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International Dairy Journal 16 (2006) 1277–1293 www.elsevier.com/locate/idairyj
Physiological, chemical and technological aspects of milk-protein-derived peptides with antihypertensive and ACE-inhibitory activity
´ R. Lopez-Fandinoa,Ã, J. Otteb, J. van Campc ˜
Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain 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-proteinderived 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. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Milk-protein-derived peptides with antihypertensive effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. ACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Assays for ACE-inhibitory and antihypertensive activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Structure-activity relationship for ACE-inhibitory peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Bioavailability and physiological relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Other possible mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Milk-derived products with antihypertensive effects in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technologies for the production of milk-protein-derived antihypertensive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Manufacture of fermented dairy products with ACE-inhibitory peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Production of antihypertensive milk protein hydrolysates in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Enrichment of hydrolysates with ACE-inhibitory peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Other strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1278 1278 1278 1278 1279 1280 1280 1281 1282 1283 1284 1285 1286
ÃCorresponding author. Tel.: +34 91 5622900; fax. +34 91 5644853.
´ E-mail address: rosina@iﬁ.csic.es (R. Lopez-Fandino). ˜ 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.06.004
. . . . . . extraction-free methods have been published recently (Li et al. . . . 1286 1287 1288 1288 1288 1289 1289 1. . Direct. . . . . Inﬂuence of oxidation . . . . . . Chemistry and stability of milk-protein-derived antihypertensive peptides . . . . . . . . . . . . . . . . Another broadly used spectrophotometric method is based on the hydrolysis of a furanocryloyl tripeptide (FA–Phe–Gly–Gly. . . Assays for ACE-inhibitory and antihypertensive activity The search for ACE inhibitory activity is the most common strategy followed in the selection of antihypertensive hydrolysates and/or peptides derived from milk proteins. . . 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. . EC 3. . . . . . . . . angiotensin II receptor blockers and ACE inhibitors are normally used. . . . . . . . . . . . . 4. . . . Lo ˜o 4. . . ACE hydrolyses an inactive decapeptide. . . . . . . the kinin–kallikrein system. . . . . . calcium channel blockers. . 5. . . vasodilators.. . . . obtained by enzymatic digestion or microbial fermentation. . 2006). . Liu. . . . . alecepril and lisinopril are used extensively in the treatment of essential hypertension despite their undesirable side effects. . In order to facilitate the characterisation of ACE inhibitory peptides. . angiotensin I. . the consumption of food products containing antihypertensive peptides has shown to signiﬁcantly reduce the blood pressure of moderately hypertensive subjects. . . . . 2005). . the neutral endopeptidase system and the endothelin-converting enzyme system. . . . . . . . . ACE ACE (peptidyldipeptide hydrolase. . . . . . . ﬂuid and electrolyte balance. . ACE also takes part of the kinin–kallikrein system as it hydrolyses bradykinin. It is based on the hydrolysis of hippuryl-His-Leu (HHL) by ACE to hippuric acid (HA) and HL. . . . .1. . . . . high blood pressure. . or their analogues. . 4.2. References . . . . . . Conclusions . etc. . . . (FitzGerald et al. . . .. . . . . . . . or concentration needed to inhibit 50% of the enzyme activity. . . .2. . . . ACE is widely distributed in many tissues. . Potential role of functional properties of milk-derived bioactive peptides . . Inﬂuence of heat processing . is a key factor in the development of cardiovascular diseases such as myocardial infarction. . . . . Acknowledgements . . . . .ARTICLE IN PRESS 1278 ´pez-Fandin et al. Since then. ACE inhibitors were ﬁrst discovered in snake venom. . . . . . . . . . . angioedema. . . . . radiochemical. . . . . . . . . Van Camp. As part of the renin–angiotensin system. . . . . . 4. the establishment of a simple.15. . . as well as from other food sources. which has a vasodilator action. . . . . . . . . . . . . . . . . . . 2. . . . . . . . . which is estimated to affect one third of the Western population. . . . . . . . . . . . . . Introduction Hypertension. 2. . . . cessation of smoking and physical activity). . .4. Special attention was paid to update the information covered in recent reviews on the subject. . . . . diuretics. The purpose of this paper is to review the current literature on dairy products with antihypertensive effects. and Verstraete (2004). . . . . & Otte. . such as hypotension. . to the potent vasoconstrictor angiotensin II. changes in life-style (reduction of overweight. . . . . . . . . . which is tedious and may overestimate ACE activity if unhydrolyzed HHL is also extracted. reduced renal function. . 2005. and to the existence of mechanisms of action other than ACE inhibition. Shi. . . . . . . efforts are being put into the production of foods with antihypertensive activity. and Walsh (2004) and Vermeirssen. . . . . . . . . . cough. . . 2. . . . . This is usually expressed as the IC50. . . These substances interfere with the different interacting biochemical pathways that control blood pressure. . The metabolic pathways associated with the control of blood pressure have been reviewed recently by FitzGerald et al. . . . . . . . . 2004). . . . Shalaby. is a risk factor for cardiovascular disease and stroke. in particular FitzGerald. . . . .e. . . . . . . . . . . . . . . . . . enalapril. Zakora. . . . . . . . In view of its prevalence and importance. . . . . . . . the renin–angiotensin system. . 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. .1) is an exopeptidase that cleaves dipeptides from the C-terminal side of various oligopeptides. . . . . . FAPGG) to . . . . . with respect to the structure-activity characteristics of angiotensin-converting enzyme (ACE) inhibitory peptides. . . The spectrophotometric method of Cushman and Cheung (1971) is most commonly utilized. . . . . . . . . . The classical approaches involve the in vitro determination of the ACE inhibitory activity of milk protein hydrolysates. namely. . . . . . . . . . . . . . . . . . synthetic ACE inhibitors such as captopril. . . . . / International Dairy Journal 16 (2006) 1277–1293 R. . . . . . . increased potassium levels. . followed by the identiﬁcation of peptide structures and the chemical synthesis of potentially active peptides. ﬂuorimetric. . & Le. . . . Murray. Milk-protein-derived peptides with antihypertensive effects Hypertension. The extent of HA release from HHL is measured after it is extracted with ethyl acetate. Despite the higher doses needed in comparison with antihypertensive drugs. dietary approaches and pharmacological treatments are broadly applied to treat hypertension. . . . . . (2004). . sensitive and reliable in vitro ACE inhibition assay is desirable. HPLC and capillary electrophoresis methods to measure ACE activity. . . . . . . . In view of the role of the diet in the prevention and treatment of the disease. .3. . . . . stroke and heart failure. . . . . . . . . . . . . . There are spectrophotometric. . . . In clinical practice. . . . in order to conﬁrm their activity. . . i. . .1. These can also be used to obtain information on the inhibitory potency of different substances (Li. . . . . . . .
The long-term feeding of the fermented milk to SHR was more effective than the pure tripeptides. Vapaatalo. 2005). Saito. exerted antihypertensive properties that increased with the calcium content of the ´ fermented product (Miguel et al. The in vivo effects are tested in spontaneously hypertensive rats (SHR). In general terms. 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. Coutinho. 2. & Verstraete. e. 2000. and showed a lower ACEinhibitory activity (IC50 ¼ 577. feeding SHR for 17 weeks a fermented milk devoid of IPP and VPP. Korpela. an ACE-inhibitory peptide obtained from Manchego cheese... & Nurminen. Meisel. probably because calcium. & Takano. whereby hydrophobic amino acids. Razaname. & Srivastava. Smagghe. The hypotensive effects caused by the short-term administration to SHR of milk protein hydrolysates. In practice. Recio. Meisel. which constitute an accepted model for human essential hypertension. 2004. 2002). differences may arise among the results of different assays due to the use of different substrates or. In addition. Sipola... Isaksson. such as LHLPLP (b-casein f133-138). b). Pripp. De Angelis. Nakamura. with slightly different conformational features. the results of those tests have highlighted an important lack of correlation between the in vitro ACE inhibitory activity and the in vivo action. ACE activity levels need to be carefully controlled to obtain comparable and reproducible values (Murray. a relationship was found between the ACE-inhibitory activity and some of the peptide characteristics (hydrophobicity and a positively charged amino acid at the C-terminal position). bradykinin. some general features have been found (FitzGerald et al. & FitzGerald. Furthermore. Stepaniak.. 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. with a unique conformer containing trans-Pro. Walsh. 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. Pihlanto-Leppala. 2004). & Cagno. are more active if present at each of the three C-terminal positions. Lo ˜o 1279 FAP and the dipeptide GG (Vermeirssen. 1995. / International Dairy Journal 16 (2006) 1277–1293 R. is open and sensitive for cleavage by ACE. gave a signiﬁcant ACE-inhibitory activity (IC50 ¼ 113. in order to inhibit ACE activity more completely (Gobbetti et al. 1994). 2004. & Juillerat. two with transPro and one with cis-Pro. Meisel.g. e. Gomez-Ruiz. 2000. as it does not take into consideration the physiological transformations that determine the bioavailability of the peptides and because there might be other mechanisms of action than ACE inhibition. Nakamura. Finckenberg.92 mM) compared which the unique conformer.g. Sakai. 1997a).3. 2005. Mutter. 2002). No relationship was found between the N-terminal structure and the ACE-inhibitory activity. 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. One preparation. 2001). Similarly. due to the use of different test conditions or ACE from different origins. The second one contained three different conformers. and Sorhaug (2004) established quantitative structure–activity relationships (QSAR) for ACE-inhibitory peptides derived from milk proteins. Yamazaki.. In addition. 2002). within the same assay. 2005). 2002. potassium and magnesium have an independent effect on blood pressure and they intensify the antihypertensive effects of IPP and VPP (Jauhiainen et al. The inﬂuence of the long-term intake of milk products on blood pressure of SHR also has been addressed. Kawai. (2004). . Peptides can adopt different conﬁgurations depending on the environmental conditions. This poses doubts on the use of the in vitro ACE inhibitory activity as the exclusive selection criterium for potential antihypertensive substances. A b-turn at the C-terminal end of bradykinin in water gives only a weak interaction with ACE (Desai. The binding to ACE is strongly inﬂuenced by the C-terminal sequence. Okubo. 2002). FitzGerald et al. & Takano.18 mM). Furthermore. 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. Kitazawa. & Van Camp. Stepaniak. Akino. 2004. Yamamoto. Pro. but containing other in vitro ACE inhibitory peptides. Quiros et al.ARTICLE IN PRESS ´pez-Fandin et al. For peptides up to six amino acids. Yamamoto. 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. that are the normotensive control of the SHR. 2006). However.. although active peptides with up to 27 amino acids have been identiﬁed (Robert.. respectively. Corsetti. ACE-inhibitory peptides usually contain 2–12 amino acids. Herregods. 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. which determine their bioactivity. ¨ ¨ The structure-activity relationship of ACE inhibitory peptides from food proteins is not well studied. as an extended or random coil structure. & Itoh. fermented products and isolated milk-derived peptides have been summarized by FitzGerald et al. Van Camp. 2004. & Belloque (2004) studied two different preparations for DKIHP (b-casein f47-51). 1997a. 1997a. In particular. the change of a trans to a cis-form of Pro in the C-terminal position of an ACEinhibitory peptide can cause signiﬁcant changes in its ´ interaction with the enzyme. However. Gobbetti.
2004a. plasma renin activity was increased in SHR that had received IPP and VPP for 14 weeks. & Nyborg. was suggested as the main mechanism. Gauthier. Nilsson. the heptapeptide lactokinin (ALPMHIR) was transported intact. ALPMHIR... & FitzGerald. carboxy and aminopeptidases). 1997). The conditions of the simulated gastrointestinal digestion (enzyme preparation.6 mM). In addition. / International Dairy Journal 16 (2006) 1277–1293 R.. However. with a low ACEinhibitory activity (IC5041000 mM). Van Camp. 1999).. Meisel. which suggests cleavage by Caco-2 cell aminopeptidases (Vermeirssen et al. only a few studies have been conducted to conﬁrm the existence of a ACE-inhibitory mechanism in vivo (Fuglsang. the active ACE-inhibitory peptide lactokinin. which supports that ACE was inhibited (Sipola.. since the transport via the short-peptide carrier. This fact has been attributed to a higher afﬁnity to the tissues and a slower elimination (Fujita & Yoshikawa. Murakami et al. Lo ˜o 2.ARTICLE IN PRESS 1280 ´pez-Fandin et al. through the intercellular junctions.. was hydrolysed by pancreatin to the potent ACE inhibitor KVLPVP (IC50 ¼ 5 mM). Raised levels of renin can be due to the lack of negative feedback by angiotensin II. ACE activity was decreased in the aorta of SHR that had taken sour milk containing the in vitro ACE inhibitors IPP and VPP (Nakamura. 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. but it may also be an indication of the existence of an additional mode of action (Vermeirssen. Van Camp et al. increasing new evidence is being provided that a different mechanism. Paracellular transport. & Takano. Ramos. the mechanistic theory of ACE inhibition of IPP and VPP remains to be conﬁrmed and other effects have to be taken into consideration.. 2003). according to Jauhiainen et al. 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. Yamamoto. Nishimura. Vermeirssen. Interestingly. 1997a). Nilsson. 2006). Tsunogai. Most food-derived peptides have lower ACE inhibitory activity in vitro than the synthetic ACE inhibitor captopril. 1996). & Verstraete. 2002). More research is needed in this respect. & Verstraete. The release of ACE-inhibitory peptides upon gastrointestinal digestion of milk proteins or protein fragments. 1996). & Takano. & Yamamoto. & Arai. In fact. & Turgeon. PepT1. & Recio. 2002). 2004). what restricts its potential to elicit a hypotensive response in humans (Rouﬁk et al. Peptides having XPP and XP may be particularly resistant to proteolysis in vivo (Mizuno. Rattray. Caco-2 cell monolayers. These studies showed that gastrointestinal digestion is an essential factor in determining ACE ´ inhibitory activity (Gomez-Ruiz. but they usually display higher in vivo activities than the efﬁcacy levels extrapolated from the in vitro activities. temperature. The tripeptide VPP was detected in the abdominal aorta of SHR 6 h after its administration in sour milk. see Vermeirssen... Masuda. 2004). However. Matsui et al. it was found that the sequence KVLPVPE (b-casein f169-175). pH and incubation time) greatly inﬂuence the degree of proteolysis and the resultant ACE-inhibitory activity (Vermeirssen. that express many intestinal enzymes and transport mechanisms.. In fact. chymotrypsin. Matsuura. the recognition by intestinal peptide transporters and the subsequent susceptibility to plasma peptidases also determine the physiological effect (Pihlanto-Leppala. 2006.. Devos. For instance. Finckenberg. Van Wijmelbeke.4. Thus. 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. For example. Van Camp et al. Rattray. . 2003). IC50 ¼ 42. 1997). led to a quick hydrolysis of the internalised peptide (Satake et al. which was probably responsible for the high antihypertensive activity of KVLPVPE in SHR (Maeno. In the case of larger sequences. Korpela et al. & Takano. Vermeirssen. 2004). was reported to be resistant to further hydrolysis by pepsin or chymotrypsin (Mullally. 2004). the susceptibility to brush border peptidases is the primary factor that decides the transport rate (Shimizu et al. 2004). Nakamura. ¨ ¨ Van Camp et al. that arises from tryptic hydrolysis of b-lactoglobulin (b-Lg f142-148. with the effort being concentrated in elucidating the pharmacokinetics and the distribution proﬁle of ACE inhibitory peptides in the different tissues (Matsui et al. a signiﬁcant amount of VPP was absorbed through Caco-2 cells.5. 2. Bioavailability and physiological relevance The physiological effects of bioactive peptides depend on their ability to reach intact their target sites. 2001. (2005). subsequent experiments revealed that ALPMHIR was susceptible to degradation on incubation with gastrointestinal and blood serum proteinases and peptidases in vitro. Van Camp. other than ACE inhibition. conﬁrming the inactivation of ACE. 2004. and one of the products increased the response to bradykinin. 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. which may involve absorption through the intestinal epithelium to get to the peripheral organs (for a review. 2002). although in concentrations too low to exert an ACE inhibitory activity. which strongly suggests that it is transepithelially transported (Masuda. Walsh et al. Decroos. 1996). Fuglsang. The action of brush-border peptidases.. 2004). have been broadly used as models for the small intestine epithelium (Shimizu. may also be involved in the blood pressurelowering effect exerted by many food-derived peptides. 2003a). 2002). Gotou. Other possible mechanisms of action Even if the hypotensive effects of milk-derived ACEinhibitory peptides have been demonstrated in SHR.
1996). not taking antihypertensive medication. a milk product Evoluss fermented with Lb. The most substantiated antihypertensive 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. 2004). 1998. lowered blood pressure after subcutaneous administration to SHR and WKY rats. Table 1). Nishimura.. 2004). 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. Bartolome. they exerted a direct action on vascular smooth muscles (Kuono. 2003. a study was conducted among patients with high-normal blood pressure and mild hypertension.actives brand. Increased production of superoxide anion and hydrogen peroxide. The antihypertensive effect of the sour milk product Calpis. Matoba. FRADHPFL. It was even suggested that these peptides might lower blood pressure through receptors expressed in the gastrointestinal tract. 2005. and decreased bioavailability of antioxidants have been demonstrated in experimental and human hypertension studies (Touyz.. 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. a mechanism of action driven by the stimulation of peripheral opioid receptors and subsequent NO release that causes vasodilation was proposed. 2004).. 2.6 mg. As shown by Nurminen et al. Hirano.. reduced NO synthesis. an effect that might be dependent or independent of ACE inhibition (Maes et al. which implies that no absorption is required (Yamada. 2004).. Volunteers consuming a 1. as this was abolished by the opioid receptor antagonist naloxone. & Vapaatalo.. peptides derived from the hydrolysis of other food proteins. and casokinin L.6 mg was observed. Walsh et al. Sugai. & Cadee.. Boeriu.5 or 3. In that study. Also. 1996). potassium and magnesium could be protective against hypertension (Nurminen. Kuboki. Similarly. & Yoshikawa.. a signiﬁcant difference in SBP between the placebo group and the VPP and IPP group receiving 3. It should be noticed that peripherally administered a-lactorphin in antihypertensive doses lacked undesirable opioid receptor-related effects in the central nervous system. & Yamamoto. ¨ Other studies have highlighted the existence of vasorelaxant opioid peptides arising from b-Lg such as b-Lg f102-105. It should be noted that the production of peptides with antioxidant properties has been reported from caseins and ´ whey proteins hydrolysed with different enzymes (Hernan´ ´ dez-Ledesma. in the case of fermented milk. Gotou. 2002) and from human casein. / International Dairy Journal 16 (2006) 1277–1293 R. 2002). It is likely that opioid receptors are involved in the antihypertensive effect. the ACE inhibitory peptides derived from dried bonito only slightly inhibited angiotensin I-induced contractions in rat-isolated aorta as compared with captopril.8 mg daily dose of IPP plus VPP exhibited a signiﬁcant decrease in SBP after 6 weeks and in those receiving either 2. & Wichers. Yamamoto. revealed a signiﬁcant decrease in systolic blood pressure (SBP) after 2 and 4 weeks of ingestion of the sour milk. Usui. which is commercialized in Japan (Calpis Co. Kasai. and the study designs have been only brieﬂy described (Pins & Keenan. Townsend. should be taken into account (Murakami et al. lactokinin (ALPMHIR) was found to inhibit the release of ET-1. Korpela. a tetrapeptide formed by in vitro proteolysis of a-lactalbumin (a-La. the high content of calcium. Ltd. Among milkderived sequences. a-lactorphin (YGLF). f50-53) with pepsin and trypsin. In this respect. Recently. YVPFPPF. Mizuno. FitzGerald et al. In addition. 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. A later trial on 46 borderline hypertensive men. However. as casoxin D. but the angiotensin I/angiotensin II ratio did not show a signiﬁcant change. & Amigo. named b-lactorphin. Davalos. Similarly. such as antinociception and sedation (Ijas et al. 2005). some of whom were taking antihypertensive medication (Hata et al. 2005). Subsequently. marketed by Calpis as AmealPeptides has been added to a new milk drink launched by Unilever under the Flora/Becel pro. and ovokinin 2-7. Strong experimental evidence indicates that oxidative stress and associated oxidative damage are mediators in cardiovascular pathologies. However.. YPFPPL (Fujita et al.ARTICLE IN PRESS ´pez-Fandin et al. RADHPF) lowered blood pressure in SHR through different modes of vasorelaxing activity (Matoba. Maeno. unlike captopril. who took different doses of a casein hydrolysate produced with Aspergillus oryzae containing IPP and VPP (Mizuno et al. but. 2001). Rival. was tested in a clinical study with mildly hypertensive patients. no signiﬁcant change was observed as compared with the placebo unfermented acidiﬁed milk group (Mizushima et al. such as YP (IC50 ¼ 720 mM. 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. & Hatae. & Yoshikawa. 1999). Onishi. when interpreting this result. the susceptibility of ALPMHIR to degradation by gastrointestinal. YLLF (Sipola et al. such as ovalbumin (ovokinin.6. 2004. Similarly. Lo ˜o 1281 For example. Japan). This product. ´ Ford. helveticus LBK-16H . McFadden... 1998). there are more examples of peptides with low ACE inhibitory activity that exert antihypertensive effects in SHR.. brush-border and serum proteinases and the ACE inhibitory activity of its degradation products. an endothelial peptide that evokes contractions in smooth muscle cells. 2002). serum levels of angiotensin I and II were measured at 4 weeks. Therefore. 2005). Fujita.1. Usui. 2004). 2004). it has been suggested that. 1999). (2000). this beneﬁt was already recorded at 3 weeks. & Takano.
(2005) 150 mL dayÀ1 (2. DMV. 2004).3 mm Hg (6 weeks) À6.15 mg IPP +1. (2005) did not observe a signiﬁcant change of diastolic blood pressure (DBP) for the test groups. (2004) Mizuno et al. & Korpela.04 mg IPP +1.4 mm Hg (4 weeks) À14. Finland or Kaiku Vitabrands. nor differences as compared with the placebo group. and must be released from the proteins by speciﬁc enzymatic hydrolysis to exert their health effects.7 mm Hg (6 weeks) À10. randomized. 2003. (2004) C12 peptide (a bovine milk protein hydrolysate containing as1-casein f2334)7alginic acid Dodeca Peptide (DP) (a tryptic hydrolysate of casein enriched in as1-casein f23-34) BioZate 1 (a WPI hydrolysate) Placebo-controlled crossover study (7-day cycles) in 10 hypertensive subjects—all receiving 5 of the possible treatments Placebo-controlled study in 18 mildly hypertensive subjects for 4 weeks.1 mm Hg (8 weeks) À4.25 mg IPP+3-3. US) were also claimed to lower blood pressure in humans (FitzGerald et al. Lo ˜o Table 1 Clinical studies with dairy products and peptides showing antihypertensive effects in humans Commercial product Description of the study Dose 95 mL day–1 (1. Seppo.1 mm Hg (6 weeks) À14. (2004) Sugai (1998) Not indicated. Placebo was unhydrolysed WPI Single dose (100 or 200 mg C12 with 877 or 1754 mg alginic acid) 200 mg dayÀ1 in water Townsend et al. 8–10 weeks. In addition LDLcholesterol was lowered by 12% (Valio Ltd. One approach is to . placebo controlled trial in 30 subjects with mild hypertension. (2002) À6. Yamamoto. Nakamura. (2003) À16 mm Hg (1st study period. However. Kanebo. probably 20 g day –1 À11 mm Hg (À7 mm Hg Pins and Keenan DBP) in comparison (2003) with control. Two other commercial products. n ¼ 39) À9 mm Hg (and— 6 mmHg DBP) at 6 h compared with 2 h. some taking antihypertensive drugs (n ¼ 15) Double-blind. & Takano.25 mg IPP+3-3. 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.. Davisco. Vaanamen. Poussa.98 mg VPP) 1. placebocontrolled trial in 17 subjects with mild hypertension (n ¼ 10) Randomized. Casein DP.42 mg VPP) 160 g dayÀ1 (1. n ¼ 59) À11 mm Hg (2nd study period. Technologies for the production of milk-protein-derived antihypertensive peptides The sequences of the ACE-inhibitory peptides mentioned above are contained within the intact milk proteins.3 mm Hg (2 weeks) À5. 2004). 1995). In principle. 2002). No effect in normotensive subjects Tuomilehto et al. Halleen. there are two approaches for releasing bioactive peptides from intact milk proteins.75 mg VPP) Two study periods with a washout period in 150 mL dayÀ1 between in 60 subjects with mild (2.4–2. Placebo was water without DP Placebo-controlled study in 30 borderline hypertensive subjects for 6 weeks. 5–7 weeks. at high alginic acid dose À6 mm Hg DBP after 4 weeks. The Netherlands) and a whey protein hydrolysate (BioZate. Matsuura et al.9 mm Hg (8 weeks) Reference Calpis (sour milk) Calpis (sour milk) ´ Ameal Peptide (casein hydrolysate) Evolus/Kaiku Vitabrand (fermented milk) Evolus/Kaiku Vitabrand (fermented milk) Evolus/Kaiku Vitabrand (fermented milk) Double-blind. Japan. Ltd. in another study consisting of two periods separated by a washout period.8 mg IPP+VPP 2.3 and À32.1 mm Hg. Jauhiainen. (1996) Mizushima et al.2 mm Hg (4 weeks) À6. a casein hydrolysate containing the peptide FFVAPFEVFGK (as1-casein f2334. & Korpela. This fermented milk was shown to increase osteoblastic bone formation in vitro (Narva. placebo-controlled trial in 39 hypertensive subjects Hata et al. & Korpela. 3.4–2.7 mm Hg (21 weeks) Seppo et al. randomized. placebo-controlled trial in 131 subjects with high-normal blood pressure and mild hypertension (n ¼ 32–33) Double-blind.6 mg IPP+VPP Effect in systolic blood pressure À9. and C12 peptide. / International Dairy Journal 16 (2006) 1277–1293 R. no statistically signiﬁcant differences were found between the sour milk and the placebo in the crossover analysis combining both phases (Tuomilehto et al.5 mg IPP+VPP 3. Suomalainen. Spain) exerted signiﬁcant antihypertensive effects in humans at daily doses of 150 mL (Seppo.7 mg VPP) Seppo et al.ARTICLE IN PRESS 1282 ´pez-Fandin et al. placebocontrolled trial in 46 hypertensive men (n ¼ 23) Single-blind. respectively. Sakai.7 mg hypertension IPP+2. Kerojoki.75 mg VPP) 150 mL dayÀ1 (2. 2004)..
delbrueckii subsp. & Recio. Moreover. The technological challenges. including dairy foods. (ii) transport systems that facilitate the uptake of the oligopeptides into the bacterial cell. thus. showing that the proteolytic system of Lactococcus should not be overlooked in the production of antihypertensive peptides. Meisel. Manufacture of fermented dairy products with ACEinhibitory peptides The production of ACE-inhibitory and antihypertensive peptides in situ in dairy products is an appealing approach.. Seppo et al. four Enterococcus faecalis strains stood out as producers of fermented milk with potent ACE inhibitory activity (IC50 ¼ 34–59 mg mLÀ1) and antihypertensive activity in SHR. as well as CHCC641 and CCCH637 from Chr.e. 2002) and LMG 11474 (Vermeirssen. Van Camp. plantarum.1. i. milk samples fermented by 4 strains of Lc. 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.. often designated PrtP (Juillard et al. the major milk proteins are degraded into a great number of peptides due to the action of indigenous milk enzymes. 1994. 2003. 2001). bulgaricus and Streptococcus thermophilus (Fuglsang et al. The ﬁrst European fermented milk drink designed to help lower blood pressure. in turn depending on storage time and conditions (Gobetti. 1999. LAB). Yamamoto et al. Lactococcus lactis subsp. & Grizzello. shown to be responsible for the already mentioned antihypertensive properties of the milk drink in vivo (see Section 2 and Table 1).e. The ﬁrst fermented milk with documented antihypertensive activity (Nakamura. Nakamura. During the fermentation of milk and maturation of cheese. 2000. 2004. which results in milk protein hydrolysates containing a great number of peptides.. et al. ¨ Ryhanen. and (ii) the production of milk protein hydrolysates with a high concentration of peptides with a speciﬁc bioactivity. LBK16 H. lactis and subsp. Yamamoto. Nakamura. Recently. 1997... 2002. 2003). Pihlanto-Leppala. lie in (i) the manufacture of fermented dairy products with a high concentration of particular bioactive peptides or their precursors. Okubo et al. Decroos et al. helveticus strain. 3. Some ACE-inhibitory peptides are products of extracellular proteinases alone.. helveticus R211... 2003b. and ﬁnally (iii) a series of intracellular peptidases. 1995.. (2006) assayed the ACE-inhibitory activity of fermented milk samples produced with 231 microorganisms isolated from raw cow’s milk samples. & Takano. & Roy. the large . The other approach is to subject isolated milk protein preparations to hydrolysis in vitro by one or a combination of enzymes. Santure. (mainly plasmin). Evoluss from Valio Ltd. Okubo et al. 1995) was marketed by the Japanese Calpis company under the tradename Amiiru S. ´ Ferranti. Lb. Muguerza et al.. and contained the same tripeptides (Tuomilehto et al. Vermeirssen. & Korhonen. Sakai et al. ¨ Tuominen. Sakai. Sakai. acidophilus. but also because of the high activity of the derived peptides (Nakamura. Lb. 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.. Finland. helveticus strains used in the production of antihypertensive fermented milk foods are Lb. Goepfert. R389 (Leclerc et al. Among them. Gauthier. 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. among them many ACE-inhibitory and antihypertensive peptides. / International Dairy Journal 16 (2006) 1277–1293 R. 1995. helveticus. Ramos. 2003). Yamamoto. Yamamoto.. 1995. 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. helveticus CP790 and a Saccharomyces cerevisiae and contains two ACE-inhibitory tripeptides. 2003. peptides relative to other peptides. Smacchi. rhamnosus. ACE-inhibitory. Sakai. bitter peptides. 2004) (Table 1). have been produced using proteolytic strains of the LAB species Lb. ¨ ¨ ¨ Fermented milks containing a particularly high number of peptides. Decroos et al. 1995). It is produced by fermentation with a combination of Lb. Lb. Gobetti. Yamamoto. Bachelard.ARTICLE IN PRESS ´pez-Fandin et al. Hansen A/S (Fuglsang et al. Rokka. Amigo. 2003b. Sakai. as well as the two species used in traditional yoghurt production Lb. that initiate the proteolytic attack. Minervini... added coagulants and microbial enzymes (especially from starter and non-starter lactic acid bacteria. which upon digestion in the gastrointestinal tract would give rise to the bioactive peptides. In a recent study (unpublished). 1994). 1999). lactis had higher ACE-inhibitory activity than the milks fermented by either of 7 strains of Lb. of casein origin.. Okubo. Nakamura. 1997. b. was fermented by another Lb.. Yamamoto et al. Goffredi. Lb. Van Camp. VPP and IPP. Other Lb. like fermented milk and cheeses. Lo ˜o 1283 exploit the proteolytic system of lactic acid bacteria to partially digest the caseins during the manufacture of dairy products.. 2004a. among them the bioactive peptides. Yamamoto et al. & Pahkala. and with a functionality that makes them suitable as ingredients in other foods.. Seppo et al. The proteolytic systems of LAB consist of (i) cell-wall proteinases (Lactocepins). & Addeo. casei. Leclerc. & Gunther. The hydrolysates (or hydrolysates enriched in particular peptides) may be applied in the manufacture of other food products. HernandezLedesma. 2003b). and with the right speciﬁcity to give higher concentrations of the active. Yamamoto. In fact. 1995. i. to provide them with the desired bioactivity. helveticus.. 2002. Lb. The strain should not be too proteolytic to destroy the product and yet to give a high proteolysis. Syvaoja. since this confers an additional positive health effect to dairy products possessing already a healthy image and having a long history of safe production. cremoris.
The highest ¨ ¨ ACE-inhibitory activity was found in the ‘‘Festivo’’ cheese ripened for 13 weeks. and subsequently fermented with Lb. 2004b). Pep X) involved in the hydrolysis of Procontaining sequences are important for the degradation of casein-derived oligopeptides because of their high content of Pro. & Marth. 1996). Johnston. ‘‘Festivo’’ cheese. Saito. ¨ Much effort is being put into expanding the knowledge about the proteolytic systems of interesting LAB. whereas others. Lactobacillus sp. Pep X is the most dominating one (Gatti. Specialized peptidases (i. Ramos.. Camembert. Bosman. acidophilus and Biﬁdobacterium sp. Coolbear.e. The cell-wall proteinases of both lactococci and lactobacilli have a very broad substrate speciﬁcity. helveticus and Lc. 2001. & Konings. Muncchetti. Deutsch. 1993). (1997) produced a fermented milk by ﬁrst inoculating a UHT treated milk with Lb casei subsp. The highest blood pressure-lowering activity was exerted by peptides from an 8 month old Gouda cheese. i. or. Edam. Many ACE-inhibitory peptides were formed in a Spanish Manchego cheese prepared speciﬁcally by inoculating the milk with Lc. 2001). although some variation within strains of Lb. 3. Singh. Peptide bonds from fragments such as 160–170 and 190–195 in b-casein are cleaved by all proteinase types. 1994). The whey was ﬁrst hydrolysed with trypsin. A number of bioactive peptides were isolated from this drink. Valence. thermolysin or other enzymes. Lazzi.. Especially powerful aminopeptidases exist in Lb.g. Lo ˜o b-casein fragments produced by the extracellular proteinase from Lb. & Roundhill. / International Dairy Journal 16 (2006) 1277–1293 R. and Aimar (1997) used a reverse order to produce fermented whey beverages. 1995) and. helveticus CP790 (Yamamoto et al. Many studies performed during the last two decades have revealed the presence of ACE-inhibitory and/or antihypertensive peptides in . from which 3 active peptides from as1-casein were isolated (as1-casein f1-6. rhamnosus (Lb. f1-7 and f1-9). such as Gouda. 1995). & Tan. Some of the aminopeptidases. thermophilus. Hagting. proteinase K. helveticus exists (Khalid. to provide the most suited LAB with the desired proteolytic capacity. followed by hydrolysis with pepsin and trypsin.2.e. A new type of lowfat cheese containing ACE-inhibitory peptides. 1995. dextranicum (20%. 2004). Havarti and Blue cheese. particularly those speciﬁc for tri. in particular RPKHPIKHQ corresponding to as1-casein f1-9. Lc. The highest ACE-inhibitory activity (IC50 ¼ 50 ng mLÀ1) corresponded to the whey initially hydrolysed by proteinase K. such as Pep N and Pep C. more likely. 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. Crawford. For example. helveticus CPN4 (Yamamoto et al. Smacchi and Gobbetti (1998). is produced in Finland with a commercial starter mixture containing strains of Lactococcus. whereas others are most likely the result of a concerted action of both proteinases and peptidases. delbrueckii subsp. Emmental. & Recio. Abubakar. GG). For example. ¨ Coker. Further progress in this area might be obtained through genetic engineering. & Creamer. 1991). are well conserved among dairy LAB. ´ Gomez-Ruiz. more than 100 different oligopeptides are released from b-casein when incubated with PI-type proteinases (Juillard et al. Itoh.. 2005). In cheese. Rokka et al. 1999). Martley. 1996). The proneness to lyse under certain conditions is thus another quality of the LAB that should be considered. Poolman. isolated a b-casein fragment from (b-casein f58-72) that inhibited ACE (IC50 ¼ 18 mM). Gagnaire. Propionibacterium. among them two b-casein fragments with ACEinhibitory activity (b-casein f177-183 and f193-202). acidophilus (Sasaki. The origin and history of the milk used and the manufacturing conditions thus affect the production of peptides. investigating the peptidase-inhibitory activity of extracts from Italian commercial cheeses. YP isolated from a yoghurt-like product fermented by Lb. many are identical to or contain sequences with proven antihypertensive activity or ACE inhibitory activity (Saito et al. 2000). Fornasari. while bonds in other regions are only cut by some proteinases (Kunji. the extent of lysis of both Lb. Among the peptides formed during maturation of commercial cheeses. & Lortal. Soda. bulgaricus and Str. in particular their activity under various conditions relevant for the fermentation of dairy products. helveticus compared e. lactis subsp. (Ryha¨ nen. 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. & Neviani. as well as probiotic strains of Lb..ARTICLE IN PRESS 1284 ´pez-Fandin et al. 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. Leuconostoc. 2004).. Mierau. among these. It should be noted that some antihypertensive dairy products have been manufactured by fermentation in combination with in vitro hydrolysis. 2000). lactis and Lb. The intracellular peptidases so far isolated from lactococci and lactobacilli are either aminopeptidases or endopeptidases. lactis (80%) and Leuconostoc mesenteroides subsp. Pihlanto-Leppala.and dipeptides are distinct to each species. and also from studies regarding the interaction between strains in environments as those prevailing in fermented milks and cheeses. Richoux. The peptide pattern of cheeses from different maturation stages is characteristic for each cheese variety (Ardo. & Pahkala. lactis cells has been shown to have a direct inﬂuence on proteolysis (Crow. Lysis of bacterial cells may also allow the intracellular peptidases to escape from their intracellular localization and to act on the large oligopeptides produced by the action of the cell-wall proteinases. as a result of lysis of the bacterial cell. These peptides are also formed during ripening of other Scandinavian cheeses (Lund & Ardo. Arai..
Pins & Keenan. Kawai.. Gauthier. 1996. Schlothauer et al. some of the active peptides may be lost (Pihlanto-Leppala et al. Maruyama et al. this technique does not take into account the protein conformation which might affect also the ACEinhibitory activity as discussed in Section 2.ARTICLE IN PRESS ´pez-Fandin et al.3. Okubo et al. whey protein concentrates and isolates. Nakamura. 1999. 2002. van der Bent. & Amigo. Schlothauer et al. ¨ ¨ Schlothauer et al.. and microbial enzymes including cell-wall proteases from LAB (Abubakar et al. compared with the 1 and 10 kDa permeates and with the original tryptic digest of whey proteins (Mullally et al.. Piilola. that also market the C12Peptide (Fitzgerald et al. For example. used membranes with cut-off between 10 and 50 kDa. Considering the size of most ACEinhibitory peptides being less than 3 kDa. 1998. Mullally et al.. 2002. Presently. many patents have been granted for the production of milk protein hydrolysates with good functionality. & Skibsted. Otte. Mullally. 2000). Yamamoto et al. 2004.. Nurminen et al. Hernandez-Ledesma. and individual whey proteins (Abubakar. which are more compact than the caseins. a-La and bovine serum albumin. Calculations performed by Pripp (2005) on the ACEinhibitory activity of b-casein after a speciﬁc theoretical hydrolysis. 3. comprising animal digestive enzymes. Mullally et al. hydrolysis of the major whey proteins. 2004. A common feature of ACE-inhibitory peptides being their relatively restricted size and relatively hydrophobic C-terminal makes fractionation based on size a promising step for pre-concentration of the active peptides. 2004). 2003.. However. 1997b. van Amerongen. Iwaniak. Maeno et al. & Korhonen. Yamamoto. 2000). & Itoh. Molle. 2004b.. Recio... 1997b). FitzGerald et al. FitzGerald and coworkers found the highest ACE-inhibitory index in the 3 kDa-permeate. also resulted in peptides with high ACE-inhibitory and/or antihypertensive activity.. The choice of the best protein substrate and enzyme combination may be assisted by the newly developed in silico calculations using dedicated software (Dziuba. ¨ ¨ Accordingly. including bioactivity. processes that are both suitable for industrial scale production. ´ 1998.. Miclo & Gaillard. (2002) in their patent. Interestingly. Maeno et al. Pihlanto-Leppala et al. FitzGerald & Meisel. In the laboratory. & Minkiewicz... Karaki et al. Pihlanto-Leppala. Mullally et al. i. With a membrane of 1 kDa. 1994).. 1998. hydrolysis with a combination of digestive enzymes or highly proteolytic and less speciﬁc enzymes.. Rokka.g. Robert et al. / International Dairy Journal 16 (2006) 1277–1293 R. Saito. 1996. 2002). helveticus (Maruyama et al. Yamamoto et al. 2000.. lactoferrin may be a good precursor if hydrolysis is performed to release many dipeptides. Van Camp. or towards the basic amino acids Lys and Arg might be beneﬁcial. 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. b-Lg. Tauzin et al. caseinates. 1997b. 1998. 2002). after hydrolysis by trypsin or an extracellular proteinase from Lb. ultraﬁltration with a cut-off of 3 or 5 kDa seems a good choice ´ ´ (Gomez-Ruiz et al. Considering the previously discussed structure-activity features of ACE-inhibitory peptides. This in silico calculation technique may be useful also for the prediction of the outcome of fermentation. as2-casein and b-casein. might be particularly useful in releasing potent ACE-inhibitory peptides (Abubakar et al. Hernandez-Ledesma et al. Concerning the whey proteins. Results from the latter study show that. Lo ˜o 1285 enzymatic digests of various milk protein preparations. individual caseins. Enzymes from various sources have been used to hydrolyse the milk proteins.. they cannot be isolated by precipitation or ion exchange chromatography. plant enzymes. though. e. Enrichment of hydrolysates with ACE-inhibitory peptides A technological challenge in the production of ACEinhibitory and antihypertensive peptides is the enrichment of fractions or the isolation of speciﬁc peptides from the total peptide mixture. 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. Meisel.. 2004. ¨ ¨ Schlothauer et al. & Korho¨ ¨ nen.. before or during proteolysis. Fitzgerald et al. Lapointe. Vermeirssen.. Kitazawa. see also Section 2). as1-casein.. 1997b. The use of high pressure to partially unfold the whey proteins. 2000. explaining the large number of ACE-inhibitory peptides obtained with trypsin. i. Not surprisingly. & Verstraete. Olsen. 1994). this has been achieved by ultraﬁltration and size exclusion chromatography. in addition to b-Lg and b-casein. 1997b.e. from Arla Foods Ingredients and Davisco Foods International as well as from DMV International and Kanebo Ltd. Ramos. The most efﬁcient inhibitors of ACE (IC50o30 mM) were formed from caseinate and the individual major caseins. enzymes with speciﬁcity towards the carboxylic side of aromatic or other hydrophobic amino acid residues. Yamamoto et al. 1995. 2002. might also increase the rate of proteolysis and alter the relative proportion of peptides (Knudsen. 1996. 1987. as far as the speciﬁcity of the microbial enzymes is known. & Fitzgerald. 1990.. This might be related to their high content of Pro. Pihlanto-Lepala et ¨ ¨ al.3.. a number of casein and whey protein hydrolysates and even individual peptides with high ACE-inhibitory activity are commercially available..e. thermolysin and proteinase K.. Since the ACE-inhibitory peptides are not characterised by a particular functional group. Pihlanto-Leppala. 1994). 1987. both of microbial origin. & Pouliot. Koskinen. Sakai. Maeno et al... such as serine phosphate groups in the caseinophosphopeptides.. Tauzin. 2000. which also gave peptide concentrates with a rather high ACEinhibition. 2003.. .. (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%. ´ 2004b. 2004. 2002. Tupasela. 2004.
Size exclusion chromatography may be used for further fractionation.. Nutraceuticals may be incorporated into fermented milk by addition to the standardized milk prior to inoculation (Awaisheh.. Righetti.e. Yamamoto. Sakai et al. Sodini. Lo ˜o Pre-concentration based on hydrophobicity using a C18 reversed-phase cartridge is also common in the laboratory (Curtis. Sturrock. 1999). Van Camp et al. or into the active site of other enzymes and receptors involved in blood pressure regulation.e. Henry. in which the biochemical assay detecting ACE inhibitory activity of peptides was coupled into the LC-MS line.4. Furthermore. among them the large fragment 49–97. For a more complete overview of the reactions in which amino acids in foods and food products are involved. the angiotensin II receptor T1 (FitzGerald et al. the product—and in particular the ACE-inhibitory peptides therein—must be stable during the ﬁnal processing. 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. and as such with their bioactivity (see Section 2. Steijns 1996. Using a continuous membrane reactor. 2005).ARTICLE IN PRESS 1286 ´pez-Fandin et al. 2000). Yamamoto & Takano. With a view to the industrial scale production of hydrolysates. However. Dennis. 2005. 2003. On-line cou˜ pling to electrospray ionization-tandem mass spectrometry ´ allows simultaneous identiﬁcation of the peptides (Gomez´ Ruiz et al. ´ Hernandez-Ledesma. Nakamura.. 1999). Ramos. Further reﬁnements. has been published (van Elswijk et al. Amigo. 1995. Jolivet.. as well as the uptake and degradation of the active peptides by the fermenting organisms should be evaluated.3). 3. Pihlanto-Leppala et al.. such as hydrolysates. Nembri.. Yamamoto et al. 1999). / International Dairy Journal 16 (2006) 1277–1293 R. packaging and storage. Using this reactor. 1997b. 2002. 2003). various initiatives have been taken towards continuous operation with increased enzyme utilization efﬁciency and increased yield and purity of the active ´ ´ peptides. 2003). followed by separation by reversed-phase HPLC (i. see Section 2). 2003). which contains many sequences with high ACEinhibitory (IC50 ¼ 4–100 mM) and antihypertensive activity (Abubakar et al. i. preparation and preservation. ¨ ¨ Reversed-phase chromatographic separation is well suited for the ﬁnal separation in the laboratory. . The question arises whether these molecular changes on amino acid level result in a partial or total loss of the bioactivity of the peptide. Hernandez-Ledesma et al. Haddadin. Bouhallab. 2004. & Recio. Nakamura.. Monnet. Pihlanto-Leppala ¨ ¨ et al. Saito et al. which contains sequences with a moderate ACE-inhibitory ´ activity (Gomez-Ruiz et al.. 2004a. 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. 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. per se or incorporated into other food products. Okubo et al. and they are not restricted to the most hydrophilic ´ fraction (Manso & Lopez-Fandino. the hydrolysate should have well-deﬁned technological functionalities not to impart the required functionality of the carrier food. the peptide bonds to be preferentially cleaved can be controlled by the enzyme and substrate concentrations and the substrate feeding ﬂow rate (Martin-Orue.. 4. Bossi. 1999). A special set-up. Sakai. MacGillivray. are prone to chemical changes occurring during food processing.. 2004). a number of tryptic peptides from b-casein were isolated in pure form. 1998. Huo... It should be possible to enrich fermented milk in the same way by milk protein hydrolysates particularly rich in ACE-inhibitory peptides. 2004b. However. Schwager. 1998. 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. Molle.. and Leonil (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. 2004). & Acharaya. Furthermore. 2003.. 2000. Robert et al. that are crucial with regard to their interaction with ACE. & Fu. Miralles. 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. since the ACEinhibitory peptides are distributed over most of the peptide proﬁle. Yamamoto. Waddell. 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. Lucas. preparation and preservation on the bioactivity of ACE-inhibitory and antihypertensive peptides is not well documented. The peptides or concentrated hydrolysates produced could be applied as functional ingredients in other foods to provide them with the desired antihypertensive activity. Despite the way of delivery. Mullally et al.. and Corrieu (2004) used this approach to incorporate casein and whey protein hydrolysates into fermented milks containing probiotic bacteria. refer to the review by Damodaran (1995). 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. 1995. & Ewart. & Robinson. Natesh. & Bouhallab.. The inﬂuence of food processing. Yamamoto & Takano. Several of the amino acids present at the C-terminal sequence of ACE-inhibitory peptides. the degradation of the active peptides during processing. 2004b. MoutevelisMinakakis et al. 2003).
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 (Leonil et al. Thr and Lys. & Finot. like pasteurization or UHT-treatment (Korhonen. of Ala. Molle. ¨ ¨ ¨ 1998). Met. it is also reported that protein digestibility is affected by the complex reactions occurring during the Maillard reaction ´ (Gilani & Sepehr. D-amino acids have been detected in various dairy products where microorganisms are involved. Cys. Since trypsin catalyses the hydrolysis of peptide bonds derived from carboxyl groups of Arg and Lys residues. Broersen et al. ´ Morgan. 1997). Alkali-treated casein is less efﬁciently hydrolyzed by pancreatic proteases such as trypsin and chymotrypsin (Berger & Possompes. Consequently the yield of bioactive peptides generated during proteolysis could be affected as well. and histidinoalanine. 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. transaminases. the additional formation of reactive a-carbonyl compounds lead to a cascade of supplementary reactions involving also other amino acids. Leonil et al. Heating at alkaline pH or above 200 1C at neutral pH. His. & de Jongh. Ser. especially in-can sterilisation. Although very speculative. as demonstrated for roasted casein and bovine serum albumin (Friedman. the resistance to chymotrypsin hydrolysis may be related to racemization of aromatic side chains.e. as such. More severe heat treatments of milk. Lys in the early stages of browning. lanthionine. partial racemization of L-amino acids into D-amino acids can occur under these conditions. supply the carbonyl component. / International Dairy Journal 16 (2006) 1277–1293 R. b). The structure of the bioactive sites in the peptides may be changed in such a way that the activity is affected directly as well. 1998) could give rise to changes in the conformation of biologically active peptides and thus their activity. & Tupasela.1. Milk proteins contain high concentrations of LAL amino acid precursors. Phe. and Lellouch (1996) reported on the effect of serine . In addition to the structural changes in Lys and other important and sensitive amino acids. The Maillard reaction (non-enzymatic browning) is known to have a signiﬁcant impact on nutritional properties in general and on amino acids in particular. ´ ´ Hamer. 2004. Inﬂuence of heat processing Thermal processing at alkaline pH can lead to changes in the structure of amino acids. Pao. especially for amino acids with a strong electron withdrawing power in the side chain. D-amino acids can be synthesized out of L-amino acids by microorganisms using amino acid oxidases. Especially the e-amino group of Lys is frequently involved in the carbonyl–amine reaction. Milk is sensitive to the Maillard reaction due to the presence of high levels of lactose and Lys-rich proteins. Amino acids supply the amino component. This may affect the sensitivity to proteolysis and.and 4-deoxydicarbonylcompounds (deoxysones). and epimerases (racemases). is hydrolysed in the acidic conditions of the stomach. and ripened cheeses (Friedman. to lysinoalanine (LAL). Asp. Raymond.. and phosphoserine. like serine phosphate groups in casein. respectively (Damodaran. 1999a). 1995). Pihlanto-Leppala. In general. and Thr. ornithoalanine. The reaction occurs between amines and carbonyl compounds forming glycosylamines. A wide species dependence in nutritional utilisation and nutritionally antagonistic and toxic manifestations of D-enantiomers and LAL have been described.g. Glu. LAL formation was also reported in thermally treated milk products. an intermediary glycosylamine is formed which rearranges to a 1-amino-1-deoxyketose (Amadori rearrangement for aldosamines) or to 2-amino2-deoxyaldose (Heyns rearrangement for ketosamines). (2004) reported that. 1999b).ARTICLE IN PRESS ´pez-Fandin et al. Rerat. Beyond the stage of 1-amino-1deoxyketose or the 2-amino-2-deoxyaldose. 1999). e. yoghurt. Vaissade. Previously. Lys is no longer biologically available. Trp (Damodaran. Lo ˜o 1287 4. 2003. Ser. lead to higher losses of available Lys compared to milder treatments. Heat alone may also induce racemization. at elevated temperatures. 1987). 1997. The alkaline induced changes in amino acids given above may affect proteolytic digestion. 1995). This electrophilic molecule reacts further with Lys. After condensation of the amine with the carbonyl compound. In addition. like fermented milk. Thus. Rickert and Imperiali (1995) reported the effect of N-glycosylation on the cis/trans isomer ratio of Pro in a particular peptide. while reducing sugars (aldoses and ketoses). and cystine in whey proteins. Voragen. In addition.. ornithine (obtained after decomposition of Arg) or His. decompose and eventually condense into insoluble brown products such as melanoidins. which. Wormarld. heat treatment of dairy proteins whether or not in combination with an alkaline treatment induces racemization and cross-linking of amino acids. Also. cystine. These products should be considered as intermediates which are further degraded to 1-. Tyr. reactive a-carbonyl compounds yielding many secondary products (Belitz & Grosch. the conformational stability of the protein is changed due to changes in the secondary structure. & Leonil. Cys. Calmes. Tyr. Bouhallab. as reported to occur during the Maillard reaction (Broersen. 3. change the yield of bioactive ACE inhibitory and antihypertensive peptides originating from these proteins. like sodium and calcium caseinates (Friedman. Cys. For a review of the literature see Friedman (1999a. ascorbic acid. and carbonyl compounds generated from lipid oxidation. such as Arg. 1999a). i. as a result of the glycosylation of b-Lg. In addition to the losses of Lys. These changes were attributed to altered intramolecular hydrogen-bonding. 2002). Rantamaki. Arg decomposes to ornithine. leads to the highly reactive dehydroalanine residue. Asn. Chymotrypsin on the other hand catalyzes hydrolysis of peptide bonds next to aromatic amino acids (Phe. it cannot be excluded that glycosylation of dairy proteins and peptides. Trp) and consequently.
/ International Dairy Journal 16 (2006) 1277–1293 R. a number of patents have been granted in this area. Obviously these oxidative changes may give rise to changes in the molecular structure of ACE-inhibitory and antihypertensive peptides. Gauthier. may also bind to the inner cavity of b-Lg. and more severely under acidic.ARTICLE IN PRESS 1288 ´pez-Fandin et al.3). 5. 2005). there might be other mechanisms of antihypertensive action different from ACE inhibition. Interactions between intact milk proteins (i.2. Gauthier. 4. a wide range of peptide sequences derived from milk proteins capable of inhibiting ACE in vitro. At pH 3. Also. These aspects should be evaluated carefully and if necessary taken into account when formulating foods containing these bioactive peptides. and depending on the ionic strength and the temperature. Conclusions As a result of an extensive research carried out during the past 20 years. such as a direct relaxation of vascular muscles and/or opioid or antioxidant activities. Similarly.e. in a study on tryptic hydrolysates of b-lactoglobulin (b-Lg). which has a large impact on the ACE-inhibitory activity of peptides containing Pro at the C-terminal (Section 2. The latter two are biologically unavailable. ´ ´ Turgeon.g.. while methionine sulfoxide can be reconverted to Met under the acidic conditions of the stomach. possibly because electrostatic repulsion prevented interaction between the positively charged b-Lg and the peptides studied. Lo ˜o O-glycosylation on cis/trans Pro isomerisation.3. and thus potentially useful in the prevention and/or treatment of hypertension. free radicals and peroxides formed during peroxidation of lipids) and the use of oxidizing agents (e. are known. Molle. His. leading to complex formation with different interfacial properties. showing opioid and ACE-inhibitory activity. thus altering their bioactivity. b-Lg) and milk protein-derived peptides have been studied by Noiseux. Met easily oxidizes to methionine sulfoxide by peroxides.and disulfoxides of L-cystine are biologically available as they reduce back to L-cystine in the body.0. The data given above illustrate that milk-derived peptides. De Clippeleer.g.0. Met. and such . This makes the intact b-Lg protein a potential carrier for bioactive peptides. severe oxidizing conditions. De Meulenaer. no peptide/b-Lg interactions were found. The hydrophobic peptides as1-casein f23-34 and b-Lg f102-105.2. & Pouliot (2001). while mono. and Leonil (1992) indicated. 2003). These properties must be taken into account in case products are to be formulated containing bioactive (ACE-inhibitory or antihypertensive) peptides. & Turgeon (2002). Trp can be oxidized under acidic. Inﬂuence of oxidation Endogenous production of oxidative compounds in foods during processing (e. quite frequently the results of tests in SHR have revealed discrepancies between the in vitro ACE inhibitory properties and the in vivo action. b-Lg f69-83. Potential role of functional properties of milk-derived bioactive peptides Milk peptides have been extensively evaluated for their interfacial properties. mainly because the in vitro methods do not take into consideration the physiological transformations that determine the bioavailability of the peptides. Cys. & Huyghebaert.and disulfone derivatives of L-cystine are biologically unavailable. 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. mono. which may be of importance for their bioavailability and bioactivity in the human body. where cationic fractions of tryptic whey protein hydrolysates having low molecular masses (o1000 Da) and isoelectric points ranging from 5. Both casein and whey protein-based hydrolysates containing a high ACE inhibitory activity have been produced. may have interfacial properties and may as well interact with other ingredients present in foods and food products. Trp and Tyr are also susce ptible to sensitized photo-oxidation. This stresses that more research is needed in order to demonstrate scientiﬁcally the physiological basis for the antihypertensive effects of milk-derived peptides.8 and 8. Dairy products and other foods containing antihypertensive and ACE-inhibitory peptides can be produced by enrichment with in vitro-produced milk protein hydrolysates. Poor interfacial properties were related to an uniform distribution of hydrophobic and hydrophilic amino acids and to the rigidity provided by disulﬁde bonds. 4. Devlieghere. However. b-Lg f130-135. 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. Gauthier. which prevents spreading at the interface. Smaller peptides are less surface active. associations or hydrophobic interactions can be formed between proteins and peptides. gave a destabilizing effect on model infant formula compared with more anionic and larger peptide fractions with higher amounts of emulsifying peptides. 2005). At pH 6. these changes can be induced particularly during the storage of the ﬁnished product unless appropriate packaging materials are used (Mestdagh et al. hydrogen peroxide) may lead to oxidation of amino acids. Dairy products are particularly vulnerable to photo-oxidation because of the presence of the photo-sensitizer riboﬂavin (Mestdagh. This conclusion was also drawn from the study of Lajoie. as a function of their amino acid sequence and their length. Interestingly. In addition. mild oxidizing conditions.8 to 10. and b-Lg f146-149 interacted with intact b-Lg. and eventually can be further oxidized to methionine sulfone and homocysteic acid.
Fujita. Hypotensive peptides from milk proteins. Desai. 35... J.. pp. M. 5. although other LAB have shown good performance. & Creamer. Crawford. is mediated by NK1 receptor. & Meisel. N. Amino acids. (2004). FitzGerald. (1987). Y. lactis M18 on cheddar cheese ripening. (1999b). Murray. Singh. Kitazawa. L. the ﬁnal extent of amino acid modiﬁcation and changes in bioactivity will depend largely on the composition of the food matrix in which the peptide is present.. F. and Bruno De Meulenaer and Sarah De Groot. Determination of angiotensin-converting enzyme inhibitory peptide Leu–Lys–Pro–Asn–Met (LKPNM) in Bonito muscle hydrolysates by LC-MS/MS. Sasaki. 50.. Vasorelaxation by casomokinin L. 48. Germany: Springer pp. G. J. K. D. Polimery. Lb. J. Peptides. 223–229. (2001). and technological developments in large scale continuous production and isolation of peptides. and histidinoalanine in food and other proteins.. for their help with the manuscript. Kurahashi. In O. FitzGerald. 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. Journal of Applied Microbiology. 78–87. & Cheung.. on the processing and preparation conditions. J. nutrition. S. 37. H.. Nahrung. (2004). Y. S. Biotechnology and Bioengineering. Rattray. E. D. L. E. International Dairy Journal.. A. (2005). Nielsen and Jes C. H.. mainly containing the tripeptides IPP and VPP. A. Journal of Dairy Science. D.. S. helveticus is often preferred for this purpose. Food chemistry (3rd ed. Fujita. M. and microbiology of lysinoalanine. Glycoforms of b-lactoglobulin with improved thermostability and preserved structural packaging.. whose antihypertensive effects have been established in humans. (2002). 15. Berger.. Suganuma. H. and proteins. nutrition and microbiology of D-amino acids. T. S... (2005). 321–430). 369. T. (2003). Martley. K. E78–E87. F. D. 15. 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