Major features associated with use of whey (adapted from Alais [62])
1. Introduction
A food can be considered as functional if, beyond its nutritional outcomes, it provides benefits upon one or more physiological functions, thus improving health while reducing the risk of illness [1]. This definition – originally proposed by the European Commission Concerted Action on Functional Food Science in Europe (FuFoSE), should be refined in that: (i) the functional effect is different from the nutritional one; and (ii) the benefit provided requires scientific consubstantiation in terms of improvement of physiological functions, or reduction of occurrence of pathological conditions. The concept of functional food emerged in Japan during the 80's, chiefly because of the need to improve the quality of life of a growing elderly population – who typically incurs in much higher health costs [2]. Nowadays, a growing consumer awareness of the relationship between nutrition and health has made the market of functional foods to boom.
Bioactive peptides can be commercially sold as nutraceuticals; a nutraceutical is an edible substance possessing health benefits that may accordingly be used to prevent or treat a disease. However, a distinction should be made between nutraceuticals taken to prevent diseases – and which are present as natural ingredients of functional foods consumed as part of the daily diet, and nutraceuticals used as adjuvants for treatment of diseases – which require pharmacologically active compounds.
Milk and dairy products have been concluded to be functional foods; a number of studies have indeed shown that many peptides from milk proteins play a role in several metabolic processes, so a considerable interest has arisen from the part of the dairy industry towards large-scale production of dairy proteins in general, and bioactive peptides in particular. Manufacture of bioactive peptides is usually carried out through hydrolysis using digestive, microbial, plant or animal enzymes, or by fermentation with lactic starter cultures. In some cases, a combination of these processes has proven crucial to obtain functional peptides of small size [3,4]. Proteins recovered from whey released by cheese manufacture already found a role as current ingredients on industrial scale. Use of these proteins (concentrated or isolated), and mainly of biologically active peptides derived therefrom as dietary supplements, pharmaceutical preparations or functional ingredients is of the utmost interest for the pharmaceutical and food industries – while helping circumventing the pollution problems associated with plain whey disposal.
2. Cheese whey
Despite having been labeled over the years as polluting waste owing to its high lactose and protein contents [5], whey is a popular protein supplement in various functional foods and the like [6]. In fact, whey compounds exhibit a number of functional, physiological and nutritional features that make them potentially useful for a wide range of applications (Table 1).
High nutritional value of protein fraction in terms of amino acid residues (e.g. Lys, Thr, Leu, Ser) Possibility of lactose production in parallel Reduction in pollution owing to biochemical oxygen demand of proteins | High dilution requiring costly dehydration High salt content (ca. 10 % of dry matter) High sugar content requiring delactosation Highly perishable raw material Widely dispersed cheese production facilities Technical innovation needed in separation (e.g. ultrafiltration and diafiltration) |
Whey can be converted into lactose-free whey powder, condensed whey, whey protein concentrates and whey protein isolates [7] – all of which are commercially available at present. In the case of bovine milk, ca. 9 L of whey is produced from 10 L of milk during cheesemaking; estimates of worldwide production of cheese in 2011 point at ca. 15 million tonnes (United States Department of Agriculture – Foreign Agricultural Service). For environmental reasons, whey cannot be dumped as such into rivers due to its high chemical and biological oxygen demands. On the other hand, whey can be hardly used as animal feed or fertilizer due to economic unfeasibility.
2.1. Physicochemical composition
There are two types of whey, depending on how it is obtained; when removal of casein is via acid precipitation at its isoelectric point (pH 4.6 at room temperature) [8], it is called acid whey; however, the most common procedure is coagulation via enzymatic action, so the product obtained is called sweet whey [9-10].
Despite containing ca. 93 % water, whey is a reservoir of milk components of a high value: it indeed contains ca. half of the nutrients found in whole milk. Said composition depends obviously on how the cheese is produced and the milk source; the compound found to higher level is lactose (4.5-5 %, w/v), followed by soluble proteins (0.6-0.8 %, w/v), lipids (0.4-0.5 %, w/v) and minerals (8-10 %, w/wdry extract) – particularly calcium, and vitamins such as thiamine, riboflavin and pyridoxin [11-13]. In fact, whey is now considered as a co-product rather than a by-product of cheese production, in view of its wide range of potential applications [13-15].
2.2. Protein composition
Milk has been recognized as one of the main sources of protein [16] in feed for young animals and food for humans of all ages [17]. Bovine milk contains ca. 3 % protein [9] – of which 80 % is caseins and 20 % is whey proteins [18]. Whey comprises a heterogeneous group of proteins that remain in the supernatant after precipitation of caseins; they are characterized by genetic polymorphisms that usually translate into replacement of one or more amino acid residues in their original peptide sequence.
Two major types of proteinaceous material can be found in whey: -lactoglobulin (-Lg) and -lactalbumin (-La); and proteose-peptone (derived from hydrolysis of -casein, -CN), small amounts of blood-borne proteins (including bovine serum albumin, BSA, and immunoglobulins, Igs), and low molecular weight (MW) peptides derived from casein hydrolysis (soluble at pH 4.6 and 20 °C) [16, 19]. Whey proteins have a compact globular structure that accounts for their solubility (unlike caseins that exist as a micellar suspension, with a relatively uniform distribution of non-polar, polar and charged groups). These proteins have amino acid profiles quite different from caseins: they have a smaller fraction of Glu and Pro, but a greater fraction of sulfur-containing amino acid residues (i.e. Cys and Met). These proteins are dephosphorylated, easily denatured by heat, insensitive to Ca2+, and susceptible to intramolecular bond formation via disulfide bridges between Cys sulfhydryl groups. Selected physicochemical parameters typical of whey proteins are tabulated in Table 2.
-Lg | 3 – 4 | 18.4 | 5.2 |
-La | 1.5 | 14.2 | 4.7 – 5.1 |
BSA | 0.3 – 0.6 | 69 | 4.7 – 4.9 |
IgG, IgA, IgM | 0.6 – 0.9 | 150 – 1000 | 5.5 – 8.3 |
Lactoperoxidase | 0.006 | 89 | 9.6 |
Lactoferrin | 0.05 | 78 | 8.0 |
Protease-peptone | 0.5 | 4 – 20 | |
Caseinomacropeptide | 7 |
2.2.1. -Lactoglobulin (-Lg)
The major protein in ruminant whey is -Lg, which represents ca. 50 % of the total whey protein inventory in cow's milk and 12 % of the total milk proteins [9, 20-21]. Although it can be found in the milk of many other mammals, it is essentially absent in human milk [22]. This is a globular protein, with 162 amino acid residues in its primary structure and a MW of 18.4 kDa. There are at least twelve genetic variants of -Lg (A, B, C, D, DR, DYAK/E, F, G, H, I, W and X) – of which A is the most common.
The monomer of -Lg has a free thiol group and two disulfide bridges – which makes -Lg exhibit a rigid spacial structure [8]; however, its conformation is pH-dependent [23] – and at temperatures above 65 °C (at pH 6.5), -Lg denatures, thus giving rise to aggregate formation [24]. Between pH values 5.2 and 7.2, that protein appears as a dimmer – with a MW of 36.0 kDa [8]. At low pH, association of monomers leads to octamer formation; and, at high temperatures, the dimer dissociates into its monomers. The solubility of -Lg depends on pH and ion strength – but it does not precipitate during milk acidification [25].
A number of useful nutritional and functional features have made -Lg become an ingredient of choice for food and beverage formulation: in fact, it holds excellent heat-gelling [26] and foaming features – which can be used as structuring and stabilizer agents in such dairy products as yogurts and cheese spreads. This protein is resistant to gastric digestion, as is stable in the presence of acids and proteolytic enzymes [22, 27-30]; hence, it tends to remain intact during passage through the stomach. It is also a rich source of Cys, an amino acid bearing a key role in stimulating synthesis of glutathione (GSH) – composed by three amino acids, Glu, Cys and Gly [31].
Many techniques have been developed for purification of β-Lg – which normally rely on its precipitation [32-35]; when large scale purification is intended, precipitation is usually complemented by ion exchange [35-36].
2.2.2. -Lactalbumin (-La)
-La appears quantitatively second in whey; it comprises ca. 20 % of all proteins in bovine whey, and 3.5 % of the total protein content of whole milk [9]. It is a calcium metalloprotein composed of 123 amino acids, with a MW of 14.4 kDa [37]; and appears as three genetic variants (A, B and C), with B being the most common [38]. Chromatographic and electrophoretic analysis within stability studies carried out at various times and temperatures (pH 6.7) indicated that -La is more heat resistant than -Lg – in part due to its denaturation being reversible below 75 °C [39]. Owing to such a relatively high thermal stability, it holds a poor capacity to gel; however, it can be easily incorporated in fluid or viscous products to increase their nutritional value. This protein is commercially used in supplements for infant formulae, because of its similarity in structure and composition to human milk proteins – coupled with its higher content of Cys, Trp, Ile, Leu and Val residues, which make it also the ingredient of choice in sport supplements [13, 40-41]. Regarding tertiary structure, -La is a compact globular protein consisting of 26 % -helix, 14 % -sheet and 60 % other motifs; it is also very similar to lysozyme [9]. This protein is one of the most studied proteins with regard to understanding the mechanism of protein stability and folding/unfolding [42]; at low pH [43], high temperature [44] or moderate concentrations of denaturants – e.g. guanidine hydrochloride [45], -La adopts a conformational structure called molten globule. A partially unfolded state, the apo-state, is formed at neutral pH upon removal of Ca2+ by ethylenediamine tetracetic acid (EDTA) [46-47]; this state preserves its secondary, but not its tertiary structure [48].
The molten globule state of -La retains a high fraction of its native secondary structure, as well as a flexible tertiary structure [45, 48-49]; it accordingly appears as an intermediate in the balance between native and unfolded states [50-51]. This structure of -La is highly heterogeneous, with proeminence of -helix driven mainly by weak hydrophobic interactions – while the -sheet domain is significantly unfolded.
2.2.3. Caseinomacropeptide (CMP)
CMP is a heterogeneous polypeptide fraction derived from cleavage of Phe105-Met106 in -casein (-CN). When milk is hydrolyzed with chymosin during cheesemaking, -CN is hydrolyzed into two portions: one remains in the cheese (
The amino acid sequence of CMP is well-known; it lacks aromatic amino acid residues (Phe, Trp and Tyr) and Arg, but several acidic and hydroxyl amino acids are present [53]. CMP from cow is soluble at pH in the range 1-10, with a minimum solubility (88 %) between pH 1 and 5 [54-55]. CMP appears to remain essentially soluble following heat treatment at 80-95 °C for 15 min at pH 4 and 7 [55]. Its emulsifying activity exhibits a minimum near the isoelectric point [54]. Dziuba and Minkiewicz [56] showed that a decrease in pH leads to a decrease in CMP volume, owing to reduction of internal electrostatic forces and steric repulsion; this apparently has a significant influence upon its emulsifying capacity.
2.2.4. Bovine serum albumin (BSA)
BSA is derived directly from the blood, and represents 0.7-1.3 % of all whey proteins [8]. Its molecule has 582 amino acid residues and a MW of 69 kDa – and contains 17 disulfide bonds and one free sulphydryl group [9]. Because of its size and higher levels of structure, BSA can bind free fatty acids and other lipids, as well as flavor compounds [57] – but this feature is severely hampered upon denaturation. Its heat-induced gelation at pH 6.5 is initiated by intermolecular thiol-disulphide interchange – similar to what happens with -Lg [58].
2.2.5. Immunoglobulins (IGs)
IGs represent 1.9-3.3 % of the total milk proteins, and are derived from blood serum [8]; they constitute a complex group, the elements of which are produced by β-lymphocytes. Igs encompass three distinct classes: IgM, IgA and IgG (IgG1 and IgG2) – with IgG1 being the major Ig present in bovine milk and colostrum [8], whereas IgA is predominant in human milk. The physiological function of Igs is to provide various types of immunity to the body; they consist of two heavy (53 kDa) and two light (23 kDa) polypeptide chains, linked by disulfide bridges [9]. The complete Ig, or antibody molecule has a MW of about 180 kDa [59]. Igs are partially resistant to proteolytic enzymes, and are in particular not inactivated by gastric acids [59].
2.2.6. Lactoferrin (LSs)
LFs are single-chain polypeptides of ca. 80 kDa, containing 1-4 glycans depending on the species. Bovine and human LFs consist of 989 and 691 amino acids, respectively [60]: the former is present to a concentration of ca. 0.1 mg mL-1 [25, 61], and is an iron-binding glycoprotein - so it is thought to play a role in iron transport and absorption in the gut of young people.
2.2.7. Proteose-peptones (PPs)
The total PP fraction (TPP) of bovine milk represents ca. 10 % of the whole whey protein content; it is accounted for by the whey protein fraction soluble after heating at 95 °C for 30 min, followed by acidification to pH 4.6 [62]. The TPP fraction is often divided in two main groups: the first one includes PPs originated from casein hydrolysis; its principal components have been labeled as 5 (PP5), 8 fast (PP8 fast) and 8 slow (PP8 slow), according to their electrophoretic mobility [62, 63]. PP3 constitutes the second group, and it is not derived from casein (it is actually found only in whey); it is known for its extreme hydrophobicity.
2.3. Functional ingredients from whey proteins
Whey proteins have unique characteristics [64] beyond their great importance in nutrition; they exhibit chemical, physical, physiological, functional and technological features also useful for food processing [14]. Based on these properties, more and more individual proteins and protein concentrates of whey have been incorporated in food at industrial scale. Therefore, whey proteins address two major issues in practice: nutritionally, they supply energy and essential amino acids, besides being important for growth and cellular repair; in terms of functionality, they play important roles upon texture, structure and overall appearance of food – e.g. gel formation, foam stability and water retention.
A few physiological properties useful in therapies have been found [65]: a number of reviews have accordingly examined to some length the bioactive properties of whey proteins in general [66-67], or of -Lg and -La in particular [26]; other authors have covered mainly such biological activities as anticarcinogenic [68] and immunomodulatory [69]. It was observed that whey proteins trigger immune responses that are significantly higher than those by diets containing casein or soy protein. Antimicrobial and antiviral actions, immune system stimulation and anticarcinogenic activity (among other metabolic features) have indeed been associated with ingestion of -Lg and -La, as well as LF, LP, BSA and CMP; the main biological activities of whey proteins are listed in Table 3.
With regard to bioactive peptides, research has undergone a notable intensification during the past decade [4, 70]. Advances in nutritional biochemistry and biomedical research have in fact helped unravel the complex relationships between nutrition and disease, thus suggesting that food proteins and peptides originated during digestion (or from
Prevention of cancer | [74] | ||
Breast and intestinal cancer; | [14, 75] | ||
Chemically-induced cancer | [76-77] | ||
Increment of gluthatione levels | [64] | ||
Increase of tumour cell vulnerability | [78-79] | ||
Antimicrobial activities | [80] | ||
Increment of satiety response | |||
Increment in plasma amino acids, cholecystokinin and glucagon-like peptide | [81] | ||
Enzyme hydrolysis | [82] | ||
Antiulcerative | |||
Prostaglandin production | [83-85] | ||
Enzyme hydrolysis | Antiulcerative | [83, 86] | |
Transporter | |||
Retinol | [9, 41, 87, 88] | ||
Palmitate | [89] | ||
Fatty acids | [90] | ||
Cellular defence against oxidative stress and detoxification | [31, 65, 91-93] | ||
Enhancement of pregastic esterase activity | [94] | ||
Transfer of passive immunity | [95] | ||
Regulation of mammary gland phosphorus metabolism | [96] | ||
Enzyme hydrolysis; Fermentation | [97-107] | ||
Enzyme hydrolysis | Antimicrobial against several gram-positive bacteria | [108-111] | |
Enzyme hydrolysis | Antimicrobial (bactericidal) | [112-113] | |
Enzyme hydrolysis | Hypocholesterolemic | [113-114] | |
Enzyme hydrolysis | Opioid agonist | [73, 97, 115] | |
Enzyme hydrolysis | [99, 116-117] | ||
Enzyme hydrolysis | Ileum contracting | [97, 99] | |
Enzyme hydrolysis | Antinociceptive | [118] | |
Prevention of cancer | |||
Enzyme hydrolysis | Intestinal cancer | [14] | |
Prevention of cancer | [119] | ||
Apoptosis of tumoral cells | [120-122] | ||
Lactose synthesis | [25, 123] | ||
Treatment of chronic stress-induced disease | [124] | ||
Antimicrobial (bactericidal) | |||
[125] | |||
Stress reduction | [123, 126] | ||
Immunomodulation | [127] | ||
Enzyme hydrolysis | Antimicrobial against several gram-positive bacteria | [108-110] | |
Enzyme hydrolysis | Opioid agonist | [97, 115, 128] | |
Enzyme hydrolysis | [26, 97-98, 101, 107] | ||
Enzyme hydrolysis | [117, 129] | ||
Enzyme hydrolysis | Ileum contracting | [97] | |
Antiulcerative | |||
Prostaglandin production | [130-132] | ||
Fatty acid binding | [13] | ||
Antioxidant | [133-134] | ||
Prevention of cancer | [135] | ||
Enzyme hydrolysis | [136-137] | ||
Enzyme hydrolysis | Ileum contracting | [138] | |
Enzyme hydrolysis | Opioid agonist | [97, 128, 139] | |
Immunomodulation | [140] | ||
Disease protection through passive immunity | [141-142] | ||
Antibacterial | [143-145] | ||
Antifungal | [146] | ||
Opioid agonist | [147] | ||
Antithrombotic | [148-153] | ||
[154-156] | |||
Antimicrobial | [56, 111, 157-160] | ||
Enzyme hydrolysis | Prebiotic | [161] | |
Increment in plasma amino acids and cholecystokinin peptide | [162-165] |
Although inactive within the primary structure of their source proteins, hydrolysis (e.g. mediated by a protease) may release peptides with specific amino acid sequences possessing biological activity. A number of chemical and biological methods of screening have accordingly been developed to aid in search for specific health effects; however, only some of those found
Scientific evidence has shown that whey proteins contain a wide range of peptides that can play crucial physiological functions and modulate some regulatory processes (see Table 3). Due to its high biological value, coupled with excellent functional properties and clean flavor, whey has earned the status of a recommended source of functional ingredients [71] – designed to reduce or control chronic diseases and promote health, thus eventually reducing the costs of health care [3, 166].
Favorable health effects have indeed been claimed for some peptides derived from food proteins – being able to affect the cardiovascular, nervous, digestive or immune systems; these encompass antimicrobial properties, blood pressure-lowering (or angiotensin-converting enzyme (ACE)-inhibitory) effects, cholesterol-lowering ability, antithrombotic and antioxidant activities, enhancement of mineral absorption and/or bioavailability thereof, cyto- or immunomodulatory effects, and opioid features. With regard to the mechanisms underlying the physiological roles of bioactive peptides, a few involve action only upon certain receptors, whereas others are enzyme inhibitors; they may also regulate intestinal absorption, and exhibit antimicrobial or antioxidant activities. Recall that oxidative metabolism is essential for survival of cells, but it generates free radicals (and other reactive oxygen species) as side effect – which may cause oxidative damage. Antioxidant activity has been found specifically in whey proteins, probably via scavenging of such radicals via Tyr and Cys amino acid residues – which is predominantly based on proton-coupled single electron or hydrogen atom transfer mechanisms; or else chelation of transition metals [167-168].
On the other hand, bioactive peptides derived from food proteins differ in general from endogenous bioactive peptides in that they can entail multifunctional features [98]. Furthermore, bioactive peptides that cannot be absorbed though the gastrointestinal tract may exert a direct role upon the intestinal lumen, or through interaction with receptors in the intestinal wall itself; some of these receptors have been implicated in such diseases as cancer, diabetes, osteoporosis, stress, obesity and cardiovascular complications.
3. Production of bioactive peptides in whey
Bioactive peptides derived from whey proteins constitute a new concept, and have open up a wide range of possibilities within the market for functional foods [4, 169]; of special interest are those released via enzymatic action – as happens during clotting in cheesemaking.
The enzymes used to bring about milk coagulation are selected protein preparations that provide in general a high clotting activity – i.e. a considerable, but selective proteolytic activity. Animal rennet obtained from the calf stomach, composed by 88-94 % and 6-12 % chymosin and pepsin, respectively, has been the coagulant of choice for cheesemaking. However, due to increased world production of cheese, the supply of animal rennet has lied below its demand; the increased prices have driven a search for alternative coagulants (including plant and microbial sources). With regard to animal rennet substitutes, pig pepsin has enjoyed a remarkable commercial success; with regard to rennet from microbial origin, the proteases from
Chymosin and the other rennet substitutes are aspartic proteases, with optimal activity at acidic pH, and possessing high degree of homology in primary and 3-dimensional structures, 3-dimensional structure and catalytic mechanism. The specificity towards the substrate is, however, rather variable; although they have a greater tendency to break peptide bonds between hydrophobic amino acids having bulky side residues, they hydrolyze a large number of bond types [172]. Of particular interest is vegetable rennet, which – with few exceptions, enjoys a still limited use worldwide. Many plant enzyme preparations proved indeed to be excessively proteolytic for manufacture of cheese, causing defects in terms of flavor and texture of the final product. These difficulties arise from the presence of non-specific enzymes that belong to complex enzyme systems (which, as such, are difficult to control). An exception to the poor suitability of vegetable coagulants is the proteinases in aqueous extracts of plants of the
Bioactive peptides derived from whey proteins can be released at industrial scale via enzyme-mediated hydrolysis with digestive enzymes – and pepsin, trypsin and chymotrypsin have been the most frequent vectors therefor [4, 169, 173]. However, whey proteins are not easily broken down by proteases in general – a realization that also explains their tendency to cause allergies upon ingestion [174]. Hence, less conventional sources of proteolytic enzymes have been sought that can cleave the whey protein backbone at specific and usual sites. This is the case of aspartic proteinases present in the flowers of
4. Recovery of proteins/peptides from whey
The relatively low concentration of proteins in whey requires concentration processes to assure high hydrolysis productivity. Development of membrane separation techniques has been essential toward this endeavor – and food industry has taken advantage of its relatively easy scale-up, as well as its being inexpensive when compared with preparative chromatographic techniques [41]. Furthermore, the absence of heat treatment allows the bioactive components to remain intact (or become only slightly affected) during processing. Recall that membrane separation allows differential concentration of a liquid, provided that the solute of interest is larger in molecular diameter than the membrane pores – so the liquid that percolates the membrane (filtrate) contains only components smaller than that size threshold [179].
The dairy industry has pioneered development of equipment and techniques for membrane filtration, which recovers whey proteins in a non-denatured state. Typical procedures include: (i) basic membrane separation, e.g. reverse osmosis, ultrafiltration and diafiltration [180-186], that permits fractionation of proteins, as well as concentration and purification thereof; (ii) nanofiltration (or ultraosmosis) that allows removal of salts or low MW contaminants; and (iii) microfiltration to remove suspended solid particles or microorganisms [179, 187]. Note that isolation of individual whey proteins on laboratory scale has resorted chiefly to salting out, ion exchange chromatography and/or crystallization [188]; such a fractionation allows fundamental studies of their immunological properties to be carried out, which are necessary to establish and support industrial interest [189-190].
5. Activity of peptides from whey upon hypertension
Hypertension is a major public health issue worldwide that affects nearly one fourth of the population; and it is usually associated with such other disorders as obesity, pre-diabetes, renal disease, atherosclerosis and heart stroke [191-194]. Its specific treatment will likely reduce the risk of incidence of cardiovascular diseases, which currently account for 30 % of all causes of death [195].
Blood pressure can be regulated through diet changes and physical exercise, as well as administration of calcium T channel antagonists, angiotensin II receptor antagonists, diuretics and ACE inhibitors [104]. A few mechanisms have been described that rationalize how peptides lower blood pressure. Traditionally, control of hypertension has focused on the renin-angiotensin system, via inhibition of ACE [173]. Captopril, enalapril and lisinopril have accordingly been used as antihypertensive drugs that act essentially as ACE inhibitors; they found a widespread application in treatment of patients with hypertension, heart failure or diabetic nephropathies [193, 196-197]. However, they bring about undesirable side effects, so safer (and, hopefully, less expensive) alternatives are urged [198-199].
In fact, increasing evidence has been provided that mechanisms other than ACE inhibition may be involved in blood pressure decrease arising from consumption of many food-derived peptides [200]; although there are few studies to date with antihypertensive peptides obtained from whey. One of them corresponds to interaction with opioid receptors that are present in the central nervous system and in peripheral tissues, while another is based on release of nitric oxide (NO) that causes vasodilatation and thus affects blood pressure. Those peptides hold the advantage of no side effects, unlike happens with such other opiates as morphine [102]. One example is α-lactorfin, a tetrapeptide derived from α-La [129, 201], for which studies showed that antihypertensive effects are mediated through the vasodilatory action of binding to opioid receptors. Furthermore, endothelium-dependent relaxation of mesenteric arteries in spontaneously hypertensive rats (SHR, which is the animal model normally accepted to study human hypertension) that was inhibited by an endothelial nitric oxide synthase (eNOS) inhibitor was also observed [202]. That peptide may even chelate minerals, and thus facilitates calcium absorption [200].
Alternative mechanisms are other routes of vasoregulator substance synthesis – e.g. kallikrein-kinin, neutral endopeptidase and endothelin-converting enzyme systems. The release of vasodilator substances like prostaglandin I2 or carbon monoxide could be implied in dependent and independent mechanisms of ACE inhibition responsible for antihypertensive effects [203-205]; an example is the peptide ALPMHIR, which inhibits release of an endothelial factor (ET-1) that causes contractions in smooth muscle cells [206].
In the last decade, production of antioxidant peptides from whey has been reported [207]. Experimental evidence – including SHR and human studies, claimed that oxidative stress is one of the causes of hypertension and several vascular diseases, via increase production of reactive oxygen species and reduction of NO synthesis and bioavailability of antioxidants [208].
Nevertheless, the most intensively studied peptides – i.e. VPP and IPP derived from caseins, showed possible mechanisms of action that could be found also in other peptides. In studies performed with rats, VPP and IPP increased plasma renin levels and activity [202]; and decreased ACE activity in the serum; they also showed endothelial function protection in mesenteric arteries [208]. The influence of VPP and IPP on gene expression of SHR abdominal aorta unfolded a significant increase of genes related with blood pressure regulation – the eNOS and connexin 40 genes [208]. Other studies have highlighted the peptide effects on the vasculature itself, showing that the antihypertensive activity of the peptide rapakinin is induced mainly by CCK1 and IP-receptor-dependent vasorelaxation; this peptide relaxes the mesenteric artery of SHR via prostaglandin I2-IP receptor, followed by CCK-CCK1 receptor pathway; other peptides improve aorta and mesenteric acetylcholine relaxation, and decrease left ventricular hypertrophy, accompanied by significant decrease in interstitial fibrosis [209]. In order to prevent hypertension, two alternative enzyme inhibitors were suggested: renin (a protease recognized as the initial compound of the renin–angiotensin system) and platelet-activating factor acetylhydrolase (PAF-AH) (a circulating enzyme secreted by inflammatory cells and involved in atherosclerosis) [208].
5.1. Inhibition of angiotensin-converting enzyme (ACE)
Since diet has a direct relationship to hypertension, the food industry (in association with research and public health institutions) has promoted development of novel functional ingredients that can contribute to keep a normal blood pressure – thus avoiding the need to take antihypertensive drugs [73, 173, 209-212]. Various investigators have accordingly hypothesized that certain peptides formed through hydrolysis of food proteins have the ability to inhibit ACE; López-Fandiño [173], FitzGerald [104, 137], Gobetti [213], Meisel [214], Korhonen and Pihlanto [4], Silva and Malcata [215], Vermeirssen [216], and Martínez-Maqueda [208] have comprehensively reviewed this subject. In general, it has been claimed that a diet rich in foods containing antihypertensive peptides is effective toward prevention and treatment of hypertension [173, 201].
ACE-inhibitory peptides may be obtained from precursor food proteins via enzymatic hydrolysis, using viable or lysed microorganisms or specific proteases [3, 73, 137, 169]. Although
In the latest two decades, various active peptides have been identified from animal proteins, including some with antihypertensive effects in animals (e.g. SHR) and even in humans [3, 73, 137, 169, 173, 201, 208, 212, 217, 299]: bovine plasma proteins [218], egg proteins [203, 219] and tuna proteins [220]; but also plant proteins, e.g. from soy [221], wine [222] and maize [223]. Nevertheless, milk proteins still appear to be the best source of ACE-inhibitory peptides.
Recall that caseins are the most abundant proteins in milk, and have an open and flexible structure that makes them susceptible to attack by proteases; hence, many ACE-inhibitors have been obtained via enzyme-mediated approaches [224-225] – e.g. casokinins. Studies on peptides with ACE-inhibitory activity obtained from whey proteins (called lactokinins) are more limited – which may be due to the rigid structure of -Lg (the major whey protein) that makes it particularly resistant to digestive enzymes. However, bioactive protein fragments with ACE-inhibitory activity have been found in whey protein hydrolyzates [107, 217, 226-228]; and Manso and López-Fandiño [155] also identified this activity in CMP hydrolyzates. Characterization of hydrolyzates of the main whey proteins – including the amino acid sequences of peptides therein that exhibit
The ACE-inhibitory activity depends on the protein substrate and the proteolytic enzymes used to break it down. ACE (i.e. a dipeptidyl carboxypeptidase) is an enzyme ubiquitous in tissues and biological fluids – where it plays an important physiological role upon regulation of the cardiovascular function, including a basic role in regulation of peripheral blood pressure via the renin-angiotensin system [229-230]. ACE inhibitors and angiotensin II receptor blockers [231-232] have been therapeutically important, since they act as efficient drugs and bring about very few collateral effects.
ACE-inhibitor peptide can reduce blood pressure in a process regulated (in part) by the renin-angiotensin system: renin — a protease secreted in response to various physiological stimuli, cleaves the protein angiotensinogen to produce the inactive decapeptide
Fermentation + trypsin + chymotrypsin | -Lg f9-14 | GLDIQK | 580 | [104, 233] | ||
Yogurt starter + trypsin + pepsin | -Lg f15-20 | VAGTWY | 1682 | [100] | ||
Fermentation with lactic acid bacteria + prozyme 6 | -Lg f17-19 | GTW | 464.4 | [105] | ||
Cardosins | -Lg f33-42 | DAQSAPLRVYc | 12.2 | 10 (5) | [107, 117] | |
Proteinase K | -Lg f78-80 | IPAc | 141 | 31 (8) | [136] | |
Cardosins | -La f16-26 | KGYGGVSLPEWc | 0.7 | 20 (5) | [107, 117] | |
Cardosins | -La f97-103 | DKVGINYc | 99.9 | [107] | ||
Cardosins | -La f97-104 | DKVGINYWc | 25.4 | 15 (5) | [107, 117] | |
Fermentation + trypsin + chymotrypsin | -La f105-110 | LAHKAL | 621 | [100] | ||
Fermentation by cheese microflora | -La f104-108 | WLAHK | 77 | [233] | ||
Neutrase | -La f105-110 | INYWL | 11 | [234] | ||
Trypsin | f7-9 | MKG | 71.8 | [103] | ||
Trypsin | f10-14 | LDIQK | 27.6 | [103] | ||
Pepsin + trypsin + chymotrypsin | f15-19 | VAGTW | 1054 | [233] | ||
Trypsin | f22-25 | LAMA | 556 | [233] | ||
Trypsin | f32-40 | LDAQSAPLR | 635 | [233] | ||
Protease N Amano | f36-42 | SAPLRVY | 8 | [235] | ||
Thermolysin | f58-61 | LQKWc | 34.7 | 18.1 (10) | [103, 236] | |
Trypsin | f81-83 | VKF | 1029 | [233] | ||
Pepsin + trypsin + chymotrypsin | f94-100 | VLDTDYK | 946 | [106, 233] | ||
Pepsin + trypsin + chymotrypsin | f102-103 | YLc | 122 | [214] | ||
Pepsin + trypsin + chymotrypsin | f102-105 | YLLFc | 172 | [113] | ||
Thermolysin | f103-105 | LLFc | 79.8 | 29 (10) | [113, 236] | |
Pepsin + trypsin + chymotrypsin | f106-111 | CMENSA | 788 | [233] | ||
Pepsin + trypsin + chymotrypsin | f142-145 | ALPMc | 928 | 21.4 (8) | [116] | |
Pepsin + trypsin + chymotrypsin | f142-146 | ALPMHc | 521 | [233] | ||
Trypsin | f142-148 | ALPMHIRc | 43 | [226] | ||
Thermolysin | f15-26 | LKGYGGVSLPEW | 83 | [237] | ||
Thermolysin | f18-26 | YGGVSLPEW | 16 | [237] | ||
Thermolysin | f20-26 | GVSLPEW | 30 | [237] | ||
Thermolysin | f21-26 | VSLPEW | 57 | [237] | ||
Synthetic | f50-51 or f18-19 | YGc | 1522 | [98] | ||
Pepsin + trypsin + chymotrypsin | f50-52 | YGL | 409 | [233] | ||
Pepsin | f50-53 | YGLFc | 733 | 23.4 (0.1) | [129, 233] | |
Synthetic | f52-53 | LFc | 349 | [26] | ||
Trypsin | f99-108 | VGINYWLAHK | 327 | [233] | ||
Trypsin | f104-108 | WLAHK | 77 | [233] | ||
Proteinase K | f208-216 | ALKAWSVARc | 3 | [238] | ||
Proteinase K | f221-222 | FP | 315 | 27 (8) | [136] | |
Trypsin | f106-112 | MAIPPKK | 28 (10) | [239] | ||
Pepsin | f20-25 | RRWQWR | 16.7 (10) | [240] | ||
Pepsin | f22-23 | WQ | 11.4 (10) | [240] |
angiotensin I. Cleavage of angiotensin I – via removal of two amino acid residues from the C-terminal end by ACE, produces the active octapeptide angiotensin II that is a potent vasoconstrictor; however, there are alternative routes to generate angiotensin II [198, 241-242]. Angiotensin II activates angiotensin II type 1 (AT1) receptor — a member of the G-protein-coupled-receptor superfamily, which plays various roles, e.g. vasoconstriction, as well as stimulation of aldosterone synthesis and release (which leads to sodium retention, and thus increases blood pressure) [198, 217, 242]. In addition, ACE acts on the kallikrein-kinin system, catalyzing degradation of the nonapeptide bradykinin – which is a vasodilator [241]. ACE-inhibitor peptides exert a hypotensive effect by preventing angiotensin II formation and degradation of bradykinin, thus reducing blood pressure in hypertensive patients [217].
Several tests on SHRs – probably the best experimental model for antihypertensive studies because they exhibit vascular reactivity and renal function similar to those in human beings [243], have been described that prove control of arterial blood pressure following a single oral administration of known ACE-inhibitory hydrolyzates or/and peptides derived from whey proteins. The antihypertensive effect associated with some of those peptides is comparable to that exhibited by VPP – an antihypertensive peptide included in functional foods that is already available in the market [117, 129, 137, 154, 201, 210-212, 242, 244-247]. To measure ACE-inhibitory activity, distinct biological, radiochromatographic, colorimetric and radioimmunologic methods have been employed – using angiotensin I as substrate. Chemical methods are sensitive, and resort to a tripeptide with a substituted amino-terminus, Z-Phe-His-Leu, as ACE-substrate – from which the dipeptide His-Leu is released and quantified by specific fluorometric procedures. A similar tripeptide used as substrate of ACE is Bz-Gly-His-Leu, or Hippuryl-His-Leu (HHL); upon incubation with the enzyme, hippuric acid is formed and the dipeptide His-Leu is released, which is subsequently measured by one of several colorimetric [248] or fluorometric methods [249], or even by capillary electrophoresis [250].
One of the most performing methods to measure ACE-inhibitory activity was developed by Cushman and Cheung [251], and is based on spectrophotometric measurement at 228 nm of hippuric acid formed by incubating the substrate HHL with ACE – in the presence of selected inhibitory substances. More recently, a modified tripeptide, furanacriloil Gly-Phe-Gly, has been chosen as substrate for a spectrophotometric method [252]. The ACE-inhibitory activity is usually measured in terms of IC50 (i.e. the concentration of inhibitory substance required to inhibit 50 % of ACE activity); a low IC50 value means that a small concentration of inhibitory substance is required to produce enzyme inhibition, so that substance displays a potent inhibitory activity.
As shown in Table 4, ACE-inhibitor peptides are produced mainly by enzymatic hydrolysis, but active sequences have also been obtained via chemical synthesis [253]. Starter and non-starter bacteria are commonly used in cheese manufacture – taking advantage namely of their proteolytic system, which contains at least 16 different peptidases that have already been characterized. Some of these bacteria were found to have ACE-inhibitory activity, or release peptides with this activity. For instance,
5.1.1. Structure/activity relationships
ACE-inhibitor peptides contain usually between 2 and 12 amino acid residues – even though larger peptides may also exhibit such an activity [173]. Ondetti [229] rationalized the interaction of competitive inhibitors for the ACE active site based on enzyme homology with carboxypeptidase A; the first ACE-inhibitor (i.e. captopril), which is one of the oral drugs widely used to treat hypertension, was designed based on this model. Recently, this model was reviewed and used to design even more potent ACE inhibitors [229, 256-257]. The base model proposes that residues of the carboxy-terminal (C-terminal) tripeptide interact with the S1, S'1 and S'2 subunits of the enzyme active site. One of the subunits has a positively charged group that forms an ionic bond with the C-terminal peptide group. The following subunit contains a group capable of interacting with the peptidic bond of the C-terminal amino acid – probably through hydrogen bonding. The third subunit has a Zn2+ atom able to carry the carbonyl group of the peptidic bond between the one before the last and the last amino acid residue of the substrate – thus making it more susceptible to hydrolysis [256].
Although the relationships between structure and activity have not been fully elucidated, ACE-inhibitory peptides possess a number of analogies with each other. The tripeptide at the C-terminus is crucial – because this is where the peptide binds to the active site of the enzyme [256]. ACE prefers substrates (or competitive inhibitors) with hydrophobic residues (e.g. Trp, Tyr, Phe and Pro) at the C-terminus, and shows poorer affinity to substrates containing dicarboxylic amino acids in the final position, or those that have a Pro residue in the one before the last position. However, presence of Pro as the last residue [258], or in the third position from the terminus [259] favors binding of peptide to enzyme, in much the same way as when Leu appears in the last position [260,261].
Bioinformatics has been used more recently to find the structural requirements of ACE-inhibitor peptides; these are termed quantitative structure/activity relationship (QSAR) models. Through a QSAR model, Pripp [262] concluded – for milk-derived peptides up to six amino acids in length, that there is a relationship between ACE-inhibitory activity and presence of a hydrophobic (or positively charged) amino acid residue in the last position of the sequence; however, no special relation was found with the structure of the N-terminus. Based on the QSAR model for peptides containing between 4 and 10 amino acid residues, Wu [263] claimed that the residue of the C-terminal tetrapeptide may determine the potency of ACE inhibition – with preference for Tyr and Cys in the first C-terminal position; His, Trp and Met in the second; Ile, Leu, Val and Met in the third; and Trp in the fourth position. Results from other QSAR-based studies aimed at finding ACE-inhibitory activity of di- and tripeptides derived from food proteins have shown that dipeptides with hydrophobic chains, as well as tripeptides with an aromatic amino acid residue at the C-terminus, a positively charged residue at the intermediate position and a hydrophobic amino acid residue at the N-terminus are likely to exhibit ACE-inhibitory power [263].
On the other hand, a biopeptide may adopt a different configuration depending on the prevailing environmental conditions; but the final structural conformation may be crucial for its ACE-inhibitory activity. The fact that the catalytic center of ACE has different structural requirements may unfold the need to develop complex mixtures of peptides, with different structural conformations, so as to produce more complete inhibition than a single peptide [264]. Meisel [265] postulated that the mechanism of ACE inhibition may involve interaction of inhibitor with the subunits that are not normally occupied by substrate, or with the anionic bond site that is different from the enzyme catalytic center. Moreover, somatic ACE has two homologous domains – each of which has an active site with distinct biochemical characteristics.
Despite the importance arising from the three amino acids in their C-terminus, it was shown that peptides with identical sequences at the C-terminus may exhibit quite different ACE-inhibitory activities from each other. One example is VRYL and VPSERYL, both identified in Manchego cheese; despite having the same C-terminal tripeptide sequence, they exhibit IC50 values of 24.1 M and 249.5 M, respectively – i.e. the latter is 10-fold less active than the former. If Val were replaced by a dicarboxylic amino acid at the fourth position of the C-terminus, e.g. via synthesis of ERVL, the IC50 measured would be 200.3 M, which corresponds to an ACE-inhibitory activity 8-fold lower than VRYL – hence demonstrating the crucial role of Val in that position for the intended bioactivity [261].
5.1.2. Bioavailability
Among the several bioactive peptides studied to date, ACE-inhibitory peptides have received particular attention because of their beneficial effects upon hypertension [226, 233, 267]. Note that such effects depend on their ability to reach the target organs without having undergone decay or transformation. Tests encompassing hypertensive animals and human clinical trials have shown that certain sequences can lower blood pressure; however, it is difficult to establish a direct link between the ability to inhibit ACE
Some peptides with ACE-inhibitory and antihypertensive activities can be transported through the intestinal mucosa via the PepT1 transporter [269]; likewise, there is evidence that other peptides may exert a direct role upon the intestinal lumen [151, 270-271]. Digestive enzymes, absorption through the intestinal tract and blood proteases can bring about hydrolysis of ACE-inhibitor peptides, thus producing fragments with lower or greater activity than their precursor sequences [216]. Hence, for ACE-inhibitor peptides exert an
Effective inclusion of ACE-inhibitory peptides in the diet consequently requires them to somehow resist the strong stomach hydrolysis that may cause loss of bioactivity [104], and afterwards be able to pass into the blood stream – where they should be resistant to peptidases therein, so as to eventually reach the target sites where they are supposed to exert their physiological effects
Besides carrying out protein degradation to varying extents, gastrointestinal digestion plays a key role in formation of ACE-inhibitory peptides [216, 277]; hence, it is relevant to assess the gastrointestinal bioavailability of any potentially interesting peptides. Several studies have accordingly provided evidence for this realization – as happened with Manchego cheese, as well as with other fermented solutions and infant formulae [100, 261, 278-281]; for instance, a potent antihypertensive peptide was released via gastrointestinal digestion from a precursor with poor ACE-inhibitory activity
Animal and human trials are therefore nuclear when assessing bioactivity of peptides; peptides that do not show
Simulated (physiological) digestion is a useful tool to assess the stability of peptides with ACE-inhibitory activity against digestive enzymes. However, the degree of hydrolysis of a given peptide depends not only on its size and nature, but also on the presence of other peptides in its vicinity [272] – which would make it difficult to test the required number of possibilities in a rather limited experimental program. Several
Some authors used whey proteins, fermented (or not at all) with
6. Concluding remarks
Processing of whey proteins yields several bioactive peptides able to trigger physiological effects in the human body. Such peptides, in concentrated form, can be commercially appealing because their claimed health-promoting features are nowadays an important driver for consumers’ food choices. Hence, they may constitute an excellent alternative for whey upgrade. Use of selective membranes to isolate, and eventually purify whey proteins and peptides has substantially increased the number and depth of studies encompassing those molecules and their hydrolysates. The technology developed is not excessively expensive, and can easily be implemented in dairy plants – of either small or large dimension. Most whey peptides bearing biological activity are released by enzymatic hydrolysis, so new alternatives to enzymes of animal origin have been under scrutiny.
This chapter focused on studies of whey peptides with antihypertensive activity – including their mechanisms of action (especially ACE inhibition), as well as the bioavailability of these peptides, and highlighting the main
Although a good deal of data have been generated encompassing food bioactive peptides, much is still left to do with whey peptides. Hence, several opportunities for further research exist, on incorporation of said ingredients in food products for human consumption. However, several scientific, technological and regulatory issues should be addressed before such peptide concentrates (and pure peptides) will have a chance to be marketed at large, aiming at both human nutrition and health.
More detailed studies are indeed welcome for a better understanding of antihypertensive mechanisms. In particular, the antihypertensive activity should be checked with extra detail – including deep studies on the blood pressure-reducing mechanisms, such as the effects of peptides on neutral endopeptidases and their putative beneficial activity upon cardiovascular diseases. The pharmacological effect of said peptides should be determined both on post- and prejunctional receptors. More extensive clinicaltrials should also be performed – after thorough bioavailability studies
Acknowledgement
This work received partial finantial support from project NEW PROTECTION – NativE, Wild PRObiotic sTrain EffecCT In Olives in briNe (ref. PTDC/AGR-ALI/117658/2010), from FCT, Portugal, coordinated by author F. X. M.
References
- 1.
Diplock A. T Aggett P. J Ashwell M Bornet F Fern E. B Roberfroid M. B Scientific concepts of functional foods in Europe consensus document. British Journal of Nutrition1999 81 1 27 - 2.
Arai S Studies on functional foods in Japan-state of the art. Biotechnology and Biochemistry1996 60 9 15 - 3.
Food-derived bioactive peptides- opportunities for designing future foods. Current Pharmaceutical DesignKorhonen H Pihlanto A 2003 9 1297 1308 - 4.
Bioactive peptides: production and functionality. International Dairy JournalKorhonen H Pihlanto A 2006 16 945 960 - 5.
Pintado M. E Pintado A. E Malcata F. X Controlled whey protein hydrolysis using two alternative proteases 1999 42 1 13 - 6.
Ingredientes y productos lácteos funcionales, bases científicas de sus efectos en la salud. In: Alimentos funcionales (ed.) Fundación Espanõla para la Ciencia y la Tecnología;Recio I López-fandino R 2005 - 7.
Production, functional properties and utilization of milk protein products. In: Fox PF. (ed.) Advanced dairy chemistry- proteins,Mulvihill D. M 1 London, UK: Elsevier;1992 369 404 - 8.
Food proteins, properties and characterization. New York, USA: Wiley- VCH Publishers;Nakai S Modler H. W 1996 - 9.
McSweeney PLH. Milk Proteins. In:Fox P. F Dairy chemistry and biochemistry London, UK: Blackie Academic and Professional;1998 - 10.
Pintado M. E Macedo A. C Malcata F. X Review: technology, chemistry and microbiology of whey cheeses Food Science and Technology International2001 7 105 116 - 11.
Whey: from waste to gold. International Food IngredientsBlenford D. E 1996 1 27 29 - 12.
Barth C. A Behnke U Nutritional significance of whey and whey components Food1997 41 2 12 - 13.
Whey components: millenia of evolution create functionalities for mammalian nutrition: what we know and what we may be overlooking. Critical Reviews in Food Science and NutritionWalzem R. L Dilliard C. J German J. B 2002 42 353 375 - 14.
le Leu RK, Regester GO, Johnson MA, Grinsted RL, Kenward RS, Smithers GW. Whey proteins as functional food ingredients? International Dairy JournalMcintosh G. H Royle P. J 1998 8 425 434 - 15.
Balagtas J. V Hutchinson F. M Krochta J. M Sumner D. A Anticipating market effects of new uses for whey and evaluating returns to research and development 2003 86 1662 1672 - 16.
Miller G. D Jarvis J. K Mcbeon L. D Handbook of Dairy Foods and Nutrition Boca Raton, USA: CRC,2000 - 17.
Proteinas em alimentos protéicos: propriedades, degradações, modificações. Varela, São PauloSgarbieri V. C 1996 139 157 - 18.
Pihlanto-leppälä A Korhonen H Bioactive peptides and proteins. Advances in Food an d Nutrition Research2003 47 175 276 - 19.
Milk Proteins In: Fundamentals of dairy chemistry. van Nostrand Reinhold. New York, USA;Whitney R. M 1988 - 20.
Law AJR Leaver J, Banks JM, Horne DS. Quantitative fractionation of whey proteins by gel permeation FPL. Milchwissenschaft1993 48 663 666 - 21.
Lactoglobulin. In: Roginski H, Fuquay JW, Fox PF (ed) Encyclopedia of Dairy Sciences. New York: Academic Press;Creamer L. K Sawyer L 2003 - 22.
The core lipocalin, bovine -lactoglobulin. Biochimica et Biophysica ActaSawyer L Kontopidis G 2000 1482 136 148 - 23.
Imafidon I. G Farkye Y. N Spanier M. A Isolation, purification, and alteration of some functional groups of major milk proteins: a review Food Science and Nutrition1997 37 663 689 - 24.
Heat denaturation of β-lactoglobulins A and B. Journal of Dairy ScienceGough P Jenness R 1961 44 1163 1168 - 25.
van Bockel. MAJS. Principles of milk properties and processes- Dairy Technology. New York, USA: Marcel Dekker;Walstra P Geurts T. J Noomen A Tellema A 1999 - 26.
Chatterton DEW Smithers G, Roupas P, Brodkorb A. Bioactivity of -lactoglobulin and -lactalbumin- technological implications for processing. International Dairy Journal2006 16 1229 1240 - 27.
Papiz M. Z Sawyer L Eliopoulos E. E North A. C Findlay J. B Sivaprasadarao R Jones T. A Newcomer M. E Kraulis P. J The structure of -lactoglobulin and it s similarity to plasma retinol-binding protein. Nature1986 324 383 385 - 28.
Mansouri A Haertle T Gerard A Gerard H Gueant J. L Retinol free and retinol complexed -lactoglobulin binding sites in bovine germ cells 1997 1357 107 114 - 29.
Plasma membrane receptor for -lactoglobulin and retinol-binding protein in murine hybridomas. BiofactorsMansouri A Gueant J. L Capiaumont J Pelosi P Nabet P Haertle T 1998 7 287 298 - 30.
Barros R. M Ferreira C. A Silva S. V Malcata F. X Quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium sulfate 2001 29 541 547 - 31.
Glutathione: an overiew of biosynthesys and modulation. Chemico-Biological Interaction- LimerickAnderson M. E 1998 111 1 14 - 32.
On the fractionation of -lactoglobulin and -lactalbumin. Biochimica et Biophysica ActaArmstrong J. M Mckenzie H. A Sawyer W. H 1967 147 60 72 - 33.
Crystal structure of the trigonal form of bovine -lactoglobulin and of its complex with retinol at 2.5 Å resolution. Journal of Molecular BiologyMonaco H. L Zanotti G Spadon P Bolognesi M Sawyer L Eliopoulos E. E 1987 197 695 706 - 34.
Felipe X Law A. J Preparative-scale fractionation of bovine, caprine and ovine whey proteins by gel permeation chromatography. Journal of Dairy Research1997 64 459 464 - 35.
De Jongh H. H Groneveld T De Groot J Mild isolation procedure discloses new protein structural properties of -lactoglobulin 2001 84 562 571 - 36.
Outinen M Tossavainen O Tupasela T Koskela P Koskinen H Rantamaki P Syvaoja E. L Antila P Kankare V Fractionation of proteins from whey with different pilot scale processes Food Science and Technology1996 29 411 417 - 37.
Lactalbumin: a calcium metalloprotein. Biochemical and Biophysical Research CommunicationHiraoka Y Segawa T Kuwajima K Sugai S Murai N 1980 95 1098 1104 - 38.
Nomenclature of proteins of cows milk- 5th revision. Journal of Dairy Science,Eigel W. N Butler J. E Ernstrom C. A Farrell H Harwalkar V. R Jenness R Whitney R. M 1984 67 1599 1631 - 39.
Law AJR Horne DS, Banks JM, Leaver J.Heat-induced changes in the whey proteins and caseins 1994 49 125 129 - 40.
Heine W. E Klein P. D Reeds P. J The importance of -lactalbumin in infantil nutrition. Journal of N utrition1991 121 277 283 - 41.
Tolkach A Kulozik U Fractionation of whey proteins and caseinomacropeptide by means of enzymatic crosslinking and membrane separation techniques 2005 67 13 20 - 42.
Chang J Bulychev A Li L A stabilized molten globule protein 2000 487 298 300 - 43.
Bolotina, Brazhnikov EV, Bychkova VE, Gilmanshin RI, Lebedev YO, Semisotnov GV, Tiktopulo EI, Ptitsyn OB.Dolgikh D. A Abaturov I. A Compact state of a protein molecule with pronounced small-scale mobility. European Biophysics Journ al1985 13 109 121 - 44.
Vanderheeren G Hanssens I Thermal unfolding of bovine alpha-lactalbumin. Comparison of circular dichroism with hydrophobicity measurements. Journal of Biological Chemistry1994 269 7090 7094 - 45.
The molten globule state as a clue for understanding the folding and cooperativity of globular-protein struture. Protein: Structure, Function and GeneticsKuwajima K 1989 6 87 103 - 46.
Kuwajima K Hiraoka Y Ikeguchi M Sugai S Comparison of the transient folding intermediates in lysozyme and alpha-lactalbumin. 1985 24 874 881 - 47.
The molten globule state of -lactalbumin. The FASEB JournalKuwajima K 1996 1 102 109 - 48.
Alpha-lactalbumin: compact state with fluctuating terciary structure? FEBS LettersDolgikh D. A Gilmanshin R. I Brazhnikov E. V Bychkova V. E Semisotnov G. V Venyaminov S Ptitsyn O. B 1981 136 311 315 - 49.
Molten globule and protein folding. Advances in Protein ChemistryPtitsyn O. B 1995 47 83 229 - 50.
Arai M Kuwajima K Role of the molten globule state in protein folding. 2000 53 209 282 - 51.
Leandro P Gomes C. M Protein misfolding in conformational disorders: rescue of folding defects and chemical chaperoning 2008 8 901 911 - 52.
Caseino-glycopeptides: characterization of a methionine residue and of the N-terminal sequence. Biochemical and Biophysical Research CommunicationsDelfour A Jolles J Alais C Jolles P 1965 19 452 455 - 53.
López-Fandiño R. k-Casein macropeptides from cheese whey, physicochemical, biological, nutritional, and technological features for possible uses. Food Reviews InternationalManso M. A 2004 20 329 355 - 54.
Solubility and emulsifying properties of -casein and its caseinomacropeptide. Journal of Food BiochemistryChobert J. M Touati A Bertrand-harb C Dalgalarrondo M Nicolas M. G 1989 13 457 473 - 55.
Characterization and functional properties of lactosyl caseinomacropeptide conjugates. Journal of Agricultural and Food Chemistry,Moreno F. J López-fandino R Olano A 2002 50 5179 5184 - 56.
Dziuba J Minkiewicz P Influence of glycosylation on micelle-stabilizing ability and biological properties of C-terminal fragments of cow’s k-casein International Dairy Journal1996 6 1017 1044 - 57.
Kinsella E Whitehead D. M Proteins in whey: chemical, physical and functional properties. 1989 343 EOF 438 EOF - 58.
De Wit J. N The use of whey protein products. In: Fox P F. (ed.) Developments in dairy chemistry 4. Functional milk proteins. Barking, UK: Elsevier Science Publishers;1989 323 345 - 59.
Korhonen H Marnila P Gill H Milk immunoglobulins and complement factors, a review. British Journal of Nutrition2000 84 75 80 - 60.
Steijns J. M Milk ingredients as nutraceuticals 2001 54 81 88 - 61.
Fonseca CSP, Brandão SCC. Propriedades anticarcinogênicas de componentes do leite. Indústria de LaticíniosFonseca M. L 1999 4 5 55 - 62.
Alais C (ed Science du lait: principes des techniques laitières 4. Paris: SEPAIC;1984 - 63.
Innocente N Corradini C Blecker C Paquot M Emulsifying properties of the total fraction and the hydrophobic fraction of bovine milk proteose-peptones 1998 8 981 985 - 64.
Parodi P. W A role for milk protein in cancer prevention. 1998 53 37 47 - 65.
Lee VWK, Smithers GW.Regester G. O Mcintosh G. H Whey proteins as nutritional and functional food ingredients 1996 48 123 127 - 66.
Functional properties ofHa E Zemel M. B whey, whey components, and essential amino acids: mechanisms underlying healt h benefits for active people (review). Journal of Nutritional Biochemistry2003 14 251 258 - 67.
Whey and whey proteins- ‘from gutter-to-gold’. International Dairy JournalSmithers G. W 2008 18 695 704 - 68.
Bouchard D Morisset D Bourbonnais Y Tremblay G. M Proteins with whey acidic-protein motifs and cancer 2006 7 167 174 - 69.
Gauthier S. F Pouliot Y Saint-sauveur D Immunomodulatory peptides obtained by the enzymatic hydrolysis of whey proteins 2006 16 1315 1323 - 70.
Xu R. J Bioactive peptides in milk and their biological and health implications 1998 14 1 16 - 71.
Prates JAM Mateus CMRP. Functional foods from animal sources and their physiologically active components. Revue de Médicine Vétérinaire2002 153 155 160 - 72.
Avanços no conhecimento sobre a função das proteínas nas dietas para desempenho físico. 5º Simpósio Latino Americano de Ciência de Alimentos- Desenvolvimento Científico e Tecnológico e Inovação na Indústria de Alimentos. Campinas-Unicamp,Amaya-farfán J 2003 - 73.
Hartmann R Meisel H Food-derived peptides with biological activity: from research to food applications 2007 18 163 169 - 74.
Gill H. S Rutherford K. J Cross M. L Bovine milk: a unique source of immunomodulatory ingredients for functional foods In: Buttriss J, Saltmarsh M. (eds.) Functional foods II- claims and evidence. Cambridge, UK: Royal Society of Chemistry Press;2000 82 90 - 75.
Ronis MJJ, Hakkak R.Badger T. M Developmental effects and health aspects of soy protein isolate, casein and whey in male and female rats. 2001 20 165 174 - 76.
Ronis MJJ, Badger TM. Diets containing whey proteins or soy protein isolate protect againstHakkak R Korourian S Shelnutt S. R Lensing S 2 12 dimethylbenzanthracene-induced mammary tumours in female rats. Cancer Epidemiology, Biomarkers and Prevention2000 - 77.
Ronis MJJ, Badger TM. Soy and whey proteins down regulate DMBA-induced liver and mammary gland CYP1 expression in female rats. Journal of NutritionRowlands J. C He L Hakkak R 2001 131 3281 3287 - 78.
Oral supplementation with whey proteins increases plasma GSH levels of HIV-infected patients. European Journal of Clinical Investigation,Micke P Beeh K. M Schlaak J. F Buhl R 2001 31 171 178 - 79.
Effects of long-term supplementation with whey proteins on plasma GSH levels of HIV-infected patients. European Journal of NutritionMicke P Beeh K. M Buhl R 2002 41 12 18 - 80.
Clare D. A Catignani G. L Swaisgood H. E Biodefense properties of milk, the role of antimicrobial proteins and peptides. 2003 9 1239 1255 - 81.
Hall W. L Millward D. J Long S. J Morgan L. M Casein and whey exert different effects on plasma amino acid profiles, gastrointestinal hormone secretion and appetite. British Journal of Nutrition2003 89 339 348 - 82.
Optimization, by response surface methodology, of degree of hydrolysis, antioxidant and ACE-inhibitory activities of whey protein hydrolyzates obtained with cardoon extract. International Dairy JournalTavares T. G Contreras M. M Amorim M Martín-álvarez P. J Pintado M. E Recio I Malcata F. X 2011 21 926 933 - 83.
Rosaneli C. F Bighetti A. E Antônio M. A Carvalho J. E Sgarbieri V. C Efficacy of a whey protein concentrate on the inhibition of stomach ulcerative lesions caused by ethanol ingestion 2002 5 221 228 - 84.
Pacheco MTB Bighetti EA, Antônio M, Carvalho JE, Possenti A, Sgarbieri VC. Antiulcerogenic activity of fraction and hydrolyzate obtained from whey protein concentrate. Brazilian Journal of Food Technology, III JIPCA2006 15 22 - 85.
Mezzaroba LFH Carvalho JE, Ponezi AN, Antônio MA, Monteiro KM, Possenti A, Sgarbieri, VC.Antiulcerative properties of bovine -lactalbumin. 2006 16 1005 1012 - 86.
Antiulcerogenic activity of peptide concentrates obtained from hydrolysis of whey protein brought about by proteases from Cynara cardunculus. International Dairy JournalTavares T. G Monteiro K. M Possenti A Pintado M. E Carvalho J. E Malcata F. X 2011 21 934 939 - 87.
Gomes AMP, Pintado ME, Malcata FX. Bovine whey proteins- overview on their main biological properties. Food Research InternationalMadureira A. R Pereira C. I 2007 40 1197 1211 - 88.
Uptake and passage of -lactoglobulin, palmitic acid and retinol across the Caco-2 monolayer. Biochimica et Biophysica Acta- BiomembranesPuyol P Dolores-perez M Sanchez L Ena J. M Calvo M 1995 1236 149 154 - 89.
Lactoglobulin binds palmitate within its central cavity. Journal of Biological ChemistryWu S. Y Pérez M. D Puyol P Sawyer L 1999 274 170 177 - 90.
Interaction of -lactoglobulin and other bovine and human whey proteins with retinol and fatty acids. Agricultural and Biological ChemistryPuyol P Pérez M. D Ena J. M Calvo M 1991 10 2515 2520 - 91.
Bounous G Batist G Gold P Whey proteins in cancer prevention 1991 57 91 94 - 92.
Baruchel S Wang T Farah R Jamali M Batist G In vivo modulation of tissue glutathione in rat mammary carcinoma model. 1995 50 1505 1508 - 93.
Bounous G Whey protein concentrate (WPC) and glutathione modulation in cancer treatment. 2000 20 4785 4792 - 94.
. Effect of -lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk. Biochimica et Biophysica Acta ,Perez MD ,Sanchez L ,Aranda P ,Ena JM ,Oria R Calvo M 1992 ;1123 151 155 . - 95.
Warme PKF Momany A, Rumball SV, Tuttle RW, Scherag HA.Computation of structures of homologous proteins. -Lactalbumin from lysozyme. 1974 13 768 782 - 96.
Farrel H. M Bede M. J Enyeart J. A Binding of p-nitrophenyl phosphate and other aromatic compounds by -Lg. 1987 70 252 258 - 97.
Bioactive peptides derived from milk proteins: ingredients for functional foods? Kieler Milchwirtschaftliche und ForschungsberichteMeisel H Schlimme E 1996 48 343 357 - 98.
Synthetic peptides corresponding to -lactalbumin and -lactoglobulin sequences with angiotensin-I-converting enzyme inhibitory activity. Biological Chemistry Hoppe-SeylerMullally M. M Meisel H Fitzgerald R. J 1996 377 259 260 - 99.
Bioactive peptide derived from in vitro proteolysis of bovine β-lactoglobulin and its effect on smooth muscle. Journal of Dairy ResearchPihlanto-leppälä A Paakkari I Rinta-koski M Antila P 1997 64 149 155 - 100.
Pihlanto-leppälä A Rokka T Korhonen H Angiotensin I converting enzyme inhibitory peptides derived from bovine milk proteins 1998 8 325 331 - 101.
Pihlanto L Bioactive peptides derived from bovine whey proteins: opioid and ACE-inhibitory peptide s. Trends in Food Science and Technology2000 11 347 356 - 102.
Ijäs H Collin M Finckenberg P Pihlanto-leppälä A Korhonen H Korpela R Vapaatalo H Nurminen M. L Antihypertensive opioid-like milk peptide -lactorphin: lacks effect on behavioural tests in mice. 2004 14 201 205 - 103.
Bioactive peptides from milk proteins. In: Pizzano R (ed.) Immunochemistry in dairy research. Kerala, India: Trivandrum;Hernández-ledesma B López-expósito I Ramos M Recio I 2006 37 60 - 104.
FitzGerald RJ Murray BA.Bioactive peptides and lactic fermentations 2006 59 118 125 - 105.
Chen G-W Tsai J-S Pan B. S Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation 2007 17 641 647 - 106.
Physicochemical characterization and in vitro digestibility of -lactoglobulin/-Lg f142-148 complexes. International Dairy JournalRoufik S Gauthier S Turgeon S 2007 17 471 480 - 107.
Novel whey-derived peptides with inhibitory activity against angiotensin-converting enzyme: in vitro activity and stability to gastrointestinal enzymes. PeptidesTavares T. G Contreras M. M Amorim M Pintado M. E Recio I Malcata F. X 2011 32 1013 1019 - 108.
Antibacterial activity of peptides and holding variants from milk proteins. International Dairy JournalExpósito I. L Récio I 2006 16 1294 1305 - 109.
Korhonen HJT, Karp M. The effect of alpha-lactalbumin and β-lactoglobulin hydrolysates on the metabolic activity of Escherichia coli JM103. Journal of Applied MicrobiologyPihlanto-leppälä A Marnila P Hubert L Rokka T 1999 87 540 545 - 110.
Pellegrini A Thomas U Bramaz N Hunziker P Fellenberg R Isolation and identification of three bactericidal domains in the bovine alpha-lactalbumin molecule. 1999 1426 439 448 - 111.
Bruck W. M Graverholt G Gibson G. R A two-stage continuous culture system to study the effect of supplemental -lactalbumin and glycomacropeptide on mixed populations of human gut bacteria challenged with enteropathogenic Escherichia coli and Salmonella serotype Typhimurium. 2003 95 44 53 - 112.
Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin. Biochimica et Biophysica ActaPellegrini A Dettling C Thomas U Hunziker P 2001 1526 131 140 - 113.
Effect of physicochemical conditions on peptide-peptide interactions in a tryptic hydrolysate of β-lactoglobulin and identification of aggregating peptides. Journal of Agricultural and Food ChemistryGroleau P. E Morin P Gauthier S. F Pouliot Y 2003 51 4370 4375 - 114.
Nagaoka S Futamura Y Miwa K Awano T Yamauchi K Kanamaru Y Kojima T Kuwata T Identification of novel hypocholesterolemic peptides derived from bovine milk -lactoglobulin 2001 281 11 17 - 115.
Antila P Paakkari I Järvinen A Mattila M. J Laukkanen M Pihlanto-leppälä A Mäntsälä P Hellman J Opioid peptides derived from in vitro proteolysis of bovine whey proteins 1991 1 215 229 - 116.
Structural analysis of a new anti-hypertensive peptide (β-lactosin B) isolated from a commercial whey product. Journal of Dairy ScienceMurakami M Tonouchi H Takahashi R Kitazawa H Kawai Y Negishi H Saito T 2004 87 1967 1974 - 117.
Tavares T. G Sevilla M. A Montero M. J Carrón R Malcata F. X Acute effects of whey peptides upon blood pressure of hypertensive rats, and relationship with their angiotensin-converting enzyme inhibitory activity Molecular Nutrition and Food Research2011 doi: mnfr.201100381. - 118.
Antinociception induced by β-lactotensin, a neurotensin agonist peptide derived from β-lactoglobulin, is mediated by NT2 and D1 receptors. Life SciencesYamauchi R Sonoda S Jinsmaa Y Yoshikawa M 2003 73 1917 1923 - 119.
De Wit J. N Nutritional and functional characteristics of whey proteins in food products Journal of Dairy Science1998 81 597 608 - 120.
C.A Zhivotovsky B Orrenius S Sabharwal H Svanborg Apoptosis induced by a human milk protein. Proceedings of the National Academy of Sciences of USA1995 92 8064 8068 - 121.
Svensson M Sabharwal H Hakansson A Mossberg A. K Lipniunas P Leffler H Svanborg C Linse S Molecular characterization of -lactalbumin folding variants that induce apoptosis in tumor cells. Journal of Biological Chemistry1999 274 6388 6396 - 122.
Conversion of -lactalbumin to a protein inducing apoptosis. Proceedings of the National Academy of Sciences of USASvensson M Hakansson A Mossberg A. K Linse S Svanborg C 2002 97 4221 4226 - 123.
Whey protein rich in -lactalbumin increases the ratio of plasma tryptophan to the sum of the other large neutral amino acids and improves cognitive performance in stress-vulnerable subjects. American Journal of Clinical NutritionMarkus C. R Olivier B Haan E 2002 75 1051 1056 - 124.
MacDonald RS. Antiproliferative effects of yoghurt fractions obtained by membrane dialysis on cultured mammalian intestinal cells. Journal of Dairy ScienceGanjam L. S Thornton W. H Marshall R. T 1997 80 2325 2329 - 125.
A folding variant of alpha-lactalbumin with bactericidal activity against Streptococcus pneumoniae. Molecular MicrobiologyHakansson A Svensson M Mossberg A. K Sabharwal H Linse S Lazou I Lönnerdal B Svanborg C 2000 35 589 600 - 126.
Panhuysen GEM, Gugten J, van Der, Alles MS, Tuiten A, Westenberg HGM, Fekkes D, Kopperschaar HF, Haan E. The bovine alpha-lactalbumin increases the plasma ratio of tryptophan to the other large neutral amino acids, and in vulnerable subjects raises brain serotonin activity, reduces cortisol concentration, and improves mood under stress. American Journal of Clinical NutritionMarkus C. R Olivier B 2000 71 1536 1544 - 127.
Dynamics of the main immunologically and nutritionally available proteins of human milk during lactation. Journal of Food Composition and AnalysisMontagne P. M Cuiliere M. L Mole C. M Bene M. C Faure G. C 2000 13 127 137 - 128.
FitzGerald RJ. Opioid peptides encrypted in milk proteins. Journal of NutritionMeisel H 2000 84 27 31 - 129.
Pihlanto-Leppälä, Piilola K, Korpela R, Tossavainen O, Coronen H, Vapaatalo H. -Lactorphin lowers blood pressure measured by radiotelemetry in normotensive and spontaneously hypertensive rats. Life ScienceNurminen M. L Sipola M Kaarto H 2000 66 1535 1543 - 130.
New biological function of bovine -lactalbumin: protective effect against ethanol- and stress-induced gastric mucosal injury in rats. Bioscience, Biotechnology and BiochemistryMatsumoto H Shimokawa Y Ushida Y Toida T Hayasawa H 2001 65 1104 1111 - 131.
Hypothetical mechanism of prostaglandin E1-induced bronchoconstriction. Medical HypothesesUchida K Tateda T Takagi S 2003 61 378 384 - 132.
Protective effect of bovine milk whey protein concentrate on the ulcerative lesions caused by subcutaneous administration of indomethacin. Journal of Medicinal FoodRosaneli C. F Bighetti A. E Antônio M. A Carvalho J. E Sgarbieri V. C 2004 7 309 314 - 133.
Mechanisms of the antioxidant activity of a high molecular weight fraction of whey. Journal of Agricultural and Food ChemistryTong L. M Sasaki S Mcclements D. J Decker E. A 2000 48 1473 1478 - 134.
Protection of albumin against the pro-oxidant actions of phenolic dietary components. Food Chemistry and ToxicologySmith C Halliwell B Aruoma O I 1992 30 483 489 - 135.
Serum albumin as a modulator of the human breast cancer cell line MCF-7. Anticancer ResearchLaursen I Briand P Lykkesfeldt A. E 1990 10 343 351 - 136.
Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion. Journal of Dairy ScienceAbubakar A Saito T Kitazawa H Kawai Y Itoh T 1998 81 3131 3138 - 137.
FitzGerald RJ Murray BA, Walsh DJ. Hypotensive peptides from milk proteins. Journal of Nutrition2004 134 980 988 - 138.
Biologically functional proteins of milk and peptides derived from milk proteins. IDF BulletinYamauchi K 1992 272 51 57 - 139.
Shiota, Chiba H, Yosliikawa M. Serophin an opioid peptide derived from bovine serum albumin. In: Brantl V. (ed.) -Casomorphins and relate peptides: recent developments. Weinheim, Germany: VCH-Verlag;Tani F 1993 49 53 - 140.
The anti-inflammatory activity of a low molecular weight component derived from the milk of hyperimmunized cows. Agents and ActionsOrmrod D. J Miller T. E 1991 32 160 166 - 141.
Hyperimmune cow colostra reduces diarrhoea due to rotavirus: a double-blind, controlled clinical trial. Acta PaediatricaMitra A. K Mahalanabis D Unicomb L Eechels R Tzipori S 1995 84 996 1001 - 142.
Immunisation of dairy cows with an Escherichia coli J5 lipopolysaccharide vaccine. Journal of Dairy ScienceTomita M Todhunter D. A Hogan J. S Smith K. L 1995 78 2178 2185 - 143.
Helicobacter pylori in children with abdominal complaints: has immune bovine colostrum some influence on gastritis? Alpe Adria Microbiology JournalOona M Rägö T Maaroos H. I Mikelsaar M Loivukene K Salminen S Korhonen H 1997 6 49 57 - 144.
Milk immunoglobulin with specific activity against purified colonization factor antigens can protect against oral challenge with enterotoxigenic Escherichia coli. Journal of Infectious DiseasesFreedman D. J Tacket C. O Delehanty A Maneval D. R Nataro J Crabb J. H 1998 177 662 667 - 145.
Effects of bovine immune- and non-immune whey preparations on the composition and pH response of human dental plaque. European Journal of Oral ScienceLoimaranta V Laine M Söderling E Vasara E Rokka S Marnila P Korhonen H Tossavainen O Tenovuo J 1999 107 244 250 - 146.
DuPont HL. Prophylactic effect of bovine anti-Cryptosporidium hyperimmune colostra immunoglobulin in healthy volunteers challenged with Cryptosporidium parvum. Clinical Infectious DiseasesOkhuysen P. C Chappell C. L Crabb J Valdez L. M Douglass E 1998 26 1324 1329 - 147.
Cholesterol-lowering and blood pressure effects of immune milk. American Journal of Clinical NutritionSharpe S. J Gamble G. D Sharpe D. N 1994 59 929 934 - 148.
The carbohydrate portions of milk glycoproteins. Journal of Dairy ResearchJolles P Fiat A. M 1979 46 187 191 - 149.
Analogy between fibrinogen and casein. Effect of an undecapeptide isolated from -casein on platelet function. European Journal of BiochemistryJolles P Levy-toledano S Fiat A. M Soria C Cillessen D Thomaidis A Dunn F. W Caen J. P 1986 158 379 382 - 150.
Demonstration of Ac-Arg-Gly-Asp-Ser-NH2 as an antiaggregatory agent in the dog by intracoronary administration. Journal of Thrombosis and HaemostasisShebuski R. J Berry D. E Bennett D. B Romoff T Storer B. L Ali F Samanen J 1989 61 183 188 - 151.
Casein peptide release and passage to the blood in humans during digestion of milk or yogurt. BiochimieChabance B Marteau P Rambaud J. C Migliore-samour D Boynard M Perrotin P Guillet R Jolles P Fiat A. M 1998 80 155 165 - 152.
Peptides affecting coagulation. British Journal of NutritionRutherford K. J Gill H 2000 84 99 102 - 153.
Platelet aggregation inhibitory activity of bovine, ovine and caprine -casein macropeptides and their tryptic hydrolyzates. Journal of Food ProtectionManso M. A Escudero C Alijo M López-fandino R 2002 65 1992 1996 - 154.
Purification and characterization of angiotensin I-converting enzyme inhibitors from sour milk. Journal of Dairy ScienceNakamura Y Yamamoto N Sakai K Okubo A Yamazaki S Takano T 1995 78 777 783 - 155.
Angiotensin I converting enzyme-inhibitory activity of bovine, ovine, and caprine -casein macropeptides and their tryptic hydrolysates. Journal of Food ProtectionManso M. A López-fandino R 2003 66 1686 1692 - 156.
Antihypertensive effect of casein hydrolysate in a placebo-controlled study in subjects with high-normal blood pressure and mild hypertension. British Journal of NutritionMizuno S Matsuura K Gotou T Nishimura S Kajimoto O Yabune M Kajimoto Y Yamamoto N 2005 94 84 91 - 157.
del Vedovo S, Prigent MJ, Guggenheim B. Specific and nonspecific inhibition of adhesion of oral actinomyces and streptococci to erythrocytes and polystyrene by caseinoglycopeptide derivatives. Infection and ImmunityNeeser J. R Chambaz A 1988 56 3201 3208 - 158.
Inhibition by lactoferrin and -casein glycomacropeptide of binding of cholera toxin to its receptor. Bioscience, Biotechnology and BiochemistryKawasaki Y Isoda H Tanimoto M Dosako S Idota T Ahiko K 1992 56 195 198 - 159.
Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutant streptococci. Journal of Dental ResearchSchupbach P Neeser J. R Golliard M Rouvet M Guggenheim B 1996 75 1779 1788 - 160.
Detection of cholera toxin-binding activity of -casein macropeptide and optimization of its production by the response surface methodology. Bioscience, Biotechnology and BiochemistryOh S Worobo R. W Kim B Rheem S Kim S 2000 64 516 522 - 161.
Growth-promoting activity of tryptic digest of caseinomacropeptide of Lactococcus lactis subsp. lactis. Le LaitBouhallab S Favrot C Maubois J. L 1993 73 73 77 - 162.
Effects of gastric digestive products from casein on CCK release by intestinal-cells in rat. Journal of Nutritional BiochemistryBeucher S Levenez F Yvon M Corring T 1994 5 578 584 - 163.
le Huerou-Luron I, Corring T. Effects of caseinomacropeptide (CMP) on digestion regulation. Reproduction, Nutrition and DevelopmentYvon M Beucher S Guilloteau P 1994 34 527 537 - 164.
Pederson NLR Nagain-Domaine C, Mahe S, Chariot J, Roze C, Tome D. Caseinomacropeptide specifically stimulates exocrine pancreatic secretation in the anesthetized rat. Peptides2000 21 1527 1535 - 165.
Dartey C Leveille G Sox T. E 2003 Compositions for appetite control and related methods. US Patent 0,059,495 A1. - 166.
A new look at an ancient concept. Chemistry and IndustryHasler C 1998 2 84 89 - 167.
Antioxidative peptides derived from milk proteins. International Dairy JournalPihlanto-leppälä A 2006 16 1306 1314 - 168.
Food protein-derived bioactive peptides: production, processing, and potential health benefits. Journal of Food ScienceUdenigwe C. C Aluko R. E 2012 R11 R24. - 169.
Milk-derived bioactive peptides: from science to applications. Journal of Functional FoodsKorhonen H 2009 1 177 187 - 170.
Relative proteolytic action of milk-cloting enzyme preparations on bovine and casein. Journal of Food ScienceUstunol Z Zeckzer T 1996 61 1136 1138 - 171.
Exogeneous enzymes in dairy technology. A review. Journal of Food BiochemistryFox P. F 1993 17 173 199 - 172.
Simões IIG Caracterização molecular da acção das cardosinas A e B sobre caseínas - e -bovinas. Tese de Mestrado, Universidade de Coimbra;1998 - 173.
Physiological, chemical and technological aspects of milk-protein-derived peptides with antihypertensive and ACE-inhibitory activity. International Dairy JournalLópez-fandino R Otte J Van Camp J 2006 16 1277 1293 - 174.
Raising the pH of the pepsin-catalysed hydrolysis of bovine whey proteins increases the antigenicity of the hydrolyzates. Clinical and Experimental AllergySchmidt D. G Meijer R. J Slangen C. J Van Beresteijn E. C 1995 25 1007 1017 - 175.
Molecular characterization of peptides released from -lactoglobulin and -lactalbumin via cardosins A and B. Journal of Dairy ScienceBarros R. M Malcata F. X 2006 89 483 494 - 176.
Hydrolysis of whey proteins by proteases extracted from Cynara cardunculus and immobilized onto highly activated supports. Enzyme and Microbial TechnologyLamas E. M Barros R. M Balcao V. M Malcata F. X 2000 28 642 652 - 177.
Modelling the kinetics of whey protein hydrolysis brought about by enzymes from Cynara cardunculus. Journal of Agricultural and Food ChemistryBarros R. M Malcata F. X 2002 50 4347 4356 - 178.
A kinetic model for hydrolysis of whey proteins by cardosin A extracted from Cynara cardunculus. Food ChemistryBarros R. M Malcata F. X 2004 88 351 359 - 179.
Current developments of microfiltration technology in the dairy industry. Le LaitSaboya L. V Maubois J. L 2000 80 541 553 - 180.
Ultrafiltration modes of operation for the separation of -lactalbumin from acid casein whey. Journal of Membrane ScienceMuller A Daufin G Chaufer B 1999 153 9 21 - 181.
Separation of -lactalbumin and -lactoglobulin using membrane ultrafiltration. Biotechnology and BioengineeringCheang B Zydney A. L 2003 83 201 209 - 182.
A two-stage ultrafiltration process for fractionation of whey protein isolate. Journal of Membrane ScienceCheang B Zydney A. L 2004 231 159 167 - 183.
Schroën CGPH, van der Sman RGM, Boom RM. Membrane fractionation of milk: state of the art and challenges. Journal of Membrane ScienceBrans G 2004 243 263 272 - 184.
Functional properties of ovine whey protein concentrates produced by membrane technology after clarification of cheese manufacture by-products. Food HydrocolloidsDíaz O Pereira C. D Cobos A 2004 18 601 610 - 185.
Investigation of ultra- and nanofiltration for utilization of whey protein and lactose. Journal of Food EngineeringAtra R Vatai G Bekassy-molnar E Balint A 2005 67 325 332 - 186.
Protein separations using membrane filtration: new opportunities for whey fractionation. International Dairy JournalZydney A. L 1998 8 243 250 - 187.
Membrane filtration of Mozzarella whey. DesalinationRektor A Vatai G 2004 162 279 286 - 188.
New opportunities from the isolation and utilization of whey proteins. Journal of Dairy ScienceSmithers G. W Ballard F. J Copeland A. D Silva K. J Dionysius D. A Francis G. L Goddard C Grieve P. A Mcintosh G. H Mitchell I. R Pearce R. J Regester G. O 1996 79 1454 1459 - 189.
Effects of purified bovine whey factors on cellular immune functions in ruminants. Veterinary Immunology and ImmunopathologyWong C. W Seow H. F Husband A. J Regester G. O Watson D. L 1997 56 85 96 - 190.
Bioactive peptide-rich concentrates from whey: pilot process characterization. Journal of Food EngineeringTavares T. G Amorim M Gomes D Pintado M. E Pereira C. D Malcata F. X 2012 110 547 552 - 191.
Eastern Stroke and Coronary Heart Disease Collaborative Research Group Blood pressure, cholesterol and stroke in Eastern Asia. Lancet1998 352 1801 807 - 192.
Blood pressure burden: vascular changes and cerebrovascular complications. Journal of HypertensionChalmers J 2000 S1 S2. - 193.
The seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. JAMAChobanian A. V Bakris G. L Black H. R Cushman W. C Green L. A Izzo J. L Jones D. W Materson B. J Oparil S Wright J. T Roccella E. J 2003 289 2560 2571 - 194.
Antihypertensive effect of bioactive peptides produced by protease-facilitated lactic acid fermentation of milk. Food ChemistryTsai J. S Chen T. J Pan B. S Gong S. D Chung M. Y 2008 106 552 558 - 195.
Murray CJL Lopez AD (eds.). The global burden of disease: a comprehensive assessment of mortality and disability from disease, injuries and risk factors in 1990 and projected to 2020. Global Burden of Disease and Injury Series,1 Cambridge: Harvard School of Public Health;1996 - 196.
Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circulation ResearchGeisterfer A. A Peach M. J Owens G. K 1988 62 749 756 - 197.
Angiotensin II induces smooth muscle cell proliferation in the normal and injured rat arterial wall. Circulation ResearchDaemen M. J Lombardi D. M Bosman F. T Schwartz S. M 1991 68 450 456 - 198.
Identification and characterization of novel angiotensin-converting enzyme inhibitors obtained from goat milk. Journal of Dairy ScienceGeerlings A Villar I. C Zarco F. H Sánchez M Vera R Gomez A. Z Boza J Duarte J 2006 89 3326 3335 - 199.
The antihypertensive effect of peptides: a novel alternative to drugs? PeptidesHong F Ming L Yi S Zhanxia L Yongquan W Chi L 2008 29 1062 1071 - 200.
Effects of bioactive substances in milk on mineral and trace element metabolism with special reference to casein phosphopeptides. British Journal of NutritionScholz-ahrens K Schrezenmeir J 2000 S147 S153. - 201.
Milk peptides and blood pressure. Journal of NutritionJauhiainen T Korpela R 2007 S-829S. - 202.
Effect of long-term intake of milk products on blood pressure in hypertensive rats. Journal of Dairy ResearchSipola M Finckenberg P Korpela R Vapaatalo H Nurminen M. L 2002 69 103 111 - 203.
Isolation and characterization of ovokinin, a bradykinin b-1 agonist peptide derived from ovalbumin. PeptidesFujita H Usui H Kurahashi K Yoshikawa M 1995 16 785 790 - 204.
A novel anti-hypertensive peptide derived from ovalbumin induces nitric oxide-mediated vasorelaxation in an isolated SHR mesenteric artery. FEBS LettersMatoba N Usui H Fujita H Yoshikawa M 1999 452 181 184 - 205.
The ACE inhibitory dipeptide Met-Tyr diminishes free radical formation in human endothelial cells via induction of heme oxygenase-1 and ferritin. Journal of NutritionErdmann K Grosser N Schipporeit K Schröder H 2006 136 2148 2152 - 206.
Huyghebaert. Influence of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR) on the release of endothelin-1 by endothelial cells. Regulatory PeptidesMaes W Van Camp J Vermeirssen V Hemeryck M Ketelslegers J. M Schrezen-meir J Van Oostveldt P 2004 118 105 109 - 207.
Identification of antioxidant and ACE-inhibitory peptides in fermented milk. Journal of the Science of Food and AgricultureHernández-ledesma B Miralles B Amigo L Ramos M Recio I 2005 85 1041 1048 - 208.
Antihypertensive peptides from food proteins: a review. Food and FunctionMartínez-maqueda D Miralles B Recio I Hernández-ledesma B 2012 3 350 361 - 209.
Long-term intake of a milk casein hydrolysate attenuates the development of hypertension and involves cardiovascular benefits. Pharmacological ResearchSánchez D Kassan M Contreras M. M Carrón R Recio I Montero M. J Sevilla M. A 2011 63 398 404 - 210.
Identification of novel antihypertensive peptides in milk fermented with Enterococcus faecalis. International Dairy JournalQuirós A Ramos M Muguerza B Delgado M. A Miguel M Aleixandre A Recio I 2007 17 33 41 - 211.
ACE-inhibitory and antihypertensive properties of a bovine casein hydrolysate. Food ChemistryMiguel M Contreras M. M Recio I Aleixandre A 2009 112 211 214 - 212.
Antihypertensive peptides: production, bioavailability and incorporation into foods. Advances in Colloid and Interface ScienceHernández-ledesma B Contreras M. M Recio I 2011 165 23 35 - 213.
Angiotensin-I-converting-enzyme-inhibitory and antimicrobial bioactive peptides. International Journal of Dairy TechnologyGobetti M Minervini F Grizzello C 2004 57 173 188 - 214.
Biochemical properties of peptides encrypted in bovine milk proteins. Current Medicinal ChemistryMeisel H 2005 12 1905 1919 - 215.
Caseins as source of bioactive peptides. International Dairy JournalSilva S. V Malcata F. X 2005 15 1 15 - 216.
Bioavailability of angiotensin I converting enzyme inhibitory peptides. British Journal of NutritionVermeirssen V Verstraete W Van Camp J 2004 92 357 366 - 217.
Bioactive peptides derived from bovine whey proteins: opioid and ACE-inhibitory peptides. Trends in Food Science and TechnologyPihlanto-leppälä A 2001 11 347 356 - 218.
Isolation of an angiotensin-converting enzyme inhibitory peptide from irradiated bovine blood plasma protein hydrolysates. Food Chemistry and Toxicology,Lee S. H Song K. B 2003 68 2469 2472 - 219.
New insights in biologically active proteins and peptides derived from hen egg. World Poultry Science JournalMine Y Kovacs-nolan J 2006 62 87 96 - 220.
Isolation and characterization of angiotensin I-converting enzyme-inhibitory peptides derived from bonito bowels. Bioscience, Biotechnology and BiochemistryMatsumura N Fujii M Takeda Y Shimizu T 1993 57 1743 1744 - 221.
Hypotensive and physiological effects of angiotensin converting enzyme inhibitory peptides derived from soy protein on spontaneously hypertensive rats. Journal of Agricultural and Food ChemistryWung J Ding X 2001 49 501 506 - 222.
Angiotensin I converting enzyme-inhibitory peptides from wine. American Journal of Enology and ViticultureTakayanagi T Yokotsuka K 1999 50 65 68 - 223.
Kaneko, Fukui F, Tanaka H, Maruyama S. Structures and activity of angiotensin-converting enzyme inhibitors in α-zein hydrolysate. Agricultural and Biological ChemistryMiyoshi S Ishikawa H 1991 55 1313 1318 - 224.
A peptide inhibitor of angiotensin I converting enzyme in the tryptic hydrolysate of casein. Agricultural and Biological ChemistryMaruyama S Suzuki H 1982 46 1393 1394 - 225.
Studies on the active site and antihypertensive activity of angiotensin I-converting enzyme inhibitors derived from casein. Agricultural and Biological ChemistryMaruyama S Mitachi H Tanaka H Tomizuka N Suzuki H 1987 51 1581 1586 - 226.
FitzGerald RJ, Meisel H. Identification of a novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine -lactoglobulin. FEBS LettersMullally M. M 1997 402 99 101 - 227.
FitzGerald RJ Meisel H. Lactokinins, whey protein derived ACE inhibitory peptides. Nährung-Food1999 43 165 167 - 228.
Preparation of ovine and caprine β-lactoglobulin hydrolysates with ACE-inhibitory activity. Identification of active peptides from caprine β-lactoglobulin hydrolysed with thermolysin. International Dairy JournalHernández-ledesma B Recio I Ramos M Amigo L 2002 12 805 812 - 229.
Design of specific inhibitors of angiotensin-converting enzyme: new class of orally active antihypertensive agents. ScienceOndetti M. A Rubin B Cushman D. W 1977 196 441 444 - 230.
Molecular basis of human hypertension: role of angiotensinogen. CellJeunemaitre X Soubrier F Kotelevtsev Y. V Lifton R. P Williams C. S Charru A Hunt S. C Hopkins P. N Williams R. R Lalouel J. M Corvol P 1992 71 169 180 - 231.
Timmermans PBMWM Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JAM, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacological Reviews1993 45 205 251 - 232.
What is the role of angiotensin-converting enzyme inhibitors in congestive heart failure and after myocardial infarction? Annals of PharmacotherapyNelson K. M Yeager B. F 1996 30 986 993 - 233.
Angiotensin-I converting enzyme inhibitory properties of whey proteins digests: concentration and characterization of active peptides. Journal of Dairy ResearchPihlanto-leppälä A Koskinen P Piilola K Tupasela T Korhonen H 2000 67 53 64 - 234.
Improved bioactive whey protein hydrolyzate. Patent PCT/NZ01/00188 (WO 02/19837 A1), New ZealandSchlothauer R. C Schollum L. M Reid J. R Harvey S. A Carr A Fanshawe R. L 2002 - 235.
Production of novel ACE inhibitory peptides from -lactoglobulin using Protease N Amano. International Dairy JournalOrtiz-chao P Gómez-ruiz J. A Rastall R. A Mills D Cramer R Pihlanto A Korhonen H Jauregi P 2009 19 69 76 - 236.
Effect of simulated gastrointestinal digestion on the antihypertensive properties of synthetic -lactoglobulin peptide sequences. Journal of Dairy ResearchHernández-ledesma B Miguel M Amigo L Aleixandre M. A Recio I 2007 74 336 339 - 237.
Fractionation and identification of ACE-inhibitory peptides from -lactalbumin and -casein produced by thermolysin-catalysed hydrolysis. International Dairy JournalOtte J Shalaby S Zakora M Nielsen M. S 2007 17 1460 1472 - 238.
Bioactive peptides derived from food proteins. Kagaku to Seibutsu (in Japanese)Chiba H Yoshikawa M 1991 29 454 458 - 239.
Vascular effects and antihypertensive properties of κ-casein macropeptide. International Dairy JournalMiguel M Manso M. A López-fandino R Alonso M. J Salaices M 2007 17 1473 1477 - 240.
Antihypertensive properties of lactoferricin B-derived peptides. Journal of Agricultural and Food ChemistryRuiz-giménez P Ibánez A Salom J. B Marcos J. F López-díez J. J Vallés S Torregrosa G Alborch E Manzanares P 2010 58 6721 6727 - 241.
Hypotensive effect of butein via the inhibition of angiotensin-converting enzyme. Biological and Pharmaceutical BulletinKang D. G Kim Y. C Sohn E. J Lee Y. M Lee A. S Yin M. H Lee H. S 2003 26 1345 1347 - 242.
Short-term effect of egg-white hydrolysate products on the arterial blood pressure of hypertensive rats. British Journal of NutritionMiguel M López F Ramos M Aleixandre A 2005 94 731 737 - 243.
Development of a strain of spontaneously hypertensive rats. Japanese Circulation JournalOkamoto K Aoki K 1963 27 282 293 - 244.
Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme. Journal of Dairy ScienceNakamura Y Yamamoto N Sakai K Takano T 1995 78 1253 1257 - 245.
Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins. Journal of Food ScienceFujita H Yokoyama K Yoshikawa M 2000 65 564 569 - 246.
Antihypertensive activity of milk fermented by Enterococcus faecalis strains isolated from raw milk. International Dairy JournalMuguerza B Ramos M Sánchez E Manso M. A Miguel M Aleixandre A Delgado M. A Recio I 2006 16 61 69 - 247.
Novel casein-derived peptides with antihypertensive activity. International Dairy JournalContreras M. M Carrón R Montero M. J Ramos M Recio I 2009 19 566 573 - 248.
Colorimetric measurement of angiotensin I-converting enzyme inhibitory activity with trinitrobenzene sulfonate. Bioscience, Biotechnology and BiochemistryMatsui T Matsufugi H. Y Osajima Y 1992 56 517 518 - 249.
A sensitive fluorometric assay for serum angiotensin converting enzyme. American Journal of Clinical PathologyFriedland J Silverstein E 1976 66 416 424 - 250.
Analysis of angiotensin-converting enzyme by capillary electrophoresis. Journal of Chromatography AShihabi Z. K 1999 853 185 188 - 251.
Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochemical PharmacologyCushman D. W Cheung H. S 1971 20 1637 1648 - 252.
Optimisation and validation of an angiotensin-converting enzyme inhibition assay for the screening of bioactive peptides. Journal of Biochemical and Biophysical MethodsVermeirssen V Van Camp J Verstraete W 2002 51 75 87 - 253.
Inhibition of angiotensin-converting enzyme by synthetic peptides of human -casein. Agricultural and Biological ChemistryKohmura M Nio N Kubo K Minoshima Y Munekata E Ariyoshi Y 1989 53 2107 2114 - 254.
A fermented milk high in bioactive peptides has a blood pressure-lowering effect in hypertensive subjects. American Journal of Clinical NutritionSeppo L Jauhiainen T Poussa T Korpela R 2003 77 326 330 - 255.
Expression of milk-derived antihypertensive peptide in Escherichia coli. Journal of Dairy ScienceLv G. S Huo G. C Fu X. Y 2003 86 1927 1931 - 256.
Enzymes of the renin-angiotensin system and their inhibitors. Annual Reviews of BiochemistryOndetti M. A Cushman D. W 1982 51 283 308 - 257.
Development and design of specific inhibitors of angiotensin-converting enzyme. American Journal of CardiologyCushman D. W Cheung H. S Sabo E. F Ondetti M. A 1982 49 1390 1394 - 258.
Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Journal of Biological ChemistryCheung H. S Wang F. L Ondetti M. A Sabo E. H Cushman D. W 1980 25 401 407 - 259.
Angiotensin I converting enzyme of calf lung. Method of assay and partial purification. BiochemistryStevens R. L Micalizzi E. R Fessler D. C Pals D. T 1972 11 2999 3007 - 260.
Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate. Journal of Agricultural and Food ChemistryKim S. K Byun H. G Park P. Y Fereidoon S 2001 49 2992 2997 - 261.
Angiotensin-converting enzyme-inhibitory activity of peptides isolated from Manchego cheese. Stability under simulated gastrointestinal digestion. International Dairy JournalGómez-ruiz J. A Ramos M Recio I 2004 14 1075 1080 - 262.
Quantitative structure-activity relationship modelling of ACE-inhibitory peptides derived from milk proteins. European Food Research and TechnologyPripp A. H Isaksson T Stepaniak L Sorhaug T 2004 219 579 583 - 263.
Structural requirements of antiotensin I-converting enzyme inhibitory peptides. Quantitative structure-activity relationship study of di- and tripeptides. Journal of Agricultural and Food ChemistryWu J. P Aluko R. E Nakai S 2006 54 732 738 - 264.
Latent bioactive peptides in milk proteins, proteolytic activation and significance in dairy processing. Critical Reviews in Food Science and NutritionGobbetti M Stepaniak L De Angelis M Corsetti A Cagno R. D 2002 42 223 239 - 265.
Biochemical properties of regulatory peptides derived from milk proteins. BiopolymersMeisel H 1997 43 119 128 - 266.
Roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin-I and bradykinin, insights from selective inhibitors. Circulation ResearchGeorgiadis D Beau F Czarny B Cotton J Yiotakis A Dive V 2003 93 148 154 - 267.
Antihypertensive peptides derived from bovine casein and whey proteins. In: Bösze, Z (ed.). Advances in experimental medicine and biology: bioactive components of milk,Saito T 606 New York, USA: Springer2008 - 268.
Food-derived peptides and intestinal functions. BiofactorsShimizu M 2004 21 43 47 - 269.
Intestinal peptide transport systems and oral drug availability. Pharmaceutical ResearchYang C. Y Dantzig A. H Pidgeon C 1999 16 1331 1343 - 270.
Gardner MLG Intestinal assimilation of intact peptides and proteins from the diet- a neglected field. Biological Reviews of the Cambridge Philosophical Society1984 59 289 331 - 271.
Effects of chain length on absortion of biologically active peptides from the gastrointestinal tract. DigestionRoberts P. R Burney J. D Black K. W Zaloga G. P 1999 60 332 337 - 272.
In vitro digestibility of bioactive peptides derived from bovine -lactoglobulin. International Dairy JournalRoufik S Gauthier S. F Turgeon S. L 2006 16 294 302 - 273.
Peptide hydrolyses in the brush borde rand soluble fractions of small intestinal mucosa of rat and man. Journal of Clinical InvestigationKim Y. S Bertwhistle W Kim Y. W 1972 51 1419 1430 - 274.
Antihypertensive peptides are present in aorta after oral administration of sour milk containing these peptides in spontaneously hypertensive rats. Journal of NutritionMasuda O Nakamura Y Takano T 1996 126 3063 3068 - 275.
Gastrointestinal enzyme production of bioactive peptides from royal jelly protein and their antihypertensive ability in SHR. Journal of Nutritional BiochemistryMatsui T Yukiyoshi A Doi S Sugimoto H Yamada H Matsumoto K 2002 13 80 86 - 276.
Des hydrolysats protéiques pour développer des aliments santé. RIA Technology VeilleLangley-danysz P 1998 581 38 40 - 277.
Antihypertensive effect of peptides obtained from Enterococcus faecalis-fermented milk in rats. Journal of Dairy ScienceMiguel M Recio I Ramos I Delgado M. A Aleixandre M. A 2006 89 3352 3359 - 278.
The impact of fermentation and in vitro digestion on the formation of angiotensin-I-converting enzyme inhibitory activity from pea and whey protein. Journal of Dairy ScienceVermeirssen V Van Camp J Decroos K Van Wijmelbeke L Verstraete W 2003 86 429 438 - 279.
Angiotensin converting enzyme inhibitory activity in commercial fermented products. Formation of peptides under simulated gastrointestinal digestion. Journal of Agricultural and Food Chemistry,Hernández-ledesma B Amigo L Ramos M Recio I 2004 52 1504 1510 - 280.
Release of angiotensin converting enzyme-inhibitory peptides by simulated gastrointestinal digestion of infant formulas. International Dairy JournalHernández-ledesma B Amigo L Ramos M Recio I 2004 14 889 898 - 281.
Stability to gastrointestinal enzymes and structure-activity relationship of -casein-peptides with antihypertensive properties. PeptidesQuirós A Contreras M. M Ramos M Amigo L Recio I 2009 30 1848 1853 - 282.
Identification of an antihypertensive peptide from casein hydrolyzate produced by a proteinase from Lactobacillus helveticus CP790. Journal of Dairy ScienceMaeno M Yamamoto N Takano T 1996 79 1316 1321 - 283.
Purification and characterization of an antihypertensive peptide from a yogurt-like product fermented by Lactobacillus helveticus CPN4. Journal of Dairy ScienceYamamoto N Maeno M Takano T 1999 82 1388 1393 - 284.
Identification and formation of angiotensin-converting enzyme-inhibitory peptides in Manchego cheese by high-performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography A,Gómez-ruiz J. A Ramos M Recio I 2004 1054 269 277 - 285.
Effect of simulated gastrointestinal digestion on the antihypertensive properties of ACE-inhibitory peptides derived from ovalbumin. Journal of Agricultural and Food ChemistryMiguel M Aleixandre M. A Ramos I López-fandino R 2006 54 726 731