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International Dairy Journal 16 (2006) 1277–1293

Review

Physiological, chemical and technological aspects of

milk-protein-derived peptides with antihypertensive and

ACE-inhibitory activity

R. Lo ´ pez-Fandin ˜ oa,Ã, J. Otteb, J. van Campc

aInstitute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, SpainbDepartment of Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark

cDepartment 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12802.5. 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

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doi:10.1016/j.idairyj.2006.06.004

ÃCorresponding author. Tel.: +3491 5622900; fax. +3491 5644853.

E-mail address: [email protected] (R. Lo ´ pez-Fandino).

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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

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 moderatelyhypertensive 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, angiotensinII 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

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

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 snakevenom. 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 classicalapproaches 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

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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 differentsubstrates 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 9mM,

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; Quiro ´ s 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

ACE-inhibitory activity (FitzGerald & Meisel, 2000;

FitzGerald et al., 2004; Gobbetti, Stepaniak, De Angelis,

Corsetti, & Cagno, 2002; Meisel, 1997a; Meisel, 2004;

Pihlanto-Leppa ¨ la ¨ , 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. Go ´ mez-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 113.18mM). 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 577.92mM) compared which the

unique conformer.

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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-inhibitorypeptides 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 (Go ´ mez-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 (IC5041000mM), was hydrolysed by

pancreatin to the potent ACE inhibitor KVLPVP

(IC50 ¼ 5mM), 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 42.6mM), 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 928mM; 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-Leppa ¨ la ¨ , 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-peptidecarrier, 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 14weeks. 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.

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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 endothelialpeptide 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 andsedation (Ija ¨ s 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 720mM; 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

whey proteins hydrolysed with different enzymes (Herna ´ n-

dez-Ledesma, Da ´ valos, Bartolome ´ , & 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, & Cadee, 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 1.8mg 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

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(Valio Ltd, Finland or Kaiku Vitabrands, Spain) exerted

significant antihypertensive effects in humans at daily doses

of 150mL (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 (À28.3 and À32.1 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

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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

pressure

Reference

Calpis Double-blind, placebo controlled trial in 30

subjects with mild hypertension, some taking

antihypertensive drugs (n ¼ 15)

95mLday –1 À9.4 mm Hg (4 weeks) Hata et al. (1996)

(sour milk) (1.04 mg IPP +1.42 mg

VPP)

À14.1mm Hg (8 weeks)

Calpis Double-blind, randomized, placebo-

controlled trial in 46 hypertensive men

(n ¼ 23)

160gdayÀ1À4.3 mm Hg (2 weeks) Mizushima et al.

(2004)(sour milk) (1.15 mg IPP +1.98 mg

VPP)

À5.2 mm Hg (4 weeks)

A ´ meal Peptide Single-blind, placebo-controlled trial in 131

subjects with high-normal blood pressure

and mild hypertension (n ¼ 32–33)

1.8mg IPP+VPP À6.3 mm Hg (6 weeks) Mizuno et al. (2005)

(casein hydrolysate) 2.5 mg IPP+VPP À6.7 mm Hg (6 weeks)

3.6mg IPP+VPP À10.1mm Hg (6 weeks)

Evolus/Kaiku Vitabrand Double-blind, randomized, placebo-

controlled trial in 17 subjects with mild

hypertension (n ¼ 10)

150 mLdayÀ1À14.9mm Hg (8 weeks) Seppo et al. (2002)

(fermented milk) (2.25 mg IPP+3-3.75 mg

VPP)

Evolus/Kaiku Vitabrand Randomized, placebo-controlled trial in 39

hypertensive subjects

150 mLdayÀ1À6.7 mm Hg (21 weeks) Seppo et al. (2003)

(fermented milk) (2.25 mg IPP+3-3.75 mg

VPP)

Evolus/Kaiku Vitabrand Two study periods with a washout period inbetween in 60 subjects with mild

hypertension

150 mLday

À1À

16 mm Hg (1st studyperiod, 8–10 weeks,

n ¼ 59)

Tuomilehto et al.(2004)(fermented milk) (2.4–2.7 mg

IPP+2.4–2.7 mg VPP)

À11 mm Hg (2nd study

period, 5–7 weeks,

n ¼ 39)

C12 peptide (a bovine milk

protein hydrolysate

containing as1-casein f23-

34)7alginic acid

Placebo-controlled crossover study (7-day

cycles) in 10 hypertensive subjects—all

receiving 5 of the possible treatments

Single dose (100 or

200mg C12 with 877 or

1754 mg alginic acid)

À9 mm Hg (and—

6mmHg DBP) at 6h

compared with 2 h, at

high alginic acid dose

Townsend et al.

(2004)

Dodeca Peptide (DP) Placebo-controlled study in 18 mildly

hypertensive subjects for 4 weeks. Placebo

was water without DP

200 mgdayÀ1 in water À6 mm Hg DBP after 4

weeks. No effect in

normotensive subjects

Sugai (1998)

(a tryptic hydrolysate of

casein enriched in as1-casein

f23-34)

BioZate 1 (a WPIhydrolysate)

Placebo-controlled study in 30 borderlinehypertensive subjects for 6 weeks. Placebo

was unhydrolysed WPI

Not indicated, probably20g day –1

À11mm Hg (À7mm HgDBP) in comparison

with control.

Pins and Keenan(2003)

In addition LDL-

cholesterol was lowered

by 12%




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 bioactivepeptides. 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, & Gu ¨ nther, 1997;

Ryha ¨ nen, Pihlanto-Leppa ¨ la ¨ , & 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

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; Herna ´ ndez-

Ledesma, Amigo, Ramos, & Recio, 2004a, b; Leclerc,

Gauthier, Bachelard, Santure, & Roy, 2002; Nakamura,

Yamamoto, Sakai, Okubo, et al., 1995; Rokka, Syva ¨ oja,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 34–59mg mLÀ1) 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 theproduction 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

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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-terminalb-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 withinstrains 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 (Ardo ¨ , 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 inhibitedACE (IC50 ¼ 18mM). 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%;

Go ´ mez-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. (Ryha ¨ -

nen, Pihlanto-Leppa ¨ la ¨ , & 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 & Ardo ¨ , 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 50ngmLÀ1) 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

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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-Leppa ¨ la ¨ , Koskinen, Piilola, Tupasela, & Korho-nen, 2000; Pihlanto-Leppa ¨ la ¨ , 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; Herna ´ ndez-Ledesma,

Recio, Ramos, & Amigo, 2002; Mullally et al., 1997b;Nurminen et al., 2000; Pihlanto-Leppa ¨ la ¨ 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

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 forthe 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 mLÀ1. 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

(Go ´ mez-Ruiz et al., 2004b; Herna ´ ndez-Ledesma et al.,

2004b; Lapointe, Molle ´ , Gauthier, & Pouliot, 2004;

Maeno et al., 1996; Mullally et al., 1997b; Nakamura,

Yamamoto, Sakai, Okubo et al., 1995; Pihlanto-Lepa ¨ la ¨ et

al., 2000). With a membrane of 1 kDa, some of the active

peptides may be lost (Pihlanto-Leppa ¨ la ¨ 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.

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Pre-concentration based on hydrophobicity using a C18

reversed-phase cartridge is also common in the laboratory

(Curtis, Dennis, Waddell, MacGillivray, & Ewart, 2002;

Herna ´ ndez-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 byreversed-phase HPLC (i.e. Pihlanto-Leppa ¨ la ¨ 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 & Lo ´ pez-Fandin ˜ o, 2003). On-line cou-

pling to electrospray ionization-tandem mass spectrometry

allows simultaneous identification of the peptides (Go ´ mez-

Ruiz et al., 2004b; Herna ´ ndez-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, Molle ´ , and Le ´ onil (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 (Go ´ mez-Ruiz et al., 2004b; Pihlanto-Le ¨ppa ¨ la

et al., 1998; Steijns 1996; Yamamoto & Takano, 1999).

Using a continuous membrane reactor, the peptide bondsto 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 4–100mM) 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 bedelivered 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).

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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 strongelectron 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 astrypsin 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.

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 (Le ´ onil 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-Leppa ¨ la ¨ , Rantama ¨ ki, & 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; Re ´ rat, 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; Le ´ onil et al., 1997, Molle ´ ,

Morgan, Bouhallab, & Le ´ onil, 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




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 infoods 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 unlessappropriate 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, Molle ´ , and Le ´ onil (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




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., & Gu ¨ nther, 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., Sa ´ nchez, 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.

Molle ´ , D., Morgan, F., Bouhallab, S., & Le ´ onil, 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., Sanchez, 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-

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-hypertensivepeptide (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-Leppa ¨ la ¨ , 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-Leppa ¨ la ¨ , A. (2001). Bioactive peptides derived from bovine whey

proteins: Opioid and ACE-inhibitory peptides. Trends in Food Science

and Technology, 11, 347–356.

Pihlanto-Leppa ¨ la ¨ , A., Koskinen, P., Piilola, K., Tupasela, T., &

Korhonen, H. (2000). Angiotensin I-converting enzyme inhibitory

properties of whey protein digests: Concentration and characterizationof active peptides. Journal of Dairy Research, 67 , 53–64.

Pihlanto-Leppa ¨ la ¨ , 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




Pritchard, G. G., & Coolbear, T. (1993). The physiology and biochemistry

of the proteolytic system in lactic acid bacteria. FEMS Microbiology

Reviews, 12, 179–206.

Quiro ´ s, 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.

Re ´ rat, A., Calmes, R., Vaissade, P., & Finot, P.-A. (2002). Nutritional andmetabolic 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 Lactobacillushelveticus NCC 2765. Journal of Agricultural and Food Chemistry, 52,

6923–6931.

Rokka, T., Syva ¨ oja, 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.

Ryha ¨ nen, E.-L., Pihlanto-Leppa ¨ la ¨ , 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 fermentedmilk 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-Leppa ¨ la ¨ , 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., & Cade ´ e, 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 milkfermented 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., Molle ´ , D., & Le ´ onil, 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 enzymeinhibitory 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






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

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.

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