin in water gives
only a weak interaction with ACE (Desai, Coutinho, &
Srivastava, 2002). Furthermore, the change of a trans to a
cis-form of Pro in the C-terminal position of an ACE-
inhibitory peptide can cause significant changes in its
interaction with the enzyme. Gómez-Ruiz, Recio, &
Belloque (2004) studied two different preparations for
DKIHP (b-casein f47-51), an ACE-inhibitory peptide
obtained from Manchego cheese. One preparation, with a
unique conformer containing trans-Pro, gave a significant
ACE-inhibitory activity (IC50 ¼ 113.18 mM). The second
one contained three different conformers, two with trans-
Pro and one with cis-Pro, and showed a lower ACE-
inhibitory activity (IC50 ¼ 577.92 mM) compared which the
unique conformer.
 ARTICLE IN PRESS
1280 R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
2.4. Bioavailability and physiological relevance
The physiological effects of bioactive peptides depend on
their ability to reach intact their target sites, which may
involve absorption through the intestinal epithelium to get
to the peripheral organs (for a review, see Vermeirssen,
Van Camp et al., 2004). The release of ACE-inhibitory
peptides upon gastrointestinal digestion of milk proteins or
protein fragments, as well as the resistance to digestion of
known ACE-inhibitory sequences has been tested in several
in vitro studies where the gastrointestinal process was
mimicked by the sequential hydrolysis with pepsin and
pancreatic enzymes (trypsin, chymotrypsin, carboxy and
aminopeptidases). These studies showed that gastrointest-
inal digestion is an essential factor in determining ACE
inhibitory activity (Gómez-Ruiz, Ramos, & Recio, 2004a;
Vermeirssen, Van Camp, Decroos, Van Wijmelbeke, &
Verstraete, 2003). For instance, it was found that the
sequence KVLPVPE (b-casein f169-175), with a low ACE-
inhibitory activity (IC5041000 mM), was hydrolysed by
pancreatin to the potent ACE inhibitor KVLPVP
(IC50 ¼ 5 mM), which was probably responsible for the high
antihypertensive activity of KVLPVPE in SHR (Maeno,
Yamamoto, & Takano, 1996).
The conditions of the simulated gastrointestinal diges-
tion (enzyme preparation, temperature, pH and incubation
time) greatly influence the degree of proteolysis and the
resultant ACE-inhibitory activity (Vermeirssen, Van
Camp, Devos, & Verstraete, 2003). The digestibility in
vitro is also determined by the length of the peptide chain
that contains the bioactive sequence and by the presence of
other peptides in the medium (Roufik, Gauthier, &
Turgeon, 2006). Thus, the active ACE-inhibitory peptide
lactokinin, ALPMHIR, that arises from tryptic hydrolysis
of b-lactoglobulin (b-Lg f142-148, IC50 ¼ 42.6 mM), was
reported to be resistant to further hydrolysis by pepsin or
chymotrypsin (Mullally, Meisel, & FitzGerald, 1997a).
However, subsequent experiments revealed that ALPM-
HIR was susceptible to degradation on incubation with
gastrointestinal and blood serum proteinases and pepti-
dases in vitro, what restricts its potential to elicit a
hypotensive response in humans (Roufik et al., 2006;
Walsh et al., 2004). Interestingly, the shorter fragment
ALPM exerted a strong hypotensive effect in SHR despite
the fact it was not an efficient ACE inhibitor in vitro
(IC50 ¼ 928 mM; Murakami et al., 2004).
The action of brush-border peptidases, the recognition
by intestinal peptide transporters and the subsequent
susceptibility to plasma peptidases also determine the
physiological effect (Pihlanto-Leppälä, 2001; Vermeirssen,
Van Camp et al., 2004). Caco-2 cell monolayers, that
express many intestinal enzymes and transport mechan-
isms, have been broadly used as models for the small
intestine epithelium (Shimizu, Tsunogai, & Arai, 1997).
The tripeptide VPP was detected in the abdominal aorta of
SHR 6 h after its administration in sour milk, which
strongly suggests that it is transepithelially transported
(Masuda, Nakamura, & Takano, 1996). Peptides having
XPP and XP may be particularly resistant to proteolysis in
vivo (Mizuno, Nishimura, Matsuura, Gotou, & Yamamo-
to, 2004). In fact, a significant amount of VPP was
absorbed through Caco-2 cells. Paracellular transport,
through the intercellular junctions, was suggested as the
main mechanism, since the transport via the short-peptide
carrier, PepT1, led to a quick hydrolysis of the internalised
peptide (Satake et al., 2002). In the case of larger
sequences, the susceptibility to brush border peptidases is
the primary factor that decides the transport rate (Shimizu
et al., 1997). For example, the heptapeptide lactokinin
(ALPMHIR) was transported intact, although in concen-
trations too low to exert an ACE inhibitory activity,
which suggests cleavage by Caco-2 cell aminopeptidases
(Vermeirssen et al., 2002). More research is needed in this
respect, with the effort being concentrated in elucidating
the pharmacokinetics and the distribution profile of ACE
inhibitory peptides in the different tissues (Matsui et al.,
2004; Matsui et al., 2002).
2.5. Other possible mechanisms of action
Even if the hypotensive effects of milk-derived ACE-
inhibitory peptides have been demonstrated in SHR, only a
few studies have been conducted to confirm the existence of
a ACE-inhibitory mechanism in vivo (Fuglsang, Rattray,
Nilsson, & Nyborg, 2003a). ACE activity was decreased in
the aorta of SHR that had taken sour milk containing the
in vitro ACE inhibitors IPP and VPP (Nakamura, Masuda,
& Takano, 1996). In addition, plasma renin activity was
increased in SHR that had received IPP and VPP for 14
weeks. Raised levels of renin can be due to the lack of
negative feedback by angiotensin II, which supports that
ACE was inhibited (Sipola, Finckenberg, Korpela et al.,
2002). However, according to Jauhiainen et al. (2005), the
mechanistic theory of ACE inhibition of IPP and VPP
remains to be confirmed and other effects have to be taken
into consideration. Fuglsang, Rattray, Nilsson, and
Nyborg (2003b) reported that the ingestion of two milks
fermented with Lactobacillus helveticus decreased the
response to an intravenous injection of angiotensin I in
unconscious normotensive rats, and one of the products
increased the response to bradykinin, confirming the
inactivation of ACE.
Most food-derived peptides have lower ACE inhibitory
activity in vitro than the synthetic ACE inhibitor captopril,
but they usually display higher in vivo activities than the
efficacy levels extrapolated from the in vitro activities. This
fact has been attributed to a higher affinity to the tissues
and a slower elimination (Fujita & Yoshikawa, 1999), but
it may also be an indication of the existence of an
additional mode of action (Vermeirssen, Van Camp
et al., 2004). In fact, increasing new evidence is being
provided that a different mechanism, other than ACE
inhibition, may also be involved in the blood pressure-
lowering effect exerted by many food-derived peptides.
 ARTICLE IN PRESS
R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
For example, the ACE inhibitory peptides derived from
dried bonito only slightly inhibited angiotensin I-induced
contractions in rat-isolated aorta as compared with
captopril, but, unlike captopril, they exerted a direct action
on vascular smooth muscles (Kuono, Hirano, Kuboki,
Kasai, & Hatae, 2005). Similarly, lactokinin (ALPMHIR)
was found to inhibit the release of ET-1, an endothelial
peptide that evokes contractions in smooth muscle cells, an
effect that might be dependent or independent of ACE
inhibition (Maes et al., 2004). However, when interpreting
this result, the susceptibility of ALPMHIR to degradation
by gastrointestinal, brush-border and serum proteinases
and the ACE inhibitory activity of its degradation
products, should be taken into account (Murakami et al.,
2004; Walsh et al., 2004).
As shown by Nurminen et al. (2000), a-lactorphin
(YGLF), a tetrapeptide formed by in vitro proteolysis of
a-lactalbumin (a-La; f50-53) with pepsin and trypsin,
lowered blood pressure after subcutaneous administration
to SHR and WKY rats. It is likely that opioid receptors are
involved in the antihypertensive effect, as this was
abolished by the opioid receptor antagonist naloxone.
Subsequently, it was demonstrated that a-lactorphin
produced an endothelium-dependent relaxation of the
mesenteric arteries of SHR that was inhibited by a nitric
oxide (NO) synthase inhibitor (Sipola et al., 2002). There-
fore, a mechanism of action driven by the stimulation of
peripheral opioid receptors and subsequent NO release that
causes vasodilation was proposed. It should be noticed that
peripherally administered a-lactorphin in antihypertensive
doses lacked undesirable opioid receptor-related effects in
the central nervous system, such as antinociception and
sedation (Ijäs et al., 2004).
Other studies have highlighted the existence of vasor-
elaxant opioid peptides arising from b-Lg such as b-Lg
f102-105, named b-lactorphin, YLLF (Sipola et al., 2002)
and from human casein, as casoxin D, YVPFPPF, and
casokinin L, YPFPPL (Fujita et al., 1996). Similarly,
peptides derived from the hydrolysis of other food
proteins, such as ovalbumin (ovokinin, FRADHPFL,
and ovokinin 2-7, RADHPF) lowered blood pressure in
SHR through different modes of vasorelaxing activity
(Matoba, Usui, Fujita, & Yoshikawa, 1999). It was even
suggested that these peptides might lower blood pressure
through receptors expressed in the gastrointestinal tract,
which implies that no absorption is required (Yamada,
Matoba, Usui, Onishi, & Yoshikawa, 2002). Among milk-
derived sequences, there are more examples of peptides
with low ACE inhibitory activity that exert antihyperten-
sive effects in SHR, such as YP (IC50 ¼ 720 mM; Yamamo-
to, Maeno, & Takano, 1999). Also, it has been suggested
that, in the case of fermented milk, the high content of
calcium, potassium and magnesium could be protective
against hypertension (Nurminen, Korpela, & Vapaatalo,
1998).
It should be noted that the production of peptides with
antioxidant properties has been reported from caseins and
1281
whey proteins hydrolysed with different enzymes (Hernán-
dez-Ledesma, Dávalos, Bartolomé, & Amigo, 2005; Rival,
Boeriu, & Wichers, 2001). Strong experimental evidence
indicates that oxidative stress and associated oxidative
damage are mediators in cardiovascular pathologies.
Increased production of superoxide anion and hydrogen
peroxide; reduced NO synthesis; and decreased bioavail-
ability of antioxidants have been demonstrated in experi-
mental and human hypertension studies (Touyz, 2004).
2.6. Milk-derived products with antihypertensive effects in
humans
Only few milk protein-derived peptides have been tested
for their in vivo antihypertensive effect in humans (Table 1;
FitzGerald et al., 2004). The concentration of the active
peptides in the studies with the C12 and DP peptide
(as1-casein f23-34) and the BioZate hydrolysate are not
given, and the study designs have been only briefly described
(Pins & Keenan, 2003; Sugai, 1998; Townsend, McFadden,
Ford, & Cadée, 2004). The most substantiated antihyper-
tensive activity in humans has been obtained for the
commercial fermented milk products and hydrolysates that
contain the ACE-inhibitory peptides IPP and VPP
(see Section 3.1; Table 1). In this respect, it should be
mentioned that a HPLC-mass spectrometry method has
been developed recently for the quantitative determination
of IPP and VPP that allows the quality control of the
antihypertensive products containing these tripeptides
(Matsuura, Mizuno, Nishimura, Gotou, & Yamamoto,
2005). The antihypertensive effect of the sour milk product
Calpis, which is commercialized in Japan (Calpis Co. Ltd.,
Japan), was tested in a clinical study with mildly hyperten-
sive patients, some of whom were taking antihypertensive
medication (Hata et al., 1996). A later trial on 46 borderline
hypertensive men, not taking antihypertensive medication,
revealed a significant decrease in systolic blood pressure
(SBP) after 2 and 4 weeks of ingestion of the sour milk.
However, no significant change was observed as compared
with the placebo unfermented acidified milk group (Mizush-
ima et al., 2004). In that study, serum levels of angiotensin I
and II were measured at 4 weeks, but the angiotensin
I/angiotensin II ratio did not show a significant change.
Recently, a study was conducted among patients with
high-normal blood pressure and mild hypertension, who
took different doses of a casein hydrolysate produced with
Aspergillus oryzae containing IPP and VPP (Mizuno et al.,
2005). Volunteers consuming a 1.8 mg daily dose of IPP
plus VPP exhibited a significant decrease in SBP after 6
weeks and in those receiving either 2.5 or 3.6 mg, this
benefit was already recorded at 3 weeks. In addition, a
significant difference in SBP between the placebo group
and the VPP and IPP group receiving 3.6 mg was observed.
This product, marketed by Calpis as AmealPeptides has
been added to a new milk drink launched by Unilever
under the Flora/Becel pro.actives brand. Similarly, a milk
product Evoluss fermented with Lb. helveticus LBK-16H
 ARTICLE IN PRESS
1282 R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293

Table 1
Clinical studies with dairy products and peptides showing antihypertensive effects in humans

Commercial product Description of the study Dose Effect in systolic blood Reference
pressure

Calpis
(sour milk)
Calpis
(sour milk)
Ámeal Peptide
(casein hydrolysate)
Evolus/Kaiku Vitabrand
(fermented milk)
Evolus/Kaiku Vitabrand
(fermented milk)
Evolus/Kaiku Vitabrand
(fermented milk)
C12 peptide (a bovine milk
protein hydrolysate
containing as1-casein f23-
34)7alginic acid
Dodeca Peptide (DP)
(a tryptic hydrolysate of
casein enriched in as1-casein
f23-34)
BioZate 1 (a WPI
hydrolysate)
Double-blind, placebo controlled trial in 30 95 mL day–1
9.4 mm Hg (4 weeks) Hata et al. (1996)
subjects with mild hypertension, some taking (1.04 mg IPP +1.42 mg
14.1 mm Hg (8 weeks)
antihypertensive drugs (n ¼ 15) VPP)
Double-blind, randomized, placebo- 160 g day
1
4.3 mm Hg (2 weeks) Mizushima et al.
controlled trial in 46 hypertensive men (1.15 mg IPP +1.98 mg
5.2 mm Hg (4 weeks) (2004)
(n ¼ 23) VPP)
Single-blind, placebo-controlled trial in 131 1.8 mg IPP+VPP
6.3 mm Hg (6 weeks) Mizuno et al. (2005)
subjects with high-normal blood pressure 2.5 mg IPP+VPP
6.7 mm Hg (6 weeks)
and mild hypertension (n ¼ 32–33) 3.6 mg IPP+VPP
10.1 mm Hg (6 weeks)
Double-blind, randomized, placebo- 150 mL day
1
14.9 mm Hg (8 weeks) Seppo et al. (2002)
controlled trial in 17 subjects with mild (2.25 mg IPP+3-3.75 mg
hypertension (n ¼ 10) VPP)
Randomized, placebo-controlled trial in 39 150 mL day
1
6.7 mm Hg (21 weeks) Seppo et al. (2003)
hypertensive subjects (2.25 mg IPP+3-3.75 mg
VPP)
Two study periods with a washout period in 150 mL day
1
16 mm Hg (1st study Tuomilehto et al.
between in 60 subjects with mild (2.4–2.7 mg period, 8–10 weeks, (2004)
hypertension IPP+2.4–2.7 mg VPP) n ¼ 59)

11 mm Hg (2nd study
period, 5–7 weeks,
n ¼ 39)
Placebo-controlled crossover study (7-day Single dose (100 or
9 mm Hg (and— Townsend et al.
cycles) in 10 hypertensive subjects—all 200 mg C12 with 877 or 6 mmHg DBP) at 6 h (2004)
receiving 5 of the possible treatments 1754 mg alginic acid) compared with 2 h, at
high alginic acid dose
Placebo-controlled study in 18 mildly 200 mg day
1 in water
6 mm Hg DBP after 4 Sugai (1998)
hypertensive subjects for 4 weeks. Placebo weeks. No effect in
was water without DP normotensive subjects
Placebo-controlled study in 30 borderline Not indicated, probably
11 mm Hg (
7 mm Hg Pins and Keenan
hypertensive subjects for 6 weeks. Placebo 20 g day –1 DBP) in comparison (2003)
was unhydrolysed WPI with control.
In addition LDL-
cholesterol was lowered
by 12%

(Valio Ltd, Finland or Kaiku Vitabrands, Spain) exerted
significant antihypertensive effects in humans at daily doses
of 150 mL (Seppo, Jauhiainen, Poussa, & Korpela, 2003;
Seppo, Kerojoki, Suomalainen, & Korpela, 2002). How-
ever, in another study consisting of two periods separated
by a washout period, no statistically significant differences
were found between the sour milk and the placebo in the
crossover analysis combining both phases (Tuomilehto
et al., 2004). This fermented milk was shown to increase
osteoblastic bone formation in vitro (Narva, Halleen,
Vaanamen, & Korpela, 2004).
It should be mentioned that the reductions in SBP
caused by the administration of IPP and VPP to humans
(Table 1) were very modest in comparison with the effects
previously reported in SHR (
28.3 and
32.1 mm Hg,
respectively, Nakamura, Yamamoto, Sakai, & Takano,
1995). Matsuura et al. (2005) did not observe a significant
change of diastolic blood pressure (DBP) for the test
groups, nor differences as compared with the placebo
group.
Two other commercial products, a casein hydrolysate
containing the peptide FFVAPFEVFGK (as1-casein f23-
34; Casein DP, Kanebo, Ltd, Japan, and C12 peptide,
DMV, The Netherlands) and a whey protein hydrolysate
(BioZate, Davisco, US) were also claimed to lower blood
pressure in humans (FitzGerald et al., 2004).
3. Technologies for the production of milk-protein-derived
antihypertensive peptides
The sequences of the ACE-inhibitory peptides men-
tioned above are contained within the intact milk proteins,
and must be released from the proteins by specific
enzymatic hydrolysis to exert their health effects. In
principle, there are two approaches for releasing bioactive
peptides from intact milk proteins. One approach is to
 ARTICLE IN PRESS
R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
exploit the proteolytic system of lactic acid bacteria to
partially digest the caseins during the manufacture of dairy
products, like fermented milk and cheeses. The other
approach is to subject isolated milk protein preparations to
hydrolysis in vitro by one or a combination of enzymes,
which results in milk protein hydrolysates containing a
great number of peptides, among them the bioactive
peptides. The hydrolysates (or hydrolysates enriched in
particular peptides) may be applied in the manufacture of
other food products, to provide them with the desired
bioactivity. The technological challenges, thus, lie in (i) the
manufacture of fermented dairy products with a high
concentration of particular bioactive peptides or their
precursors, which upon digestion in the gastrointestinal
tract would give rise to the bioactive peptides, and (ii) the
production of milk protein hydrolysates with a high
concentration of peptides with a specific bioactivity, and
with a functionality that makes them suitable as ingredients
in other foods, including dairy foods.
3.1. Manufacture of fermented dairy products with ACE-
inhibitory peptides
The production of ACE-inhibitory and antihypertensive
peptides in situ in dairy products is an appealing approach,
since this confers an additional positive health effect to
dairy products possessing already a healthy image and
having a long history of safe production. During the
fermentation of milk and maturation of cheese, the major
milk proteins are degraded into a great number of peptides
due to the action of indigenous milk enzymes, (mainly
plasmin), added coagulants and microbial enzymes
(especially from starter and non-starter lactic acid
bacteria; LAB).
The single most effective way to increase the number of
bioactive peptides in fermented dairy products is to
ferment or co-ferment with highly proteolytic strains of
LAB. The challenges in this approach using LAB in dairy
products lie in choosing the right strains or combination of
strains with optimal proteolytic activity and lysis tendency
at the right time. The strain should not be too proteolytic
to destroy the product and yet to give a high proteolysis,
and with the right specificity to give higher concentrations
of the active, ACE-inhibitory, peptides relative to other
peptides, i.e., bitter peptides. Moreover, the content of
potent ACE-inhibitory peptides seems to rely on a balance
between their formation and further breakdown into
inactive peptides and amino acids, in turn depending on
storage time and conditions (Gobetti, Minervini, &
Grizzello, 2004; Meisel, Goepfert, & Günther, 1997;
Ryhänen, Pihlanto-Leppälä, & Pahkala, 2001).
Fermented milks containing a particularly high number
of peptides, among them many ACE-inhibitory and
antihypertensive peptides, have been produced using
proteolytic strains of the LAB species Lb. helveticus, Lb.
casei, Lb. plantarum, Lb. rhamnosus, Lb. acidophilus,
Lactococcus lactis subsp. lactis and subsp. cremoris, as
1283
well as the two species used in traditional yoghurt
production Lb. delbrueckii subsp. bulgaricus and Strepto-
coccus thermophilus (Fuglsang et al., 2003b; Gobetti,
Ferranti, Smacchi, Goffredi, & Addeo, 2000; Hernández-
Ledesma, Amigo, Ramos, & Recio, 2004a, b; Leclerc,
Gauthier, Bachelard, Santure, & Roy, 2002; Nakamura,
Yamamoto, Sakai, Okubo, et al., 1995; Rokka, Syväoja,
Tuominen, & Korhonen, 1997; Seppo et al., 2003;
Vermeirssen, Van Camp, Decroos et al., 2003; Yamamoto
et al., 1994, 1999). Recently, Muguerza et al. (2006)
assayed the ACE-inhibitory activity of fermented milk
samples produced with 231 microorganisms isolated
from raw cow’s milk samples. Among them, four
Enterococcus faecalis strains stood out as producers of
fermented milk with potent ACE inhibitory activity
(IC50 ¼ 34–59 mg mL
1) and antihypertensive activity in
SHR.
The first fermented milk with documented antihyperten-
sive activity (Nakamura, Yamamoto, Sakai, Okubo et al.,
1995; Nakamura, Yamamoto, Sakai, & Takano, 1995) was
marketed by the Japanese Calpis company under the
tradename Amiiru S. It is produced by fermentation with a
combination of Lb. helveticus CP790 and a Saccharomyces
cerevisiae and contains two ACE-inhibitory tripeptides,
VPP and IPP, of casein origin, shown to be responsible for
the already mentioned antihypertensive properties of the
milk drink in vivo (see Section 2 and Table 1). The first
European fermented milk drink designed to help lower
blood pressure, Evoluss from Valio Ltd., Finland, was
fermented by another Lb. helveticus strain, LBK16 H, and
contained the same tripeptides (Tuomilehto et al., 2004)
(Table 1). Other Lb. helveticus strains used in the
production of antihypertensive fermented milk foods are
Lb. helveticus R211, R389 (Leclerc et al., 2002) and LMG
11474 (Vermeirssen, Van Camp, Decroos et al., 2003), as
well as CHCC641 and CCCH637 from Chr. Hansen A/S
(Fuglsang et al., 2003b). In fact, Lb. helveticus has been the
preferred fermenting organism in the pursuit of an effective
ACE-inhibitory milk product due to its generally higher
proteolytic activity compared to other LAB (Fuglsang
et al., 2003b; Yamamoto et al., 1994), but also because of
the high activity of the derived peptides (Nakamura,
Yamamoto, Sakai, Okubo et al., 1995; Nakamura,
Yamamoto, Sakai et al., 1995; Yamamoto et al., 1999;
Seppo et al., 2002, 2003). In a recent study (unpublished),
milk samples fermented by 4 strains of Lc. lactis had higher
ACE-inhibitory activity than the milks fermented by either
of 7 strains of Lb. helveticus, showing that the proteolytic
system of Lactococcus should not be overlooked in the
production of antihypertensive peptides.
The proteolytic systems of LAB consist of (i) cell-wall
proteinases (Lactocepins), often designated PrtP (Juillard
et al., 1995), that initiate the proteolytic attack, (ii)
transport systems that facilitate the uptake of the
oligopeptides into the bacterial cell, and finally (iii) a series
of intracellular peptidases. Some ACE-inhibitory peptides
are products of extracellular proteinases alone, i.e. the large
 ARTICLE IN PRESS
1284 R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
b-casein fragments produced by the extracellular protei-
nase from Lb. helveticus CP790 (Yamamoto et al., 1994),
whereas others are most likely the result of a concerted
action of both proteinases and peptidases, i.e. YP isolated
from a yoghurt-like product fermented by Lb. helveticus
CPN4 (Yamamoto et al., 1999). This dipeptide was
probably released by Pep X acting on the C-terminal
b-casein oligopeptides liberated during early hydrolysis by
a PI-type cell-wall proteinase (Pritchard & Coolbear, 1993).
The cell-wall proteinases of both lactococci and lacto-
bacilli have a very broad substrate specificity. For example,
more than 100 different oligopeptides are released from
b-casein when incubated with PI-type proteinases (Juillard
et al., 1995). Peptide bonds from fragments such as
160–170 and 190–195 in b-casein are cleaved by all
proteinase types, while bonds in other regions are only
cut by some proteinases (Kunji, Mierau, Hagting, Pool-
man, & Konings, 1996).
The intracellular peptidases so far isolated from lacto-
cocci and lactobacilli are either aminopeptidases or
endopeptidases. Some of the aminopeptidases, such as
Pep N and Pep C, are well conserved among dairy LAB,
whereas others, particularly those specific for tri- and
dipeptides are distinct to each species. Specialized pepti-
dases (i.e. Pep X) involved in the hydrolysis of Pro-
containing sequences are important for the degradation of
casein-derived oligopeptides because of their high content
of Pro. Especially powerful aminopeptidases exist in Lb.
helveticus compared e.g., Lc. lactis and Lb. acidophilus
(Sasaki, Bosman, & Tan, 1995) and, among these, Pep X is
the most dominating one (Gatti, Fornasari, Lazzi, Munc-
chetti, & Neviani, 2004), although some variation within
strains of Lb. helveticus exists (Khalid, Soda, & Marth,
1991).
The small peptides produced by endopeptidases in the
bacterial cells may be excreted into the milk product by
some sort of exchange of these peptides over the cell
membrane (Kunji et al., 1996), or, more likely, as a result
of lysis of the bacterial cell. Lysis of bacterial cells may also
allow the intracellular peptidases to escape from their
intracellular localization and to act on the large oligopep-
tides produced by the action of the cell-wall proteinases. In
cheese, the extent of lysis of both Lb. helveticus and Lc.
lactis cells has been shown to have a direct influence on
proteolysis (Crow, Martley, Coolbear, & Roundhill, 1995;
Valence, Deutsch, Richoux, Gagnaire, & Lortal, 2000).
The proneness to lyse under certain conditions is thus
another quality of the LAB that should be considered.
The peptide pattern of cheeses from different maturation
stages is characteristic for each cheese variety (Ardö, 2001;
Coker, Crawford, Johnston, Singh, & Creamer, 2005). The
origin and history of the milk used and the manufacturing
conditions thus affect the production of peptides. Among
the peptides formed during maturation of commercial
cheeses, such as Gouda, Edam, Emmental, Camembert,
Havarti and Blue cheese, many are identical to or contain
sequences with proven antihypertensive activity or ACE
inhibitory activity (Saito et al., 2000). The highest blood
pressure-lowering activity was exerted by peptides from an
8 month old Gouda cheese, in particular RPKHPIKHQ
corresponding to as1-casein f1-9. Smacchi and Gobbetti
(1998), investigating the peptidase-inhibitory activity
of extracts from Italian commercial cheeses, isolated a
b-casein fragment from (b-casein f58-72) that inhibited
ACE (IC50 ¼ 18 mM). Many ACE-inhibitory peptides were
formed in a Spanish Manchego cheese prepared specifically
by inoculating the milk with Lc. lactis subsp. lactis (80%)
and Leuconostoc mesenteroides subsp. dextranicum (20%;
Gómez-Ruiz, Ramos, & Recio, 2004b). A new type of low-
fat cheese containing ACE-inhibitory peptides, ‘‘Festivo’’
cheese, is produced in Finland with a commercial starter
mixture containing strains of Lactococcus, Leuconostoc,
Propionibacterium, Lactobacillus sp. as well as probiotic
strains of Lb. acidophilus and Bifidobacterium sp. (Ryhä-
nen, Pihlanto-Leppälä, & Pahkala, 2001). The highest
ACE-inhibitory activity was found in the ‘‘Festivo’’ cheese
ripened for 13 weeks, from which 3 active peptides from
as1-casein were isolated (as1-casein f1-6, f1-7 and f1-9).
These peptides are also formed during ripening of other
Scandinavian cheeses (Lund & Ardö, 2004).
Much effort is being put into expanding the knowledge
about the proteolytic systems of interesting LAB, in
particular their activity under various conditions relevant
for the fermentation of dairy products. Further progress in
this area might be obtained through genetic engineering, to
provide the most suited LAB with the desired proteolytic
capacity, and also from studies regarding the interaction
between strains in environments as those prevailing in
fermented milks and cheeses.
It should be noted that some antihypertensive dairy
products have been manufactured by fermentation in
combination with in vitro hydrolysis. For example, Rokka
et al. (1997) produced a fermented milk by first inoculating
a UHT treated milk with Lb casei subsp. rhamnosus (Lb.
GG), followed by hydrolysis with pepsin and trypsin.
A number of bioactive peptides were isolated from this
drink, among them two b-casein fragments with ACE-
inhibitory activity (b-casein f177-183 and f193-202). Saito,
Abubakar, Itoh, Arai, and Aimar (1997) used a reverse
order to produce fermented whey beverages. The whey was
first hydrolysed with trypsin, proteinase K, thermolysin or
other enzymes, and subsequently fermented with Lb.
delbrueckii subsp. bulgaricus and Str. thermophilus. The
highest ACE-inhibitory activity (IC50 ¼ 50 ng mL
1) corre-
sponded to the whey initially hydrolysed by proteinase K.
3.2. Production of antihypertensive milk protein
hydrolysates in vitro
In vitro hydrolysis of milk proteins allows the selection
of the protein substrate and enzyme specificity to optimise
the yield of bioactive peptides. Many studies performed
during the last two decades have revealed the presence of
ACE-inhibitory and/or antihypertensive peptides in
 ARTICLE IN PRESS
R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
enzymatic digests of various milk protein preparations, i.e.
caseinates, individual caseins, whey protein concentrates
and isolates, and individual whey proteins (Abubakar,
Saito, Kitazawa, Kawai, & Itoh, 1998; Fitzgerald et al.,
2004; Karaki et al., 1990; Maeno et al., 1996; Maruyama
et al., 1987; Mullally, Meisel, & Fitzgerald, 1997b;
Pihlanto-Leppälä, Koskinen, Piilola, Tupasela, & Korho-
nen, 2000; Pihlanto-Leppälä, Rokka, & Korhonen, 1998;
Schlothauer et al. 2002; Tauzin, Miclo & Gaillard, 2002;
Yamamoto et al., 1994). Enzymes from various sources
have been used to hydrolyse the milk proteins, comprising
animal digestive enzymes, plant enzymes, and microbial
enzymes including cell-wall proteases from LAB (Abuba-
kar et al., 1998; FitzGerald et al., 2004; Maeno et al., 1996;
Mullally et al., 1997b; Yamamoto et al., 1994). The most
efficient inhibitors of ACE (IC50o30 mM) were formed
from caseinate and the individual major caseins, as1-casein,
as2-casein and b-casein, after hydrolysis by trypsin or an
extracellular proteinase from Lb. helveticus (Maruyama
et al., 1987; Robert et al., 2004; Tauzin et al., 2002;
Yamamoto et al., 1994). This might be related to their high
content of Pro. Interestingly, hydrolysis of the major whey
proteins, b-Lg, a-La and bovine serum albumin, also
resulted in peptides with high ACE-inhibitory and/or
antihypertensive activity. Concerning the whey proteins,
which are more compact than the caseins, hydrolysis with a
combination of digestive enzymes or highly proteolytic and
less specific enzymes, e.g., thermolysin and proteinase K,
both of microbial origin, might be particularly useful in
releasing potent ACE-inhibitory peptides (Abubakar et al.,
1998; FitzGerald & Meisel, 1999; Hernández-Ledesma,
Recio, Ramos, & Amigo, 2002; Mullally et al., 1997b;
Nurminen et al., 2000; Pihlanto-Leppälä et al., 2000;
Schlothauer et al., 2002). The use of high pressure to
partially unfold the whey proteins, before or during
proteolysis, might also increase the rate of proteolysis
and alter the relative proportion of peptides (Knudsen,
Otte, Olsen, & Skibsted, 2002).
Considering the previously discussed structure-activity
features of ACE-inhibitory peptides, enzymes with speci-
ficity towards the carboxylic side of aromatic or other
hydrophobic amino acid residues, or towards the basic
amino acids Lys and Arg might be beneficial, explaining
the large number of ACE-inhibitory peptides obtained with
trypsin. The choice of the best protein substrate and
enzyme combination may be assisted by the newly
developed in silico calculations using dedicated software
(Dziuba, Iwaniak, & Minkiewicz, 2003; Vermeirssen, van
der Bent, Van Camp, van Amerongen, & Verstraete, 2004).
Results from the latter study show that, in addition to b-Lg
and b-casein, lactoferrin may be a good precursor if
hydrolysis is performed to release many dipeptides.
Calculations performed by Pripp (2005) on the ACE-
inhibitory activity of b-casein after a specific theoretical
hydrolysis, showed that initial hydrolysis after Pro residues
would increase the apparent bioavailable ACE-inhibitory
activity of b-casein after gastrointestinal proteolysis by a
1285
factor of 10. This in silico calculation technique may be
useful also for the prediction of the outcome of fermenta-
tion, as far as the specificity of the microbial enzymes is
known. However, this technique does not take into account
the protein conformation which might affect also the ACE-
inhibitory activity as discussed in Section 2.3.
Not surprisingly, many patents have been granted for
the production of milk protein hydrolysates with good
functionality, including bioactivity. For example,
Schlothauer et al. (2002) have patented a method for
hydrolysis of a whey protein isolate (WPI) using Neutrase
or other proteases at 50 1C and neutral pH to a DH of less
than 10%. The WPI hydrolysate thus produced contained
a number of ACE inhibitory peptides originating from
b-Lg and a-La with IC50 below 25 mg mL
1. Presently, a
number of casein and whey protein hydrolysates and even
individual peptides with high ACE-inhibitory activity are
commercially available, i.e. from Arla Foods Ingredients
and Davisco Foods International as well as from DMV
International and Kanebo Ltd., that also market the C12-
Peptide (Fitzgerald et al., 2004; Pins & Keenan, 2003; see
also Section 2).
3.3. Enrichment of hydrolysates with ACE-inhibitory
peptides
A technological challenge in the production of ACE-
inhibitory and antihypertensive peptides is the enrichment
of fractions or the isolation of specific peptides from the
total peptide mixture. Since the ACE-inhibitory peptides
are not characterised by a particular functional group,
such as serine phosphate groups in the caseinophospho-
peptides, they cannot be isolated by precipitation or
ion exchange chromatography. A common feature of
ACE-inhibitory peptides being their relatively restricted
size and relatively hydrophobic C-terminal makes fractio-
nation based on size a promising step for pre-concentration
of the active peptides. In the laboratory, this has been
achieved by ultrafiltration and size exclusion chromato-
graphy, processes that are both suitable for industrial
scale production. Considering the size of most ACE-
inhibitory peptides being less than 3 kDa, ultrafiltration
with a cut-off of 3 or 5 kDa seems a good choice
(Gómez-Ruiz et al., 2004b; Hernández-Ledesma et al.,
2004b; Lapointe, Mollé, Gauthier, & Pouliot, 2004;
Maeno et al., 1996; Mullally et al., 1997b; Nakamura,
Yamamoto, Sakai, Okubo et al., 1995; Pihlanto-Lepälä et
al., 2000). With a membrane of 1 kDa, some of the active
peptides may be lost (Pihlanto-Leppälä et al., 2000).
Accordingly, FitzGerald and coworkers found the highest
ACE-inhibitory index in the 3 kDa-permeate, compared
with the 1 and 10 kDa permeates and with the original
tryptic digest of whey proteins (Mullally et al., 1997b).
Schlothauer et al. (2002) in their patent, though, used
membranes with cut-off between 10 and 50 kDa, which also
gave peptide concentrates with a rather high ACE-
inhibition.
 ARTICLE IN PRESS
1286 R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
Pre-concentration based on hydrophobicity using a C18
reversed-phase cartridge is also common in the laboratory
(Curtis, Dennis, Waddell, MacGillivray, & Ewart, 2002;
Hernández-Ledesma, Miralles, Amigo, Ramos, & Recio,
2005; Mullally et al., 1997b; Saito et al., 2000; Yamamoto
et al., 1999). Size exclusion chromatography may be used
for further fractionation, followed by separation by
reversed-phase HPLC (i.e. Pihlanto-Leppälä et al., 2000).
Reversed-phase chromatographic separation is well suited
for the final separation in the laboratory, since the ACE-
inhibitory peptides are distributed over most of the peptide
profile, and they are not restricted to the most hydrophilic
fraction (Manso & López-Fandiño, 2003). On-line cou-
pling to electrospray ionization-tandem mass spectrometry
allows simultaneous identification of the peptides (Gómez-
Ruiz et al., 2004b; Hernández-Ledesma et al., 2004a;
Robert et al., 2004). A special set-up, in which the
biochemical assay detecting ACE inhibitory activity of
peptides was coupled into the LC-MS line, has been
published (van Elswijk et al., 2003).
With a view to the industrial scale production of
hydrolysates, various initiatives have been taken towards
continuous operation with increased enzyme utilization
efficiency and increased yield and purity of the active
peptides. Bouhallab, Mollé, and Léonil (1993) suggested
the use of a membrane reactor with a 3 kDa cut-off
cellulosic membrane for the continuous tryptic hydrolysis
of b-casein and recovery of the fragment 193–209, which
contains sequences with a moderate ACE-inhibitory
activity (Gómez-Ruiz et al., 2004b; Pihlanto-Lëppäla
et al., 1998; Steijns 1996; Yamamoto & Takano, 1999).
Using a continuous membrane reactor, the peptide bonds
to be preferentially cleaved can be controlled by the
enzyme and substrate concentrations and the substrate
feeding flow rate (Martin-Orue, Henry, & Bouhallab,
1999). Righetti, Nembri, Bossi, and Mortarino (1997)
reported the use of a multi-compartment reactor operating
under an electrical field for the continuous harvest of
peptides from b-casein according to their pI. Using this
reactor, a number of tryptic peptides from b-casein were
isolated in pure form, among them the large fragment
49–97, which contains many sequences with high ACE-
inhibitory (IC50 ¼ 4–100 mM) and antihypertensive activity
(Abubakar et al., 1998; Nakamura, Yamamoto, Sakai,
Okubo et al., 1995; Nakamura, Yamamoto, Sakai et al.,
1995; Yamamoto & Takano, 1999).
The peptides or concentrated hydrolysates produced
could be applied as functional ingredients in other foods to
provide them with the desired antihypertensive activity.
Nutraceuticals may be incorporated into fermented milk by
addition to the standardized milk prior to inoculation
(Awaisheh, Haddadin, & Robinson, 2005). Lucas, Sodini,
Monnet, Jolivet, and Corrieu (2004) used this approach to
incorporate casein and whey protein hydrolysates into
fermented milks containing probiotic bacteria. It should be
possible to enrich fermented milk in the same way by milk
protein hydrolysates particularly rich in ACE-inhibitory
peptides. However, the degradation of the active peptides
during processing, as well as the uptake and degradation of
the active peptides by the fermenting organisms should be
evaluated.
3.4. Other strategies
Further optimization of the antihypertensive potential of
milk-derived peptides may be obtained by molecular
modelling of the peptide into the active sites of ACE
(Brew, 2003; Natesh, Schwager, Sturrock, & Acharaya,
2003), or into the active site of other enzymes and receptors
involved in blood pressure regulation, i.e. the angiotensin
II receptor T1 (FitzGerald et al., 2004; Moutevelis-
Minakakis et al., 2003; see Section 2). Furthermore, the
bioavailability of the active peptides may be optimized by
targeting to specific peptide receptors or cross-linking to
protein-transduction domains or to specific peptide carriers
(Vermeirssen, Van Camp et al., 2004).
ACE-inhibitory peptides can be produced also by genetic
engineering in a GRAS micro-organism and subsequently
delivered in situ in the small intestine (Lv, Huo, & Fu,
2003). Further refinements, like the inclusion of a signal
peptide for excretion and an inducible promoter that can
be switched on at the right place in the gastro-intestinal
tract may target the delivery of a high concentration of
active peptides at the right place.
4. Chemistry and stability of milk-protein-derived
antihypertensive peptides
The bioactive peptides derived from milk proteins can be
delivered in the form of dairy products or functional
ingredients, such as hydrolysates, per se or incorporated
into other food products. Despite the way of delivery, the
product—and in particular the ACE-inhibitory peptides
therein—must be stable during the final processing,
packaging and storage. Furthermore, the hydrolysate
should have well-defined technological functionalities not
to impart the required functionality of the carrier food.
Several of the amino acids present at the C-terminal
sequence of ACE-inhibitory peptides, that are crucial with
regard to their interaction with ACE, and as such with
their bioactivity (see Section 2.3), are prone to chemical
changes occurring during food processing, preparation and
preservation. The question arises whether these molecular
changes on amino acid level result in a partial or total loss
of the bioactivity of the peptide. The influence of food
processing, preparation and preservation on the bioactivity
of ACE-inhibitory and antihypertensive peptides is not
well documented. However, some reactions potentially
occurring in dairy products may be relevant to the changes
in molecular structure of amino acids in these bioactive
peptides and thus may give rise to changes in their
bioactivity. For a more complete overview of the reactions
in which amino acids in foods and food products are
involved, refer to the review by Damodaran (1995).
 ARTICLE IN PRESS
R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
4.1. Influence of heat processing
Thermal processing at alkaline pH can lead to changes in
the structure of amino acids, such as Arg, Ser, Thr and Lys.
Arg decomposes to ornithine. In addition, partial racemi-
zation of L-amino acids into D-amino acids can occur under
these conditions, especially for amino acids with a strong
electron withdrawing power in the side chain, i.e. Asp, Ser,
Cys, Glu, Phe, Asn, and Thr. Heat alone may also induce
racemization, as demonstrated for roasted casein and
bovine serum albumin (Friedman, 1999a). In addition,
D-amino acids can be synthesized out of L-amino acids by
microorganisms using amino acid oxidases, transaminases,
and epimerases (racemases). Thus, D-amino acids have
been detected in various dairy products where microorgan-
isms are involved, like fermented milk, yoghurt, and
ripened cheeses (Friedman, 1999a).
Heating at alkaline pH or above 200 1C at neutral pH, of
Ala, Cys, cystine, and phosphoserine, leads to the highly
reactive dehydroalanine residue. This electrophilic mole-
cule reacts further with Lys, Cys, ornithine (obtained after
decomposition of Arg) or His, to lysinoalanine (LAL),
lanthionine, ornithoalanine, and histidinoalanine, respec-
tively (Damodaran, 1995). Milk proteins contain high
concentrations of LAL amino acid precursors, like serine
phosphate groups in casein, and cystine in whey proteins.
LAL formation was also reported in thermally treated milk
products, like sodium and calcium caseinates (Friedman,
1999b).
The alkaline induced changes in amino acids given above
may affect proteolytic digestion. Alkali-treated casein is
less efficiently hydrolyzed by pancreatic proteases such as
trypsin and chymotrypsin (Berger & Possompes, 1987).
Since trypsin catalyses the hydrolysis of peptide bonds
derived from carboxyl groups of Arg and Lys residues, it
has been hypothesized that the decreased sensitivity for
trypsin hydrolysis is due to formation of LAL from Lys
and decomposition of the Arg residue. Chymotrypsin on
the other hand catalyzes hydrolysis of peptide bonds next
to aromatic amino acids (Phe, Tyr, Trp) and consequently,
the resistance to chymotrypsin hydrolysis may be related to
racemization of aromatic side chains. A wide species
dependence in nutritional utilisation and nutritionally
antagonistic and toxic manifestations of D-enantiomers
and LAL have been described. For a review of the
literature see Friedman (1999a, b).
In general, heat treatment of dairy proteins whether or
not in combination with an alkaline treatment induces
racemization and cross-linking of amino acids. This may
affect the sensitivity to proteolysis and, as such, change the
yield of bioactive ACE inhibitory and antihypertensive
peptides originating from these proteins. The structure of
the bioactive sites in the peptides may be changed in such a
way that the activity is affected directly as well.
The Maillard reaction (non-enzymatic browning) is
known to have a significant impact on nutritional proper-
ties in general and on amino acids in particular.
1287
The reaction occurs between amines and carbonyl com-
pounds forming glycosylamines, which, at elevated tem-
peratures, decompose and eventually condense into
insoluble brown products such as melanoidins. Amino
acids supply the amino component, while reducing sugars
(aldoses and ketoses), ascorbic acid, and carbonyl com-
pounds generated from lipid oxidation, supply the carbo-
nyl component. After condensation of the amine with the
carbonyl compound, an intermediary glycosylamine is
formed which rearranges to a 1-amino-1-deoxyketose
(Amadori rearrangement for aldosamines) or to 2-amino-
2-deoxyaldose (Heyns rearrangement for ketosamines).
These products should be considered as intermediates
which are further degraded to 1-, 3- and 4-deoxydicarbo-
nylcompounds (deoxysones), reactive a-carbonyl com-
pounds yielding many secondary products (Belitz &
Grosch, 1999). Especially the e-amino group of Lys is
frequently involved in the carbonyl–amine reaction. Lys in
the early stages of browning, is hydrolysed in the acidic
conditions of the stomach. Beyond the stage of 1-amino-1-
deoxyketose or the 2-amino-2-deoxyaldose, Lys is no
longer biologically available. In addition to the losses of
Lys, the additional formation of reactive a-carbonyl
compounds lead to a cascade of supplementary reactions
involving also other amino acids, e.g. Met, Tyr, His, Trp
(Damodaran, 1995).
Milk is sensitive to the Maillard reaction due to the
presence of high levels of lactose and Lys-rich proteins.
Lys-residues in b-Lg have been shown to interact with
lactose giving specific lactosyl b-Lg conjugates during heat
treatment of milk and whey (Léonil et al., 1997). More
severe heat treatments of milk, especially in-can sterilisa-
tion, lead to higher losses of available Lys compared to
milder treatments, like pasteurization or UHT-treatment
(Korhonen, Pihlanto-Leppälä, Rantamäki, & Tupasela,
1998). In addition to the structural changes in Lys and
other important and sensitive amino acids, it is also
reported that protein digestibility is affected by the
complex reactions occurring during the Maillard reaction
(Gilani & Sepehr, 2003; Rérat, Calmes, Vaissade, & Finot,
2002). Consequently the yield of bioactive peptides
generated during proteolysis could be affected as well.
Although very speculative, it cannot be excluded that
glycosylation of dairy proteins and peptides, as reported to
occur during the Maillard reaction (Broersen, Voragen,
Hamer, & de Jongh, 2004; Léonil et al., 1997, Mollé,
Morgan, Bouhallab, & Léonil, 1998) could give rise to
changes in the conformation of biologically active peptides
and thus their activity. Broersen et al. (2004) reported that,
as a result of the glycosylation of b-Lg, the conformational
stability of the protein is changed due to changes in the
secondary structure. These changes were attributed to
altered intramolecular hydrogen-bonding. Previously,
Rickert and Imperiali (1995) reported the effect of
N-glycosylation on the cis/trans isomer ratio of Pro in a
particular peptide. Also, Pao, Wormarld, Raymond,
and Lellouch (1996) reported on the effect of serine
 ARTICLE IN PRESS
1288 R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
O-glycosylation on cis/trans Pro isomerisation, which has a
large impact on the ACE-inhibitory activity of peptides
containing Pro at the C-terminal (Section 2.3).
4.2. Influence of oxidation
Endogenous production of oxidative compounds in
foods during processing (e.g. free radicals and peroxides
formed during peroxidation of lipids) and the use of
oxidizing agents (e.g. hydrogen peroxide) may lead to
oxidation of amino acids. Met easily oxidizes to methionine
sulfoxide by peroxides, and eventually can be further
oxidized to methionine sulfone and homocysteic acid. The
latter two are biologically unavailable, while methionine
sulfoxide can be reconverted to Met under the acidic
conditions of the stomach. Similarly, mono- and disulf-
oxides of L-cystine are biologically available as they reduce
back to L-cystine in the body, while mono- and disulfone
derivatives of L-cystine are biologically unavailable.
Trp can be oxidized under acidic, mild oxidizing condi-
tions, and more severely under acidic, severe oxidizing
conditions. Cys, His, Met, Trp and Tyr are also susce
ptible to sensitized photo-oxidation. Dairy products are
particularly vulnerable to photo-oxidation because of
the presence of the photo-sensitizer riboflavin (Mestdagh,
De Meulenaer, De Clippeleer, Devlieghere, & Huyghe-
baert, 2005).
Obviously these oxidative changes may give rise to
changes in the molecular structure of ACE-inhibitory
and antihypertensive peptides, thus altering their bioactiv-
ity. Interestingly, these changes can be induced particularly
during the storage of the finished product unless
appropriate packaging materials are used (Mestdagh
et al., 2005).
4.3. Potential role of functional properties of milk-derived
bioactive peptides
Milk peptides have been extensively evaluated for their
interfacial properties. These properties must be taken into
account in case products are to be formulated containing
bioactive (ACE-inhibitory or antihypertensive) peptides.
Turgeon, Gauthier, Mollé, and Léonil (1992) indicated, in
a study on tryptic hydrolysates of b-lactoglobulin (b-Lg),
that peptides with good interfacial properties exhibit
discrete hydrophobic regions (three or five residues)
separated by polar residues (two or three residues) with a
minimum weight allowing this distribution. Poor inter-
facial properties were related to an uniform distribution of
hydrophobic and hydrophilic amino acids and to the
rigidity provided by disulfide bonds, which prevents
spreading at the interface. Smaller peptides are less surface
active. This conclusion was also drawn from the study of
Lajoie, Gauthier, & Pouliot (2001), where cationic frac-
tions of tryptic whey protein hydrolysates having low
molecular masses (o1000 Da) and isoelectric points ran-
ging from 5.8 to 10.2, gave a destabilizing effect on
model infant formula compared with more anionic
and larger peptide fractions with higher amounts of
emulsifying peptides. Larger (2 kDa) and negatively
charged peptides are presumably absorbed at the fat
globules interface where they can generate electrostatic
repulsions which prevent flocculation of the fat globules
(Gauthier & Pouliot, 2003). Also, associations or hydro-
phobic interactions can be formed between proteins and
peptides, leading to complex formation with different
interfacial properties. Interactions between intact milk
proteins (i.e. b-Lg) and milk protein-derived peptides have
been studied by Noiseux, Gauthier, & Turgeon (2002). At
pH 3.0, no peptide/b-Lg interactions were found, possibly
because electrostatic repulsion prevented interaction be-
tween the positively charged b-Lg and the peptides studied.
At pH 6.8 and 8.0, and depending on the ionic strength and
the temperature, b-Lg f130-135, b-Lg f69-83, and b-Lg
f146-149 interacted with intact b-Lg. The hydrophobic
peptides as1-casein f23-34 and b-Lg f102-105, showing
opioid and ACE-inhibitory activity, may also bind to the
inner cavity of b-Lg. This makes the intact b-Lg protein a
potential carrier for bioactive peptides, which may be of
importance for their bioavailability and bioactivity in the
human body.
The data given above illustrate that milk-derived
peptides, as a function of their amino acid sequence and
their length, may have interfacial properties and may as
well interact with other ingredients present in foods and
food products. These aspects should be evaluated carefully
and if necessary taken into account when formulating
foods containing these bioactive peptides.
5. Conclusions
As a result of an extensive research carried out during
the past 20 years, a wide range of peptide sequences derived
from milk proteins capable of inhibiting ACE in vitro, and
thus potentially useful in the prevention and/or treatment
of hypertension, are known. However, quite frequently
the results of tests in SHR have revealed discrepancies
between the in vitro ACE inhibitory properties and the in
vivo action, mainly because the in vitro methods do not
take into consideration the physiological transformations
that determine the bioavailability of the peptides. In
addition, there might be other mechanisms of antihyper-
tensive action different from ACE inhibition, such as a
direct relaxation of vascular muscles and/or opioid or
antioxidant activities. This stresses that more research is
needed in order to demonstrate scientifically the physiolo-
gical basis for the antihypertensive effects of milk-derived
peptides.
Dairy products and other foods containing antihyperten-
sive and ACE-inhibitory peptides can be produced by
enrichment with in vitro-produced milk protein hydrolysates.
Both casein and whey protein-based hydrolysates containing
a high ACE inhibitory activity have been produced, a
number of patents have been granted in this area, and such
 ARTICLE IN PRESS
R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
hydrolysates are now commercially available from major
dairy companies. Further progress in this area might be
obtained by the use of in silico calculations, and technolo-
gical developments in large scale continuous production and
isolation of peptides. Alternatively, the active peptides
encrypted in milk proteins can be liberated during fermenta-
tion of milk by the action of the proteolytic system of the
fermenting organism. Many fermented milk and cheese
products containing ACE-inhibitory peptides have been
manufactured with proteolytic LAB, and a number of milk
products, mainly containing the tripeptides IPP and VPP,
whose antihypertensive effects have been established in
humans, are commercially available. Lb. helveticus is often
preferred for this purpose, although other LAB have shown
good performance. Further progress in this area may come
from elucidation of the specificity of microbial proteolytic
systems in the integrated environments prevailing in dairy
products.
Studies on the interactions of ACE-inhibitory and
antihypertensive peptides with other food components
during processing and preparation and effects of these
interactions on their bioactivity are scarce. In view of the
presence of reactive amino acids in the active C-terminal
zone (e.g., Lys, Arg) of several ACE-inhibitory and
antihypertensive peptides, it cannot be excluded that the
effects of processing and preparation described above may
influence the final activity of these peptides in real food
systems. However, the final extent of amino acid modifica-
tion and changes in bioactivity will depend largely on the
composition of the food matrix in which the peptide is
present, on the processing and preparation conditions, and
finally also on the peptide structure itself. More research is
needed to clarify these issues.
Acknowledgements
The authors thank Mette S. Nielsen and Jes C. Knudsen,
from the Department of Food Science, the Royal
Veterinary and Agricultural University, Denmark, and
Bruno De Meulenaer and Sarah De Groot, from the
Department of Food Safety and Food Quality, Faculty of
Bio-Science Engineering, Ghent University, Belgium, for
their help with the manuscript.
References
Abubakar, A., Saito, T., Kitazawa, H., Kawai, Y., & Itoh, T. (1998).
Structural analysis of new antihypertensive peptides derived from
cheese whey protein by proteinase K digestion. Journal of Dairy
Science, 81, 3131–3138.
Ardö, Y. (2001). Cheese ripening. General mechanisms and specific cheese
varieties. Bulletin of the International Dairy Federation, 369, 7–12.
Awaisheh, S. S., Haddadin, M. S. Y., & Robinson, R. K. (2005).
Incorporation of selected nutraceuticals and probiotic bacteria into a
fermented milk. International Dairy Journal, 15, 1184–1190.
Belitz, H.-D., & Grosch, W. (1999). Food chemistry (2nd Ed). Berlin,
Germany: Springer pp. 257–267.
1289
Berger, J., & Possompes, B. (1987). Effects of severely alkali-treated casein
on in vitro and in vivo digestibility and selected intestinal enzyme-
activities. Nutrition Reports International, 35, 69–78.
Bouhallab, S., Mollé, D., & Léonil, J. (1993). Continuous hydrolysis of
b-casein in a membrane reactor: Preparation of a bioactive peptide.
Biotechnology Letters, 15, 697–702.
Brew, K. (2003). Structure of human ACE gives new insights into inhibitor
binding and design. Trends in Pharmacological Sciences, 24, 391–394.
Broersen, K., Voragen, A. G. J., Hamer, R. J., & de Jongh, H. H. J.
(2004). Glycoforms of b-lactoglobulin with improved thermostability
and preserved structural packaging. Biotechnology and Bioengineering,
86, 78–87.
Coker, C. J., Crawford, R. A., Johnston, K. A., Singh, H., & Creamer,
L. K. (2005). Towards the classification of cheese varieties and
maturity on the basis of statistical analysis of proteolytic data—
A review. International Dairy Journal, 15, 631–643.
Crow, V. L., Martley, F. G., Coolbear, T., & Roundhill, S. J. (1995). The
influence of phage-assisted lysis of Lactococcus lactis subsp. lactis M18
on cheddar cheese ripening. International Dairy Journal, 5, 451–472.
Curtis, J. M., Dennis, D., Waddell, D. S., MacGillivray, T., & Ewart, H.
S. (2002). Determination of angiotensin-converting enzyme inhibitory
peptide Leu–Lys–Pro–Asn–Met (LKPNM) in Bonito muscle hydro-
lysates by LC-MS/MS. Journal of Agricultural and Food Chemistry, 50,
3919–3925.
Cushman, D. W., & Cheung, H. S. (1971). Spectrophotometric assay and
properties of the angiotensin converting enzyme of rabbit lung.
Biochemical Pharmacology, 20, 1637–1648.
Damodaran, S. (1995). Amino acids, peptides, and proteins. In O. R.
Fennema (Ed.), Food chemistry (3rd ed, pp. 321–430). New York,
USA: Marcel Dekker, Inc.
Desai, P., Coutinho, E., & Srivastava, S. (2002). Conformational diversity
of T-kinin in DMSO, water and HFA. European Journal of Medicinal
Chemistry, 37, 135–146.
Dziuba, J., Iwaniak, A., & Minkiewicz, P. (2003). Computer-aided
characteristics of proteins as potential precursors of bioactive peptides.
Polimery, 48, 50–53.
FitzGerald, R. J., & Meisel, H. (1999). Lactokinins: Whey protein-derived
ACE inhibitory peptides. Nahrung, 43, 165–167.
FitzGerald, R. J., & Meisel, H. (2000). Milk protein derived inhibitors of
angiotensin-I-converting enzyme. British Journal of Nutrition, 84, S33–S37.
FitzGerald, R. J., Murray, B. A., & Walsh, G. J. (2004). Hypotensive
peptides from milk proteins. Journal of Nutrition, 134, 980S–988S.
Friedman, M. (1999a). Chemistry, nutrition and microbiology of D-amino
acids. Journal of Agricultural and Food Chemistry, 47, 3457–3479.
Friedman, M. (1999b). Chemistry, biochemistry, nutrition, and micro-
biology of lysinoalanine, lanthionine, and histidinoalanine in food and
other proteins. Journal of Agricultural and Food Chemistry, 47,
1295–1319.
Fuglsang, A., Rattray, F. P., Nilsson, D., & Nyborg, C. N. (2003a).
Characterization of new milk-derived inhibitors of angiotensin
converting enzyme in vitro and in vivo. Journal of Enzyme Inhibition
Medicinal Chemistry, 18, 407–412.
Fuglsang, A., Rattray, F. P., Nilsson, D., & Nyborg, C. N. (2003b). Lactic
acid bacteria: Inhibition of angiotensin converting enzyme in vitro and
in vivo. Antonie van Leeuwenhoek, 83, 27–34.
Fujita, H., Suganuma, H., Usui, H., Kurahashi, K., Nakagiri, R., Sasaki,
R., et al. (1996). Vasorelaxation by casomokinin L, a derivative of
b-casomorphin and casoxin D, is mediated by NK1 receptor. Peptides,
17, 635–639.
Fujita, H., & Yoshikawa, M. (1999). LKPNM: A prodrug-type ACE-
inhibitory peptide derived from fish protein. Immunopharmacology, 44,
123–127.
Gatti, M., Fornasari, M. E., Lazzi, C., Muncchetti, G., & Neviani,
E. (2004). Peptidase activity in various species of dairy thermophilic
lactobacilli. Journal of Applied Microbiology, 96, 223–229.
Gauthier, S. F., & Pouliot, Y. (2003). Functional and biological properties
of peptides obtained by enzymatic hydrolysis of whey proteins. Journal
of Dairy Science, 86, E78–E87.
 ARTICLE IN PRESS
1290 R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
Gilani, G. S., & Sepehr, E. (2003). Protein digestibility and quality in
products containing antinutritional factors are adversely affected by
old age in rats. Journal of Nutrition, 133, 220–225.
Gobetti, M., Ferranti, P., Smacchi, E., Goffredi, F., & Addeo, F. (2000).
Production of angiotensin-I-converting-enzyme-inhibitory peptides in
fermented milks started by Lactobacillus delbrueckii subsp. bulgaricus
SS1 and Lactococcus lactis subsp. cremoris FT4. Applied and
Environmental Microbiology, 66, 3898–3904.
Gobbetti, M., Stepaniak, L., De Angelis, M., Corsetti, A., & Cagno, R. D.
(2002). Latent bioactive peptides in milk proteins: proteolytic
activation and significance in dairy processing. Critical Reviews in
Food Science and Nutrition, 42, 223–239.
Gobetti, M., Minervini, F., & Grizzello, C. (2004). Angiotensin
I-converting-enzyme-inhibitory and antimicrobial bioactive peptides.
International Journal of Dairy Technology, 57, 173–188.
Gómez-Ruiz, J. A., Ramos, M., & Recio, I. (2004a). Angiotensin
converting enzyme-inhibitory activity of peptides isolated from
Manchego cheese. Stability under simulated gastrointestinal digestion.
International Dairy Journal, 14, 1075–1080.
Gómez-Ruiz, J. A., Ramos, M., & Recio, I. (2004b). Identification and
formation of angiotensin-converting enzyme-inhibitory peptides in
Manchego cheese by high-performance liquid chromatography-tan-
dem mass spectrometry. Journal of Chromatography A, 1054, 269–277.
Gómez-Ruiz, J. A., Recio, I., & Belloque, J. (2004). ACE-inhibitory
activity and structural properties of peptide Asp-Lys–Ile–His–Pro [b-
CN f(47–51)]. Study of the peptide forms synthesized by different
methods. Journal of Agricultural and Food Chemistry, 52, 6315–6319.
Hata, Y., Yamamoto, M., Ohni, M., Nakajima, K., Nakamura, Y., &
Takano, T. (1996). A placebo-controlled study of the effect of sour
milk on blood pressure in hypertensive subjects. American Journal of
Clinical Nutrition, 64, 767–771.
Hernández-Ledesma, B., Recio, I., Ramos, M., & Amigo, L. (2002).
Preparation of ovine and caprine b-lactoglobulin hydrolysates with
ACE-inhibitory activity. Identification of active peptides from caprine
b-lactoglobulin hydrolysed with thermolysin. International Dairy
Journal, 12, 805–812.
Hernández-Ledesma, B., Amigo, L., Ramos, M., & Recio, I. (2004a).
Angiotensin converting enzyme inhibitory activity in commercial
fermented products. Formation of peptides under simulated gastro-
intestinal digestion. Journal of Agricultural and Food Chemistry, 52,
1504–1510.
Hernández-Ledesma, B., Amigo, L., Ramos, M., & Recio, I. (2004b).
Release of angiotensin converting enzyme-inhibitory peptides by
simulated gastrointestinal digestion of infant formulas. International
Dairy Journal, 14, 889–898.
Hernández-Ledesma, B., Dávalos, A., Bartolomé, B., & Amigo, L. (2005).
Preparation of enzymatic hydrolysates from a-lactalbumin and
b-lactoglobulin. Identification of active peptides by HPLC-MS/MS.
Journal of Agricultural and Food Chemistry, 53, 588–593.
Hernández-Ledesma, B., Miralles, B., Amigo, L., Ramos, M., & Recio,
I. (2005). Identification of antioxidant and ACE-inhibitory peptides in
fermented milk. Journal of the Science of Food and Agriculture, 85,
1041–1048.
Ijäs, H., Collin, M., Finckenberg, P., Pihlanto-Leppälä, A., Korhonen, H.,
Korpela, R., et al. (2004). Antihypertensive opioid-like milk peptide a-
lactorphin: Lack of effect on behavioural test in mice. International
Dairy Journal, 14, 201–205.
Jauhiainen, T., Collin, M., Narva, M., Cheng, Z. J., Poussa, T.,
Vapaatalo, H., et al. (2005). Effect of long-term intake of milk
peptides and minerals on blood pressure and arterial function in
spontaneously hypertensive rats. Milchwissenschaft, 60, 358–363.
Juillard, V., Laan, H., Kunji, E. R. S., Jeronimus-Stratingh, C. M., Bruins, A.
P., & Konings, W. N. (1995). The extracellular PI-type proteinase of
Lactococcus lactis hydrolyses b-casein into more than one hundred different
oligopeptides. Journal of Bacteriology, 177, 3472–3478.
Karaki, H., Doi, K., Sugano, S., Uchiwa, H., Sugai, R., Murakami, U.,
et al. (1990). Antihypertensive effect of tryptic hydrolysate of milk
casein in spontaneously hypertensive rats. Comparative Biochemistry
and Physiology, 96(part C), 367–371.
Khalid., N. M., Soda, M. E., & Marth, E. H. (1991). Peptide hydrolases of
Lactobacillus helveticus and Lactobacillus delbrueckii ssp. bulgaricus.
Journal of Dairy Science, 74, 29–45.
Knudsen, J. C., Otte, J., Olsen, K., & Skibsted, L. H. (2002). Effect of high
hydrostatic pressure on the conformation of b-lactoglobulin A as
assessed by proteolytic peptide profiling. International Dairy Journal,
12, 791–803.
Korhonen, H., Pihlanto-Leppälä, A., Rantamäki, P., & Tupasela,
T. (1998). Impact of processing on bioactive proteins and peptides.
Trends in Food Science and Technology, 9, 307–319.
Kunji, E. R. S., Mierau, I., Hagting, A., Poolman, B., & Konings, W. N.
(1996). The proteolytic systems of lactic acid bacteria. Antonie van
Leeuwenhoek, 70, 187–221.
Kuono, K., Hirano, S., Kuboki, H., Kasai, M., & Hatae, K. (2005).
Effects of dried bonito (Katsuobushi) and captopril, an angiotensin
I-converting enzyme inhibitor, on rat isolated aorta: a possible
mechanism of antihypertensive action. Bioscience Biotechnology and
Biochemistry, 69, 911–915.
Lajoie, N., Gauthier, S. F., & Pouliot, Y. (2001). Improved storage
stability of model infant formula by whey peptide fractions. Journal of
Agricultural and Food Chemistry, 49, 1999–2007.
Lapointe, J.-F., Mollé, D., Gauthier, S. F., & Pouliot, Y. (2004). Effect of
calcium on thermolysin hydrolysis of b-casein tryptic peptides.
International Dairy Journal, 14, 185–193.
Leclerc, P.-L., Gauthier, S. F., Bachelard, H., Santure, M., & Roy,
D. (2002). Antihypertensive activity of casein-enriched milk fermented
by Lactobacillus helveticus. International Dairy Journal, 12, 995–1004.
Léonil, J., Mollé, D., Fauquant, J., Maubois, J. L., Pearce, R. J., &
Bouhallab, S. (1997). Characterization by ionization mass spectro-
metry of lactosyl b-lactoglobulin conjugates formed during heat
treatment of milk and whey and identification of one lactose-binding
site. Journal of Dairy Science, 80, 2270–2281.
Li, G.-H., Liu, H., Shi, Y.-H., & Le, G.-W. (2005). Direct spectro-
photometric measurement of angiotensin I-converting enzyme
inhibitory activity for screening bioactive peptides. Journal of
Pharmaceutical and Biomedical Analysis, 37, 219–224.
Lucas, A., Sodini, I., Monnet, C., Jolivet, P., & Corrieu, G. (2004).
Probiotic cell counts and acidification in fermented milks supplemen-
ted with milk protein hydrolysates. International Dairy Journal, 14,
47–53.
Lund, M., & Ardö, Y. (2004). Purification and identification of water-
soluble phosphopeptides from cheese using Fe(III) affinity chromato-
graphy and mass spectrometry. Journal of Agricultural and Food
Chemistry, 52, 6616–6622.
Lv, G. S., Huo, G. C., & Fu, X. Y. (2003). Expression of milk-derived
antihypertensive peptide in Escherichia coli. Journal of Dairy Science,
86, 1927–1931.
Maeno, M., Yamamoto, N., & Takano, T. (1996). Identification of an
antihypertensive peptide from casein hydrolysate produced by a
proteinase from Lactobacillus helveticus CP790. Journal of Dairy
Science, 79, 1316–1321.
Maes, W., Van Camp, J., Vermeirssen, V., Hemeryck, M., Ketelslegers,
J. M., Schrezenmeir, J., et al. (2004). Influence of the lactokinin
Ala–Leu–Pro–Met–His–Ile–Arg (ALPMHIR) on the release of en-
dothelin-1 by endothelial cells. Regulatory Peptides, 118, 105–109.
Manso, M. A., & López-Fandiño, R. (2003). Angiotensin I converting
enzyme-inhibitory activity of bovine, ovine and caprine k-casein
macropeptides and their tryptic hydrolysates. Journal of Food
Protection, 66, 1686–1692.
Martin-Orue, C., Henry, G., & Bouhallab, S. (1999). Tryptic hydrolysis of
k-caseinomacropeptide: Control of the enzymatic reaction in a
continous membrane reactor. Enzyme and Microbial Technology, 24,
173–180.
Maruyama, S., Mitachi, H., Awaya, J., Kurono, M., Tomizuka, N., &
Suzuki, H. (1987). Angiotensin I-converting enzyme inhibitory activity
 ARTICLE IN PRESS
R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
of the C-terminal hexapeptide of as1-casein. Agricultural and
Biological Chemistry, 51, 2557–2561.
Masuda, O., Nakamura, Y., & Takano, T. (1996). Antihypertensive
peptides are present in aorta after oral administration of sour milk
containing these peptides to spontaneously hypertensive rats. Journal
of Nutrition, 126, 3063–3068.
Matoba, N., Usui, H., Fujita, H., & Yoshikawa, M. (1999). A novel anti-
hypertensive peptide derived from ovalbumin induces nitric oxide-
mediated vasorelaxation in an isolated SHR mesenteric artery. FEBS
Letters, 452, 181–184.
Matsui, T., Imamura, M., Oka, H., Osajima, K., Kimoto, K.-I.,
Kawasaki, T., et al. (2004). Tissue distribution of antihypertensive
dipeptide, Val–Tyr, after its ingle oral administration to spontaneously
hypertensive rats. Journal of Peptide Science, 10, 535–545.
Matsui, T., Tamaya, K., Seki, E., Osajima, K., Matsumoto, K., &
Kawasaki, T. (2002). Absorption of Val–Tyr in vitro angiotensin
I-converting enzyme inhibitory activity into the circulating blood
system of mild hypertensive subjects. Biological and Pharmaceutical
Bulletin, 25, 1228–1230.
Matsuura, K., Mizuno, S., Nishimura, S., Gotou, T., & Yamamoto, N.
(2005). Quantitative analysis of the antihypertensive peptides
Val–Pro–Pro and Ile–Pro–Pro in casein hydrolyzate using an
Aspergillus oryzae protease: an LC-MS method. Milchwissenschaft,
60, 24–27.
Meisel, H. (1997a). Biochemical properties of regulatory peptides derived
from milk proteins. Biopolymers, 43, 119–128.
Meisel, H. (1997b). Biochemical properties of bioactive peptides derived
from milk proteins: Potential nutraceuticals for food and pharmaceu-
tical applications. Livestock Production Science, 50, 125–138.
Meisel, H. (2004). Multifunctional peptides encrypted in milk proteins.
BioFactors, 21, 55–61.
Meisel, H., Goepfert, A., & Günther, S. (1997). ACE-inhibitory activities
in milk products. Milchwissenschaft, 52, 307–311.
Mestdagh, F., De Meulenaer, B., De Clippeleer, J., Devlieghere, F., &
Huyghebaert, A. (2005). Protective influence of several packaging
materials on light-oxidation of milk. Journal of Dairy Science, 88,
499–510.
Miguel, M., Muguerza, B., Sánchez, E., Delgado, M. A., Recio, I.,
Ramos, M., et al. (2005). Long-term blood pressure-lowering effect of
milk fermented by Enterococcus faecalis CECT 5728 in spontaneously
hypertensive rats. British Journal of Nutrition, 93, 1–9.
Mizuno, S., Matsuura, K., Gotou, T., Nishimura, S., Kajimoto, O.,
Yabune, M., et al. (2005). Antihypertensive effect of casein hydro
lysate in a placebo-controlled study in subjects with high-normal
blood pressure and mild hypertension. British Journal of Nutrition, 94,
84–91.
Mizuno, S., Nishimura, S., Matsuura, K., Gotou, T., & Yamamoto,
N. (2004). Release of short and proline-rich antihypertensive peptides
from casein hydrolysate with an Aspergillus oryzae protease. Journal of
Dairy Science, 87, 3183–3188.
Mizushima, S., Ohshige, K., Watanabe, J., Kimura, M., Kadowaki, T.,
Nakamura, Y., et al. (2004). Randomized controlled trial of sour milk
on blood pressure in borderline hypertensive men. American Journal of
Hypertension, 17, 701–706.
Mollé, D., Morgan, F., Bouhallab, S., & Léonil, J. (1998). Selective
detection of lactosylated peptides in hydrolysates by liquid chromato-
graphy/electrospray tandem mass spectrometry. Analytical Biochem-
istry, 259, 152–161.
Moutevelis-Minakakis, P., Gianni, M., Stougiannou, H., Zoumpoulakis,
P., Zoga, A., Vlahakos, A. D., et al. (2003). Design and synthesis of
novel antihypertensive drugs. Bioorganic & Medicinal Chemistry
Letters, 13, 1737–1740.
Muguerza, B., Ramos, M., Sánchez, E., Manso, M. A., Miguel, M.,
Aleixandre, A., et al. (2006). Antihypertensive activity of milks
fermented by Enterococcus faecalis strains isolated from raw milk.
International Dairy Journal, 16, 61–69.
Mullally, M. M., Meisel, H., & FitzGerald, R. J. (1997a). Identification of
a novel angiotensin-I-converting enzyme inhibitory peptide corre-
1291
sponding to a tryptic fragment of bovine b-lactoglobulin. FEBS
Letters, 402, 99–101.
Mullally, M. M., Meisel, H., & Fitzgerald, R. J. (1997b). Angiotensin-I-
converting enzyme inhibitory activities of gastric and pancreatic
proteinase digests of whey proteins. International Dairy Journal, 7,
299–303.
Murakami, M., Tonouchi, H., Takahashi, R., Kitazawa, H., Kawai, Y.,
Negishi, H., et al. (2004). Structural analysis of a new anti-hypertensive
peptide (b-lactosin B) isolated from a commercial whey product.
Journal of Dairy Science, 87, 1967–1974.
Murray, B. A., Walsh, D. J., & FitzGerald, R. J. (2004). Modification of
the furanacryloyl-L-phenylalanylglycylglycine assay for determination
of angiotensin-I-converting enzyme inhibitory activity. Journal of
Biochemical and Biophysical Methods, 59, 127–137.
Nakamura, Y., Masuda, O., & Takano, T. (1996). Decrease of tissue
angiotensin I-converting enzyme activity upon feeding sour milk in
spontaneously hypertensive rats. Bioscience, Biotechechnology and
Biochemistry, 60, 488–489.
Nakamura, Y., Yamamoto, N., Sakai, K., Okubo, A., Yamazaki, S., &
Takano, T. (1995). Purification and characterization of angiotensin-I-
converting enzyme inhibitors from a sour milk. Journal of Dairy
Science, 78, 777–783.
Nakamura, Y., Yamamoto, N., Sakai, K., & Takano, T. (1995).
Antihypertensive effect of sour milk and peptides isolated from it a
that are inhibitors to angiotensin-I-converting enzyme. Journal of
Dairy Science, 78, 1253–1257.
Narva, M., Halleen, J., Vaanamen, K., & Korpela, R. (2004). Effects of
Lactobacillus helveticus fermented milk on bone cells in vitro. Life
Sciences, 75, 1727–1734.
Natesh, R., Schwager, S. L. U., Sturrock, E. D., & Acharaya, K. R.
(2003). Crystal structure of the human angiotensin-converting
enzyme–lisinopril complex. Nature, 421, 551–554.
Noiseux, I., Gauthier, S. F., & Turgeon, S. L. (2002). Interactions between
bovine b-lactoglobulin and peptides under different physico
chemical conditions. Journal of Agricultural and Food Chemistry, 50,
1587–1592.
Nurminen, M.-L., Korpela, R., & Vapaatalo, H. (1998). Dietary factors in
the pathogenesis and treatment of hypertension. Annals of Medicine,
30, 143–150.
Nurminen, M.-L., Sipola, M., Kaarto, H., Pihlanto-Leppälä, A., Piilola,
K., Korpela, R., et al. (2000). a-lactorphin lowers blood pressure
measured by radiotelementry in normotensive and spontaneously
hypertensive rats. Life Sciences, 66, 1535–1543.
Pao, Y.-L., Wormarld, M. R., Raymond, A. D., & Lellouch, A. C. (1996).
Effect of serine O-glycosylation on cis-trans proline isomerisation.
Biochemical and Biophysical Research Communications, 219,
157–162.
Pihlanto-Leppälä, A. (2001). Bioactive peptides derived from bovine whey
proteins: Opioid and ACE-inhibitory peptides. Trends in Food Science
and Technology, 11, 347–356.
Pihlanto-Leppälä, A., Koskinen, P., Piilola, K., Tupasela, T., &
Korhonen, H. (2000). Angiotensin I-converting enzyme inhibitory
properties of whey protein digests: Concentration and characterization
of active peptides. Journal of Dairy Research, 67, 53–64.
Pihlanto-Leppälä, A., Rokka, T., & Korhonen, H. (1998). Angiotensin I
converting enzyme inhibitory peptides derived from bovine milk
proteins. International Dairy Journal, 8, 325–331.
Pins, J. J., & Keenan, J. M. (2003). The antihypertensive effects of a
hydrolyzed whey protein isolate supplement (Biozate 1): A pilot study.
FASEB Journal, 17(Suppl.), A1110.
Pripp, A. H., Isaksson, T., Stepaniak, L., & Sorhaug, T. (2004).
Quantitative structure-activity relationship modelling of ACE-inhibi-
tory peptides derived from milk proteins. European Food Research and
Technology, 219, 579–583.
Pripp, H. A. (2005). Initial proteolysis of milk proteins and its effect
on formation of ACE-inhibitory peptides during gastrointestinal
proteolysis: A bioinformatic, in silico, approach. European Food
Research and Technology, 221, 712–716.
 ARTICLE IN PRESS
1292 R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
Pritchard, G. G., & Coolbear, T. (1993). The physiology and biochemistry
of the proteolytic system in lactic acid bacteria. FEMS Microbiology
Reviews, 12, 179–206.
Quirós, A., Ramos, M., Muguerza, B., Delgado, M. A., Miguel, M.,
Aleixandre, A., et al. (2006). Identification of novel antihypertensive
peptides in milk fermented with Enterococus faecalis. International
Dairy Journal, in press, doi:10.1016/j.idairyj.2005.12.011.
Rérat, A., Calmes, R., Vaissade, P., & Finot, P.-A. (2002). Nutritional and
metabolic consequences of the early Maillard reaction of heat treated
milk in pig. European Journal of Nutrition, 41, 1–11.
Rickert, K. W., & Imperiali, B. (1995). Analysis of the conserved
glycosylation site in the nicotinic acetylcholine-receptor-potential roles
in complex assembly. Chemistry & Biology, 2, 751–775.
Righetti, P. G., Nembri, F., Bossi, A., & Mortarino, M. (1997). Continous
enzymatic hydrolysis of b-casein and isoelectric collection of some of
the biologically active peptides in an electric field. Biotechnology
Progress, 13, 258–264.
Rival, S. G., Boeriu, C. G., & Wichers, H. J. (2001). Caseins and casein
hydrolyzates. 2. Antioxidant properties and relevance to lipoxygenase
inhibition. Journal of Agricultural and Food Chemisty, 49, 295–302.
Robert, M.-C., Razaname, A., Mutter, M., & Juillerat, M. A. (2004).
Identification of angiotensin-I-converting enzyme inhibitory peptides
derived from sodium caseinate hydrolysates produced by Lactobacillus
helveticus NCC 2765. Journal of Agricultural and Food Chemistry, 52,
6923–6931.
Rokka, T., Syväoja, E.-L., Tuominen, J., & Korhonen, H. (1997). Release
of bioactive peptides by enzymatic proteolysis of Lactobacillus GG
fermented UHT milk. Milchwissenschaft, 52, 675–678.
Roufik, S., Gauthier, S. F., & Turgeon, S. L. (2006). In vitro digestibility
of bioactive peptides derived from bovine b-lactoglobulin. Interna-
tional Dairy Journal, 16, 294–302.
Ryhänen, E.-L., Pihlanto-Leppälä, A., & Pahkala, E. (2001). A new type
of ripened, low-fat cheese with bioactive properties. International Dairy
Journal, 11, 441–447.
Saito, T., Abubakar, A., Itoh, T., Arai, I., & Aimar, M. V. (1997).
Development of a new type of fermented cheese whey beverage with
inhibitory effects against angiotensin-converting enzyme. Tohoku
Journal of Agricultural Research, 48, 15–23.
Saito, T., Nakamura, T., Kitazawa, H., Kawai, Y., & Itoh, T. (2000).
Isolation and structural analysis of antihypertensive peptides that exist
naturally in gouda cheese. Journal of Dairy Science, 83, 1434–1440.
Sasaki., M., Bosman, B. W., & Tan, P. S. T. (1995). Comparison of
proteolytic activities in various lactobacilli. Journal of Dairy Research,
62, 601–610.
Satake, M., Enjoh, M., Nakamura, Y., Takano, T., Kawamura, Y., Arai,
S., et al. (2002). Transepithelial transport of the bioactive tripeptide
Val–Pro–Pro in human intestinal Caco-2 cell monolayers. Bioscience,
Biotechnology and Biochemistry, 66, 378–384.
Schlothauer, R.-C., Schollum, L.M., Reid, J.R., Harvey, S.A., Carr, A., &
Fanshawe, R.L. (2002). Improved bioactive whey protein hydrolysate.
PCT/NZ01/00188(WO 02/19837 A1). New Zealand.
Seppo, L., Jauhiainen, T., Poussa, T., & Korpela, R. (2003). A fermented
milk high in bioactive peptides has a blood pressure-lowering effect in
hypertensive subjects. American Journal of Clinical Nutrition, 77, 326–330.
Seppo, L., Kerojoki, O., Suomalainen, T., & Korpela, R. (2002). The effect
of a Lactobacillus helveticus LBK-16H fermented milk on hypertension:
A pilot study on humans. Milchwissenschaft, 57, 124–127.
Shalaby, S.M., Zakora, M., & Otte, J. (2006). Performance of two
commonly used Angiotensin-converting enzyme inhibition assays
using synthetic peptide substrates. Journal of Dairy Research, in press.
Shimizu, M., Tsunogai, M., & Arai, S. (1997). Transepithelial transport of
oligopeptides in the human intestinal cell, Caco-2. Peptides, 18, 681–687.
Sipola, M., Finckenberg, P., Korpela, R., Vapaatalo, H., & Nurminen,
M.-L. (2002). Effect of long-term intake of milk products on blood
pressure in hypertensive rats. Journal of Dairy Research, 69, 103–111.
Sipola, M., Finckenberg, P., Vapaatalo, H., Pihlanto-Leppälä, A.,
Korhonen, H., Korpela, R., et al. (2002). a-lactorphin and
b-lactorphin improve arterial function in spontaneously hypertensive
rats. Life Sciences, 71, 1245–1253.
Smacchi, E., & Gobbetti, M. (1998). Peptides from several Italian cheeses
inhibitory to proteolytic enzymes of lactic acid bacteria, Pseudomonas
fluorescens ATCC 948 and to the angiotensin I-converting enzyme.
Enzyme and Microbial Technology, 22, 687–694.
Steijns, J. (1996). Dietary proteins as the source of new healthy promoting
bio-active peptides with special attention to glutamine peptide. Food
Technolgy Europe, 3, 80–84.
Sugai, R. (1998). ACE inhibitors and functionl foods. Bulletin of the IDF,
336, 17–20.
Tauzin, J., Miclo, L., & Gaillard, J.-L. (2002). Angiotensin-I-converting
enzyme inhibitory peptides from tryptic hydrolysate of bovine
as2-casein. FEBS Letters, 531, 369–374.
Touyz, R. M. (2004). Reactive oxygen species, vascular oxidative stress
and redox signaling in hypertension: what is the clinical significance?
Hypertension, 44, 248–252.
Townsend, R. R., McFadden, C. B., Ford, V., & Cadée, J. A. (2004).
A randomized, double-blind, placebo-controlled trial of casein protein
hydrolysaet (C12 peptide) in human essential hypertension. American
Journal of Hypertension, 17, 1056–1058.
Tuomilehto, J., Lindstrom, J., Hyyrynen, J., Korpela, R., Karhunen,
M. L., Mikkola, L., et al. (2004). Effect of ingesting sour milk
fermented using Lactobacillus helveticus bacteria producing tripeptides
on blood pressure in subjects with milk hypertension. Journal of
Human Hypertension, 18, 795–802.
Turgeon, S. L., Gauthier, S. F., Mollé, D., & Léonil, J. (1992). Interfacial
properties of tryptic peptides of b-Lactoglobulin. Journal of Agricul-
tural and Food Chemistry, 40, 669–675.
Valence, F., Deutsch, S. M., Richoux, R., Gagnaire, V., & Lortal,
S. (2000). Autolysis and related proteolysis in Swiss cheese for two
Lactobacillus helveticus strains. Journal of Dairy Research, 67,
261–271.
van Elswijk, D. A., Diefenbach, O., van der Berg, S., Irth, H., Tjaden, U. R.,
& Van Der Greef, J. (2003). Rapid detection and identification of
angiotensin-converting enzyme inhibitors by on-line liquid chromato-
graphy-biochemical detection, coupled to electrospray mass spectro-
metry. Journal of Chromatography A, 1020, 45–58.
Vercruysse, L., Smagghe, G., Herregods, G., & Van Camp, J. (2005). ACE
inhibitory activity in enzymatic hydrolysates of insect protein. Journal
of Agricultural and Food Chemistry, 53, 5207–5211.
Vermeirssen, V., Deplancke, B., Tappenden, K. A., Van Camp, J.,
Gaskins, H. R., & Verstraete, W. (2002). Intestinal transport of the
lactokinin Ala–Leu–Pro–Met–His–Ile–Arg through a Caco-2 Bbe
monolayer. Journal of Peptide Science, 8, 95–100.
Vermeirssen, V., Van Camp, J., & Verstraete, W. (2002). Optimisation and
validation of an angiotensin-converting enzyme inhibition assay for
the screening of bioactive peptides. Journal of Biochemical and
Biophysical Methods, 51, 75–87.
Vermeirssen, V., Van Camp, J., Decroos, K., Van Wijmelbeke, V., &
Verstraete, W. (2003). The impact of fermentation and in vitro
digestion on the formation of angiotensin-I-converting enzyme
inhibitory activity from pea and whey protein. Journal of Dairy
Science, 86, 429–438.
Vermeirssen, V., Van Camp, J., Devos, L., & Verstraete, W. (2003). Release
of Angiotensin I converting enzyme (ACE) inhibitory activity during in
vitro gastrointestinal digestion: from batch experiment to semicontin-
uous model. Journal of Agricultural and Food Chemistry, 51, 5680–5687.
Vermeirssen, V., Van Camp, J., & Verstraete, W. (2004). Bioavailability of
angiotensin I converting enzyme inhibitory peptides. British Journal of
Nutrition, 92, 357–366.
Vermeirssen, V., van der Bent, A., Van Camp, J., van Amerongen, A., &
Verstraete, W. (2004). A quantitative in silico analysis calculates the
angiotensin I converting enzyme (ACE) inhibitory activity in pea and
whey protein digests. Biochimie, 86, 213–239.
Walsh, D. J., Bernard, H., Murray, B. A., MacDonald, J., Pentzien,
A.-K., Wright, G. A., et al. (2004). In vitro generation and stability of
 ARTICLE IN PRESS
R. López-Fandiño et al. / International Dairy Journal 16 (2006) 1277–1293
the lactokinin b-lactoglobulin fragment (142–148). Journal of Dairy
Science, 87, 3845–3857.
Yamada, Y., Matoba, N., Usui, H., Onishi, K., & Yoshikawa, M. (2002).
Design of a highly potent anti-hypertensive peptide based on
ovokinin(2-7). Bioscience, Biotechnology and Biochemistry, 66,
1213–1217.
Yamamoto, N., Akino, A., & Takano, T. (1994). Antihypertensive effect of
the peptides derived from casein by an extracellular proteinase from
1293
Lactocobacillus helveticus CP790. Journal of Dairy Science, 77,
917–922.
Yamamoto, N., Maeno, M., & Takano, T. (1999). Purification and
characterization of an antihypertensive peptide from a yogurt-like
product fermented by Lactobacillus helveticus CPN4. Journal of Dairy
Science, 82, 1388–1393.
Yamamoto, N., & Takano, T. (1999). Antihypertensive peptides derived
from milk proteins. Nahrung, 43, 159–164.