The scientific evidence for the role of milk protein-derived bioactive peptides in humans: A Review

Journal of Functional Foods 17 (2015) 640–656

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The scientific evidence for the role of milk protein-derived bioactive peptides in humans: A Review Alice B. Nongonierma, Richard J. FitzGerald * Department of Life Sciences and Food for Health Ireland (FHI), University of Limerick, Limerick, Ireland

A R T I C L E

I N F O

A B S T R A C T

Article history:

Milk proteins are a good source of bioactive peptides (BAPs). BAPs can positively affect various

Received 12 March 2015

health biomarkers in vitro. The role of milk protein-derived BAPs in humans was reviewed

Received in revised form 11 June

herein. To date, a limited number of BAPs have been identified in the gastrointestinal tract

2015

of humans and in the circulation at concentrations which may be sufficient to mediate in

Accepted 11 June 2015

vivo bioactivity. While the outcomes of several human intervention studies have shown prom-

Available online

ising results, some studies have failed to demonstrate additional health benefits. This may be related to a low potency and bioavailability of the BAPs evaluated as well as confound-

Keywords:

ing factors (i.e. genotype, health status and other sources of inter-individual variability).

Bioactive peptides

Universal guidelines for the evaluation of BAPs in humans such as adequately powered double-

Milk

blind randomised clinical trials are needed.

Human studies

© 2015 Elsevier Ltd. All rights reserved.

Bioavailability

Contents 1. 2.

Introduction ...................................................................................................................................................................................... 641 Milk protein-derived BAPs identified in the circulation of humans ........................................................................................ 642

* Corresponding author. Department of Life Sciences and Food for Health Ireland (FHI), University of Limerick, Limerick, Ireland. Tel.: +353 (0) 61 202598; fax: +353 (0) 61 331490. E-mail address: [email protected] (R.J. FitzGerald). Abbreviations: A, alanine; ACE, angiotensin converting enzyme; ACP, amorphous calcium phosphate; ACPF, amorphous calcium phosphate fluoride; Ala, alanine; Arg, arginine; BAP, bioactive peptide; BCAA, branched-chain amino acids; BMI, body mass index; BP, blood pressure; C, cysteine; Ca, calcium; CCK, cholecystokinin; CK, creatine kinase; CMP, caseinomacropeptide; CMPA, cow’s milk protein allergy; CN, casein; CNH, casein hydrolysate; CPP, caseinophosphopeptides; CVD, cardiovascular disease; Cys, cysteine; D, aspartic acid; DBP, diastolic blood pressure; DPP-IV, dipeptidyl peptidase IV; E, glutamic acid; EFSA, European Food Safety Authority; F, phenylalanine; Fe, iron; G, glycine; GI, gastrointestinal transit; GIP, gastric inhibitory polypeptide; GIT, gastrointestinal transit; GLP-1, glucagon-like peptide 1; Glu, glutamic acid; Gly, glycine; GSH, glutathione; H, histidine; hsCRP, high-sensitivity C-reactive protein; I, isoleucine; IgA, immunoglobulin A; IL-10, interleukin-10; IL-6, interleukin-6; IL-8, interleukin-8; Ile, isoleucine; ITF, inulin type fructan; K, lysine; L, lysine; LDH, lactate dehydrogenase; Leu, leucine; LF, lactoferrin; LTP, lactotripeptides; Lys, lysine; M, methionine; N, asparagine; P, proline; Phe, phenylalanine; PKU, phenylketonuria; ppm, part per million; Pro, proline; PYY, peptide YY; Q, glutamine; R, arginine; ROS, reactive oxygen species; S, serine; SBP, systolic blood pressure; SGID, simulated gastrointestinal digestion; Ser, serine; Ser(P), phosphorylated serine; T, threonine; T2D, type 2 diabetes; TAC, total antioxidant capacity; TFCA, true fractional Ca absorption; Thr, threonine; TNF-α, tumour necrosis factor-α; Tr-1, type 1-regulatory T; Trp, tryptophan; Tyr, tyrosine; V, valine; Val, valine; W, tryptophan; WHO, World Health Organization; WP, whey protein; WPC, whey protein concentrate; WPH, whey protein hydrolysate; WPI, whey protein isolate; Y, tyrosine; Zn, zinc http://dx.doi.org/10.1016/j.jff.2015.06.021 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 17 (2015) 640–656

3. 4. 5. 6. 7. 8.

9.

1.

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ACE inhibitory peptides .................................................................................................................................................................. Mineral binding peptides ................................................................................................................................................................ Antidiabetic peptides ...................................................................................................................................................................... Satiating peptides ............................................................................................................................................................................ Immunomodulatory and antimicrobial peptides ....................................................................................................................... Other bioactivities ............................................................................................................................................................................ 8.1. Opioid peptides ..................................................................................................................................................................... 8.2. Antioxidant peptides ............................................................................................................................................................ 8.3. Populations with specific needs: allergy and phenylketonuria (PKU) .......................................................................... Conclusions and perspectives on the efficacy of milk BAPs in humans ................................................................................ Acknowledgement ........................................................................................................................................................................... References .........................................................................................................................................................................................

Introduction

Milk is the primary source of nutrients for young mammalians. It is recognised as being nutritionally balanced and has therefore attracted a lot of scientific interest over the years. Various properties of intact milk proteins have been reported including satiating, antimicrobial, mineral binding, antilipidaemic and anticancer properties (Anderson & Moore, 2004; Chatterton, Smithers, Roupas, & Brodkorb, 2006; Clare & Swaisgood, 2000; Cross et al., 2007; Nakamura et al., 2013). Following their digestion in the gastrointestinal tract (GIT), milk proteins are broken down to smaller components consisting of free amino acids and peptides, which may be used as the building blocks for a wide range of proteins in the body (Wada & Lönnerdal, 2014). Over the past few decades, it has been shown that specific protein fragments, called bioactive peptides (BAPs), may beneficially modulate certain health related biomarkers, at least in vitro. To date, there have been many studies demonstrating the potentially beneficial effect in vitro of food protein-derived BAPs on biomarkers associated with the metabolic syndrome and bone health (Hernández-Ledesma, García-Nebot, Fernández-Tomé, Amigo, & Recio, 2014; Panchaud, Affolter, & Kussmann, 2012; Udenigwe & Aluko, 2012). These include BAPs with antihypertensive, antidiabetic, antiobesity, antioxidant, immunomodulatory and mineral binding properties (FitzGerald & Meisel, 2003b; Korhonen & Pihlanto, 2006; Nongonierma & FitzGerald, 2012; Pihlanto, 2006; Power, Jakeman, & FitzGerald, 2013). In some instances, the in vivo effects of these milk protein-derived BAPs have been demonstrated in small animals (Bernabucci & Nardone, 2014; García-Tejedor et al., 2014; Gaudel et al., 2013; Sánchez-Rivera et al., 2014; Schellekens et al., 2014; Yamada et al., 2015) and also in humans (Akhavan, Luhovyy, Brown, Cho, & Anderson, 2010; Geerts et al., 2011; Jauhiainen et al., 2009, 2010; Morifuji et al., 2010; Usinger et al., 2010; Xu, Qin, Wang, Li, & Chang, 2008). Several studies have suggested that milk protein-derived BAPs may be used as preventative/prophylactic agents to alleviate symptoms of various diseases in humans. Although various drugs exist to cure/slow down the progress of specific diseases in humans, their side-effects may sometimes outweigh their benefits (Fekete, Givens, & Lovegrove, 2015; Li-Chan, 2015; Saadi, Saari, Anwar, Hamid, & Ghazali, 2015). In this context, food protein-derived peptides and specifi-

643 645 646 646 647 648 648 648 649 649 650 650

cally milk protein-derived BAPs have potential as natural alternatives to drugs for disease management (Carrasco-Castilla, Hernández-Álvarez, Jiménez-Martínez, Gutiérrez-López, & Dávila-Ortiz, 2012; Li-Chan, 2015; Saadi et al., 2015; Zambrowicz, Timmer, Polanowski, Lubec, & Trziszka, 2013). For example, no cytotoxic effects have been shown in small animals following the consumption of angiotensin converting enzyme (ACE) inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro (Bernard, Nakamura, Bando, & Mennear, 2005; Maeno, Nakamura, Mennear, & Bernard, 2005; Ponstein-Simarro Doorten, vd Wiel, & Jonker, 2009). Similarly, the antimicrobial (lactoferrin- LF f(1– 11)) peptides have been evaluated as being safe for human consumption (Brouwer, Rahman, & Welling, 2011; van der Velden, van Iersel, Blijlevens, & Donnelly, 2009). Milk protein-derived BAPs are not biologically active when present within the parent protein. They may be released by different means which include enzymatic hydrolysis of the proteins using various proteolytic/peptidolytic preparations, microbial fermentation, chemical or physical hydrolytic means and also naturally during the gastrointestinal (GI) digestion of milk proteins (Nongonierma & FitzGerald, 2011; Saadi et al., 2015). Milk protein-derived BAPs are therefore naturally present in a wide range of dairy products and foods containing dairybased ingredients. Depending on their sequence, these BAPs may reach the small intestine intact and be absorbed as is or they may be degraded by GI enzymes or serum peptidases in the circulation. Several studies have investigated the stability and bioavailability of milk BAPs, mainly following in vitro simulated gastrointestinal digestion (SGID) treatments (Nongonierma & FitzGerald, 2013; Stuknyte˙, Cattaneo, Masotti, & De Noni, 2015; Walsh et al., 2004) or permeation through Caco-2 cell monolayers (Picariello et al., 2013; Quirós, Dávalos, Lasunción, Ramos, & Recio, 2008; Shimizu, Tsunogai, & Arai, 1997; Vermeirssen et al., 2002). However, only a limited number of studies have been carried out in relation to the stability and bioavailability of BAPs in humans (Foltz et al., 2007; Jauhiainen et al., 2007, 2009). This may, in some instances, be related to analytical limitations associated with the detection of peptides and notably short peptides (Le Maux, Nongonierma, & FitzGerald, 2015; Panchaud et al., 2012) in relatively complex physiological fluids. In addition, because BAPs are naturally found within the body, notably in digestate fluids (Barbé et al., 2014; Boutrou et al., 2013; Chabance et al., 1998), the serum (Foltz et al., 2007) and different tissues (Jauhiainen et al., 2007) at relatively low levels

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Journal of Functional Foods 17 (2015) 640–656

(<10 ppm), it may in certain cases be challenging to demonstrate the contribution of specific BAP sequences to a target bioactive property. Quantification of a number of casein (CN)derived peptides in the jejunum of human subjects showed that their average concentration was 17 and 900 µM for the opioid peptide β-casomorphin-7 (β-CN f(60–66)) and the ACE inhibitory peptide β-CN f(108–113), respectively. The half maximal inhibitory concentrations of β-casomorphin-7 and β-CN f(108–113) were estimated at 100 µM (opioid agonist activity) and 423 µg/L (0.6 µM, ACE inhibition), respectively. This led the authors to conclude that these peptides were likely to display a biological activity in vivo (Boutrou et al., 2013). Interestingly, to date, no short peptides (di- and tripeptides) have been detected in the GIT of humans. This may be due to analytical limitations associated with MS peptide identification (Boutrou, Henry, & Sanchez-Rivera, 2015). The bioactive response of a single peptide may be masked by the presence of other components originating from the diet (Hansen, Sandström, Jensen, & Sørensen, 1997). Furthermore, the difficulty of defining adequate control subjects has often been highlighted in human intervention studies. It appears easier to obtain more relevant controls with animals as their diet can be more tightly regulated than that of human subjects. Moreover, a number of studies have highlighted the contribution of confounding factors (variables which are not controlled) such as genotype on the biological/physiological outcome(s) of dairy-derived dietary components in humans (Abdullah et al., 2014; Cicero, Gerocarni, Laghi, & Borghi, 2011; Fekete, Givens, & Lovegrove, 2013). This may explain the interindividual variability of responses to specific interventions with bioactive components. In many instances, a direct correlation between ingestion of specific milk protein-derived BAPs and their beneficial effects on specific health biomarkers appears difficult to establish. Consequently it has become challenging for different regulatory bodies to grant health claim approval to specific ingredients. This is notably the case for milk protein-derived opioid and ACE inhibitory peptides from milk proteins even though these BAPs have been quite extensively studied (De Noni et al., 2009; EFSA, 2010, 2011). Different meta-analyses have been performed on human trial data obtained with milk protein-derived BAPs. This is particularly the case for ACE inhibitory peptides (Cicero, Aubin, Azais-Braesco, & Borghi, 2013; Cicero et al., 2011; Fekete et al., 2013; Qin et al., 2013; Turpeinen, Järvenpää, Kautiainen, Korpela, & Vapaatalo, 2013; Xu et al., 2008) and caseinophosphopeptides (CPPs) (Yengopal & Mickenautsch, 2009). However, these metaanalyses are very focused and do not take a general overview incorporating the potential multifunctional effects of milk protein-derived BAPs. Since it has been clearly demonstrated that BAPs can have multifunctional properties (Ballard et al., 2013; Mandal et al., 2014), it appeared essential to take a broader view when reviewing the scientific evidence in connection with the in vivo effects of milk protein-derived BAPs. Boutrou et al. (2015) have recently reviewed the BAPs originating from milk which have been identified in the intestinal contents of humans and animals. Although the presence of milk protein-derived BAPs has been demonstrated in the GIT and in the plasma of humans, it is still unclear if these peptides display a biological effect in vivo. Specific peptides are able to reach the blood circulation without being degraded and may be found in physi-

ologically relevant amounts (Foltz et al., 2007). However, to our knowledge, only a limited number of reviews have critically assessed the effect of milk BAPs on metabolic health in humans (Artym & Zimecki, 2013; Hernández-Ledesma et al., 2014; McGregor & Poppitt, 2013). The aim of this review was to assess current literature related to the manner in which different milk protein-derived BAP sequences may affect a wide range of health biomarkers in humans. Different biomarkers/targets which appear crucial in human health have been incorporated in the present review. The evidence linking the in vitro activity of milk protein-derived BAPs and their physiological effects in humans will be discussed, taking specific examples from ACE inhibitory, mineral binding, antidiabetic, satiating, immunomodulatory, opioid and antioxidant activities along with their role in nutrition for populations with specific needs. Finally, recommendations to improve human intervention studies with milk protein-derived BAPs with health promoting effects will be suggested.

2. Milk protein-derived BAPs identified in the circulation of humans To date, there are a limited number of scientific studies which have reported the presence of peptides in the circulation of humans (Chabance et al., 1995, 1998; Foltz et al., 2007; Gardner, 1983). Subsequent to ingestion of milk or dairy products, milk protein-derived peptides have been identified in biological fluids of humans including plasma, serum and milk (Foltz et al., 2007; Koch et al., 1988; Kost et al., 2009; Renlund et al., 1993; Righard, Carlsson-Jonsson, & Nyberg, 2014) as outlined in Table 1. To date, the peptides identified in biological fluids from humans mostly originate from CNs. Dipeptides and peptides as large as caseinomacropeptide (CMP; 64 amino acid length) have been identified in the plasma, serum and milk of humans (Table 1). Some of the peptides detected in human plasma have previously been reported for their ACE inhibitory (Foltz et al., 2007) and opioid (Koch et al., 1988; Kost et al., 2009; Renlund et al., 1993; Righard et al., 2014) properties. For example, the ACE inhibitory peptides Leu-Trp, Phe-Tyr, Ile-Tyr, Ile-Trp, Ala-Trp, ValTyr, Leu-Pro-Pro and Ile-Pro-Pro, were detected in the circulation of humans following the ingestion of a lactotripeptide (LTP) enriched yoghurt beverage (Foltz et al., 2007). CMP from bovine and human milk has also been detected in the plasma of newborns (Chabance et al., 1995). An average concentration of 16 and 21 µg/mL of CMP was reported in the plasma of newborns fed with human milk and cow’s milk-based infant formula, respectively. The question remains whether these concentrations are sufficient to mediate a physiological response. The presence of intact CMP in the plasma of infants has been explained by the relatively higher permeability of their gut, the low level of proteolytic enzymes in their stomach and the inhibitory role of CMP on gastric secretion (Chabance et al., 1995). Other human studies have reported the detection of peptides in the plasma (Chabance et al., 1998). Fragments corresponding to αs1-CN f(1–23), αs1-CN f(1–21) and κ-CN f(106–126) have been identified following the ingestion of milk or yoghurt by human subjects (Chabance et al., 1998). The low number of peptides found within the plasma compared to the GIT (Boutrou

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Table 1 – Peptide sequences detected in human biological fluids (plasma, serum and milk) following the ingestion of bovine milk or dairy products. Parent protein

Peptide fragment

Peptide sequencea

Locus

Bioactive properties

Reference

αs1-CN

1–21 1–23 51–58 51–58 51–58 51–57 60–66 106–117 106–169 81–82 and 399–400 Diverse Diverse Diverse (α-La and BSA) Diverse Diverse Diverse (β- and κ-CN) Diverse Diverse Diverse Diverse

RPKHPIKHQGLPQEVLNENLL RPKHPIKHQGLPQEVLNENLLRF (isracidin) YPFVEPIP (human β-casomorphin-8) YPFVEPIP (human β-casomorphin-8) YPFVEPIP (human β-casomorphin-8) YPFVEPI (human β-casomorphin-7) YPFPGPI (bovine β-casomorphin-7) MAIPPKKNQDKT Glycomacropeptide IY LW FY IW

Plasma Plasma Plasma Milk Serum and milk Plasma Plasma Plasma Plasma Serum Serum Serum Serum

n.d. Antibacterial Opioid Opioid Opioid Opioid Opioid n.d. Antithrombic ACE inhibitor ACE inhibitor ACE inhibitor ACE inhibitor

(Chabance et al., 1998) (Chabance et al., 1998) (Koch et al., 1988) (Renlund et al., 1993) (Righard et al., 2014) (Kost et al., 2009) (Kost et al., 2009) (Chabance et al., 1998) (Chabance et al., 1995) (Foltz et al., 2007) (Foltz et al., 2007) (Foltz et al., 2007) (Foltz et al., 2007)

AW VY IPP

Serum Serum Serum

ACE inhibitor ACE inhibitor ACE inhibitor

(Foltz et al., 2007) (Foltz et al., 2007) (Foltz et al., 2007)

LPP IL VL LL

Serum Plasma Plasma Plasma

ACE inhibitor n.d. n.d. n.d.

(Foltz et al., 2007) (Morifuji et al., 2010) (Morifuji et al., 2010) (Morifuji et al., 2010)

β-CN

κ-CN LF CN and whey

a Peptide sequence with the one letter amino acid code. n.d.: not disclosed; ACE: angiotensin converting enzyme; BSA: bovine serum albumin; CN: casein; α-La: α-lactalbumin; LF: lactoferrin.

et al., 2013; Chabance et al., 1998) was attributed to the possible degradation of peptides which may occur following the hydrolytic action of GI enzymes and intestinal cell peptidases. Of the κ-CN peptides detected in the stomach, duodenum or plasma in the study of Chabance et al. (1998), a number have been reported to have antithrombic, antiaggregation and antihypertensive activities. A large number of studies have been conducted in small animals which have shown a physiological effect of milk proteinderived BAPs. Although only a few studies have been conducted in humans, this number has been on the increase for the last few years. This can be explained by the growing interest by the scientific, industrial and healthcare communities in the application of natural components for health enhancement. This is particularly relevant in the context where metabolic diseases and their associated complications are on the increase worldwide. BAPs from milk or other food proteins, alone or in combination with drugs (Nongonierma & FitzGerald, 2015), may find application as natural alternatives to drugs to act as preventative agents of various diseases in humans (Li-Chan, 2015; Saadi et al., 2015). This could help avoid the side-effects seen with many drugs.The effects of milk protein-derived BAPs having antihypertensive, opioid, satiating and blood serum glucose regulating activities have been particularly studied in humans. However, in many cases, a direct link between in vitro and in vivo bioactivity is difficult to establish.

3.

ACE inhibitory peptides

Milk protein-derived antihypertensive peptides and particularly ACE inhibitory peptides have been evaluated in numerous

human intervention studies (Fekete et al., 2013). This may be linked with the fact that cardiovascular diseases (CVDs) are the major cause of death in humans globally (WHO, 2013a). High blood pressure (BP) is a modifiable risk factor for CVD. Mechanisms other than ACE inhibition have also been reported as being associated with the observed hypotensive effects of milk BAPs (endothelin converting enzyme inhibition, opioidinduced BP regulation, renin inhibition, immunomodulation and antioxidation) (Fekete et al., 2013; Norris & FitzGerald, 2013). Three groups of milk BAPs with ACE inhibitory activity have been extensively evaluated in humans; these include the CNderived LTPs (Ile-Pro-Pro and Val-Pro-Pro commercialised as Evolus® and Ameal S®), Ser-Lys-Val-Tyr-Pro and the αs1-CNderived C12 peptide (Phe-Phe-Val-Ala-Pro-Phe-Pro-Glu-ValPhe-Gly-Lys) (Fekete et al., 2013). The LTPs appear to be the most studied peptides in humans to date (Fekete et al., 2015). A number of meta-analyses on LTPs are available in the scientific literature describing the main outcomes of the consumption of these peptides in humans (Cicero et al., 2011, 2013; Fekete et al., 2015; Qin et al., 2013; Turpeinen et al., 2013; Xu et al., 2008). The main mechanism of action involved in the BP reducing effect of the LTPs is thought to involve ACE inhibition, arginase inhibition (relevant to the nitric oxide system) and endothelium-derived hyperpolarisation factor-related mechanisms (Turpeinen et al., 2013). A summary of the 6 metaanalyses carried out to date on the effect of LTPs on BP in humans is outlined in Table 2. Based on the inclusion criteria, in the most recent meta-analysis, 33 human interventions conducted between 1996 and 2012 were taken into account (Fekete et al., 2015). It was shown that the main outcome of the human interventions to date is a significant reduction in

644

1.8–10.2 Europeans 14 (15 sets) 2002–2012

1277 (씹 and 씸)

10.5 (average) Japanese and Europeans 33 (in 30 papers) 1996–2012

2200 (1184 씹 and 1016 씸)

n.d. Japanese and Europeans 28 (in 24 papers) 1996–2011

1919 (씹 and 씸)

2–52 (9.4 ± 13.3) Japanese and Europeans 18 1996–2010

1691 (씹 and 씸)

2.0–10.2 Japanese and Europeans 1532 (씹 and 씸) 19 1996–2010

DBP: diastolic blood pressure; SBP: systolic blood pressure; LTP: lactotripeptide; n.d.: not determined; 씹: males; 씸: females. a Statistical significance of the results, *: statistically different (P < 0.05) results compared to the control and ns: not significantly different (P > 0.05) from the control.

(Cicero et al., 2013)

(Fekete et al., 2015)

(Qin et al., 2013)

SBP: −1.30* DBP: −0.57ns SBP: −0.94ns DBP: −0.46ns n.d.

(Cicero et al., 2011) n.d.

(Turpeinen et al., 2013) n.d.

(Xu et al., 2008) n.d.

SBP: −4.8* DBP: −2.2* SBP: −4.0* DBP: −1.9* SBP: −3.73* DBP: −1.97* SBP: −1.66* DBP: −0.76* SBP: −2.95* DBP: −1.51* SBP: −1.28* DBP: −0.59* 2.6–5.6 Japanese and Europeans 9 (12 trials) 1996–2005

623 (씹 and 씸)

Reference Average reduction of 24 h ambulatory blood pressure (mmHg)a Average reduction of office blood pressure (mmHg)a LTP dose (mg/day) Ethnicity Total number of participants Number of studies included Period of study

Table 2 – Summary of the meta-analyses carried out with lactotripeptides (LTPs; Val-Pro-Pro and Ile-Pro-Pro) with angiotensin converting enzyme (ACE) inhibitory properties: effect on blood pressure reduction in humans.

Journal of Functional Foods 17 (2015) 640–656

systolic BP (SBP, −2.95 mmHg) and diastolic BP (DBP, −1.51 mmHg, Table 2) (Fekete et al., 2015). Analysis of the subgroup characteristics allowed identification of various parameters which had a differential impact on BP. A higher reduction in SBP and DBP was seen in (a) subjects with higher baseline BP, (b) lower body mass index (BMI) and (c) younger age (Cicero et al., 2013; Fekete et al., 2015; Qin et al., 2013; Xu et al., 2008). The study design also affected the BP outcomes with a positive effect of BP reduction seen in studies (a) ≤8 weeks in duration, (b) carried out with a lower LTP dose (≤10 mg/day), (c) LTPs obtained by fermentation, (d) with a Japanese vs. European population and (e) double-blind randomised clinical trials (Cicero et al., 2011; Fekete et al., 2015; Qin et al., 2013; Turpeinen et al., 2013). The racial effects have been explained by both genetic differences and also environmental factors. Within the environmental factors, diet is thought to play an important role in the responsiveness of different populations. For instance, the lower consumption of milk/dairy products in Japan compared to Europe may, in part, explain the differences observed (Fekete et al., 2015). Other parameters may influence the greater decrease in BP seen in the Japanese population; these comprise factors which have been shown to positively impact on BP reduction, including a lower BMI and a lower LTP dose (7.57 vs. 11.68 mg/day for the Japanese and European populations, respectively) (Fekete et al., 2015). A higher responsiveness of Japanese subjects to drugs involving the renin–angiotensin system and differences in the pharmacokinetics of the LTPs in Asians in comparison to Caucasians have also been proposed to explain these differences (Cicero et al., 2011). In addition, it has been suggested that peptides with different cis/trans conformations may have been employed in the different human studies and these may have a different biological activity (Fekete et al., 2015; Turpeinen et al., 2013). All six meta-analyses showed a significant reduction in SBP and DBP in humans consuming the LTPs. However, the decrease in SBP and DBP was modest compared to that usually reported with BP reducing drugs (Baguet, Legallicier, Auquier, & Robitail, 2007). In a meta-analysis carried out on BP reducing drugs, it has been stated that a modest reduction of BP may positively impact on risk reduction of CVD. After a one year follow-up with BP reducing drugs, a reduction of the SBP (−10 mmHg) and DBP (−5 mmHg) was predicted to lower the risk of coronary artery disease and stroke by approximately 20 and 32%, respectively (Law, Morris, & Wald, 2009). Interestingly, the LTP Ile-Pro-Pro has been detected in the circulation of humans following ingestion of an LTPenriched yoghurt beverage (Foltz et al., 2007). Significantly higher levels of Ile-Pro-Pro were detected after consumption of the LTP drink compared to the placebo (an isocaloric beverage), 897 ± 157 and 555 ± 0.09 pmol/L, respectively. Furthermore, these levels were increased when the yoghurt was consumed with a meal (Foltz et al., 2007). Despite the detection of ACE inhibitory milk protein-derived peptides in human serum and the positive outcomes of several meta-analyses, the effects observed in humans on BP reduction are moderate. In addition, due to confounding factors in humans studies, conflicting results are found in the literature, making it difficult to conclude on the role of milk protein-derived BAPs as antihypertensive agents.

Journal of Functional Foods 17 (2015) 640–656

4.

Mineral binding peptides

CPPs have been proposed to play a role in improving the bioavailability of dietary metal ions. It has been suggested that the mineral binding properties of CPPs are linked with a specific sequence, the “acidic motif”, i.e., Ser(P)-Ser(P)-Ser(P)-Glu-Glu (Bouhallab & Bouglé, 2004). To date, the relationship between CPP intake and Ca absorption has still not been clearly demonstrated in the scientific literature (FitzGerald, 1998; FitzGerald & Meisel, 2003a; Hartmann & Meisel, 2007; Meisel & FitzGerald, 2003; Meisel & Frister, 1988; Pinto et al., 2012). Owing to their anionic properties, CPPs are thought to be resistant to GI digestion (Kitts, 2005). This may help explain the fact that they have been found in the GIT and faeces of rats and humans (Hartmann & Meisel, 2007; Kasai, Iwasaki, Tanaka, & Kiriyama, 1995). To date, most of the human studies carried out with CPPs in humans have focused on their teeth remineralising properties in the context of dental caries (for review, see: Nongonierma & FitzGerald, 2012; Pinto et al., 2012; Reynolds, 2008). Many studies concluded in a beneficial effect of CPPs on dental enamel remineralisation in humans. For dental applications, amorphous calcium phosphate (ACP), the precursor of hydroxyapatite, has been combined to CPPs to form CPP–ACP complexes. These complexes have been formulated in a dental ingredient, sold under the trade name Recaldent™, aimed at remineralising the enamel surface and reducing the rate of enamel decalcification. Recaldent™ is thought to increase Ca and phosphate solubility as well as hydroxide ion content (Azarpazhooh & Limeback, 2008; Cross, Huq, & Reynolds, 2007; Reynolds, 1998; Rose, 2000; Shen et al., 2011). Several products are found on the marketplace which are composed of Recaldent™; these include Trident white sugar gum, Recaldent™ sugar free gum, Recaldent mints™ and MI Paste or Tooth mousse (Azarpazhooh & Limeback, 2008; Gupta & Prakash, 2011). CPP–ACP is understood to act through localising Ca and phosphate ions in the plaque and via their buffering capacity properties (Kitasako et al., 2010; Reynolds, Cai, Shen, & Walker, 2003). Despite the large amount of promising data generated from in vitro, in situ and also in vivo studies, contradictory results are also found in the scientific literature (Nongonierma & FitzGerald, 2012; Peters, 2010), suggesting no beneficial effect of CPPs as anticariogenic agents in humans (Altenburger, Gmeiner, Hellwig, Wrbas, & Schirrmeister, 2010; Beerens, van der Veen, van Beek, & ten Cate, 2010; Brochner et al., 2011). This may be linked to the set-up of the human trials, i.e., differences in CPP dose, residence time, treatment applied to the control group and CPP interactions with other components (Nongonierma & FitzGerald, 2012). The presence of CPPs in the GIT of humans has been demonstrated (Hartmann & Meisel, 2007; Meisel et al., 2003). CPPs were detected at higher concentrations following the ingestion of milk compared to that of CPPs. It was concluded that milk proteins may have a protective effect preventing further degradation of CPPs in the digestive tract (Meisel et al., 2003). A limited number of human studies carried out with CPPs have evaluated their mineral bioavailability en-

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hancement effects. Ca and Zn absorption was evaluated in 22 young adults (20–30 y) in the presence of CPPs and commercial whole grain or rice-based cereal infant products (Hansen et al., 1997). Although no effect was seen for fractional Ca absorption in the presence of CPPs with the rice meal, differences in the absorption (+30%) were reported. There was an increase in Zn fractional absorption and Zn absorption in the presence of CPPs with rice. No significant effect of CPPs on fractional absorption and absorption of Ca and Zn was seen with the whole rice, possibly due to the presence of phytates (Hansen et al., 1997). Similarly, another human study conducted with 31 adults (19–30 y) ingesting white bread with different levels of Ca and phytates did not show any positive role of CPPs on Ca and Zn absorption (Hansen et al., 1997). This lack of an effect of CPPs on the bioavailability of dietary Ca and Zn may arise from different factors such as the instability of the CPPs during digestion or an inadequate dosage of the CPPs. Radioisotopically labelled milk supplemented with CPP bound Fe (Fe-β-CN f(1–25)) or a control Fe (FeSO4) was administered once to 10 young females (20–30 y). There was a significant increase in tissue (liver, spleen and sacrum) Fe uptake but not its absorption with Fe-β-CN f(1–25) compared to FeSO4 (Ait-Oukhatar et al., 2002). Another in vivo study consisted of a randomised trial with 15 human subjects which were administered Ca lactate drinks with or without CPPs (control). The true fractional Ca absorption (TFCA, representing the proportion of Ca in the urine relative to that ingested) differed between the groups. Surprisingly, there was a decrease in the TFCA in the test groups receiving two different types of commercially available CPP preparations (TFCA = 22.7 and 23.5) compared to that of the control (TFCA = 26.0) group (Teucher et al., 2006). CPPs did not further enhance Ca absorption in healthy adults (25–36 y) as compared to a control milk drink (López-Huertas et al., 2006). Similar results were obtained with post-menopausal women, showing no effect of CPP supplementation on Ca absorption (Narva, Karkkainen, Poussa, Lamberg-Allardt, & Korpela, 2003). The effect of night time consumption of fermented milk supplemented with Ca and Ca enhancers (inulin type fructans-ITF and CPPs) on bone resorption was studied in 85 post-menopausal women (Adolphi et al., 2009). In the presence of ITF and CPPs, an increase in urinary Ca excretion was seen, which was linked with an enhanced Ca absorption. It was shown that CPP supplementation was effective in increasing Ca bioavailability in post-menopausal women with low Ca absorption in contrast with women with a high Ca absorption. However, the Ca enhancers did not further improve the reduced bone resorption observed following the consumption of fermented milk (Heaney, Saito, & Orimo, 1994). The different effects of CPPs on mineral bioavailability in humans could be explained by the presence of chelating agents (i.e. phytates, oxalates and tannins), the food matrix, CPP preparation, CPP dose and CPP : mineral ratio (Erba, Ciappellano, & Testolin, 2002; FitzGerald, 1998; FitzGerald & Meisel, 2003a; Gueguen & Pointillart, 2000; Meisel & FitzGerald, 2003; Meisel & Frister, 1988) as well as the health status of the human subjects, i.e., in terms of mineral deficiencies (Adolphi et al., 2009), Ca malabsorption (Heaney et al., 1994) and Vit D levels (Narva et al., 2003).

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Antidiabetic peptides

Consumption of milk protein-derived BAPs has been linked with serum glucose regulatory properties in humans. Different mechanisms may include an insulinotropic activity, incretin secretagogue action, as well as activity on different metabolic enzymes involved in the regulation of serum glucose such as dipeptidyl peptidase IV (DPP-IV), α-amylase and α-glucosidase (Lacroix & Li-Chan, 2014). No in vivo data appear to have been reported to date on the DPP-IV and α-glucosidase inhibitory activity of milk protein hydrolysates in humans. To our knowledge, milk protein hydrolysates have mostly been shown to regulate serum glucose in humans through their insulinotropic activity. The individual components which are responsible for this insulinotropic activity of milk protein hydrolysate have not been clearly identified in humans. It is thought that the active components may comprise a combination of branched-chain amino acids (BCAA) and milk protein-derived peptides (Luhovyy, Akhavan, & Anderson, 2007; Morifuji et al., 2010). Free amino acids can directly act at the β-cell level to release insulin through various mechanisms including membrane depolarisation, mitochondrial signalling affecting insulin secretion, etc. (Newsholme, Gaudel, & McClenaghan, 2010). The insulinotropic properties of milk-derived components have been particularly attributed to whey-derived peptides (Frid, Nilsson, Holst, & Björck, 2005; Luhovyy et al., 2007; Oseguera-Toledo, González de Mejía, Reynoso-Camacho, Cardador-Martínez, & Amaya-Llano, 2014). However, it has been shown that both whey protein hydrolysates (WPHs) and casein hydrolysates (CNHs) are able to induce an insulinotropic response in humans. Most milk protein-derived hydrolysates which have been evaluated in humans appear to exist as proprietary products. In contrast, a human trial was recently conducted with a whey protein isolate (WPI) hydrolysate generated at a laboratory-scale with Flavourzyme™ (Novozymes Japan, Chiba, Japan) (Goudarzi & Madadlou, 2013). The insulinotropic effects of milk protein hydrolysates have been seen both with (Frid et al., 2005; Geerts et al., 2011; Luhovyy et al., 2007) or without carbohydrates (Akhavan et al., 2010; Morifuji et al., 2010; Power, Hallihan, & Jakeman, 2009) and in the absence or presence of Leu (Geerts et al., 2011; Manders, Koopman, et al., 2006; Manders, Praet, et al., 2006), an amino acid which has been shown to have a strong insulinotropic activity. The insulinotropic activity of WPHs has been related to intestinal amino acid absorption and the increased plasma concentration of free amino acids (Leu, Ile, Phe, Arg, Tyr, Thr, Val, Ala and Lys), BCAA-containing dipeptides (e.g. Ile-Leu, LeuLeu and Val-Leu) and possibly cyclic dipeptides (Koopman et al., 2009; Morifuji et al., 2010; Stanstrup et al., 2014; van Loon, Saris, Verhagen, & Wagenmakers, 2000). Contradictory results exist, where no direct correlation between plasma BCAA and the consumption of an insulinotropic WPH could be established (Power et al., 2009). Consumption of 10 g of a commercial WPH (Hilmar™ 8350; Hilmar Ingredients, Canada) by humans did not induce a reduction in post-meal serum glucose, although insulin secretion was increased (Akhavan et al., 2010). This was explained by the fact that the WPH did not affect gut hormone secretion (i.e., cholecystokinin – CCK, glucagon-like peptide 1

– GLP-1 and gastric inhibitory polypeptide – GIP) and consequently gastric emptying. In addition, the response observed in humans with various WPHs is possibly dependent on their free amino acid and peptide compositions. The extent of protein hydrolysis may dictate in vivo plasma amino acid level following ingestion of protein hydrolysates (Morifuji et al., 2010). A dose–response for insulin secretion was shown with a WPH generated at a laboratory-scale (Goudarzi & Madadlou, 2013). WPH doses ≤0.1 g/kg did not induce an insulinotropic response in humans. The higher dose tested for WPH (0.4 g/kg) was associated with a reduction in the 2 h post-prandial serum glucose concentration (<7.8 mM) (Goudarzi & Madadlou, 2013). Most of the in vivo work with CNHs appears to have been carried out with the proprietary CNH InsuVida™ previously named Insuvital™ (DSM, Delft, The Netherlands). Different doses of CNH were tested with or without carbohydrates and with or without free amino acids (Leu and Phe). The outcomes of the 7 human interventions carried out with InsuVida™ are summarised in Table 3. Most of the studies carried out to date have shown a beneficial effect of the CNH on the reduction of serum glucose in type 2 diabetic (T2D) subjects (Geerts et al., 2011; Manders, Koopman, et al., 2006; Manders, Praet, et al., 2006; Manders et al., 2005; Manders et al., 2014). In contrast, no significant effect of the CNH was found in two studies. In one case this was related to the relatively low amount (6 g) of CNH ingested (Jonker et al., 2011). In the second study, a relatively high dose (0.4 g/kg) of CNH was ingested (Manders, Praet, Vikstrom, Saris, & van Loon, 2007). The reasons behind the lack of an observed reduction in serum glucose in the study of Manders et al. (2007) are not clearly understood. Lower doses of the CNH have been tested in humans with the rationale of reducing high protein loads which may be detrimental to T2D subjects who are at risk of renal complications. Reducing the ingested dose (15 g) of CNH by half as compared to previous studies (Manders, Koopman, et al., 2006; Manders, Praet, et al., 2006) still resulted in a reduction in serum glucose (Geerts et al., 2011). Similarly, reduction of the CNH dose to 12 g induced a reduction in serum glucose, however, this was not seen on ingestion of 6 g CNH (Jonker et al., 2011). Addition of Leu to the CNH induced a large increase in plasma insulin concentration but only had a minor impact on postprandial serum glucose level (Geerts et al., 2011; Manders, Koopman, et al., 2006).

6.

Satiating peptides

The development of new solutions to combat obesity is necessary due to the global increase in obesity incidence and its link with health complications including dyslipidaemia, CVD, impaired glucose metabolism, etc. (Gustafson, McMahon, Morrey, & Nan, 2001; Nguyen et al., 2012; Schrezenmeir & Jagla, 2000; WHO, 2013b). Reducing food intake and increasing energy expenditure are a means to control body weight. A reduction of food intake may be facilitated by increasing satiety. Satiety, which arises from various signals between the gut and brain, corresponds to the hunger alleviation seen after food intake. It is thought to involve stimulation of gut hormone (CCK, peptide YY (PYY) and GLP-1) receptors, a slower gastric emptying, an increased diet-induced thermogenesis and possibly opioid

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Table 3 – Summary of the glycaemic management outcomes of the randomised double blind human studies conducted with the InsuVidaTM (DSM, Delft, The Netherlands) casein hydrolysate. Samples evaluated

Human subjects

In vivo outcomes % decrease of blood glucose concentrationa

% increase of blood insulin concentrationa

(1) = 0.7 g/kg CHO (2) = (1) + 0.35 g/kg CNH mixed with Leu and Phe (1) = 0.7 g/kg CHO (2) = (1) + 0.3 g/kg CNH (3) = (2) + 0.1 g/kg Leu

59–64 y 씹 T2D: 59 Healthy: 9 57–62 y 씹 T2D: 10 Healthy: 10

T2D: −15.12% Healthy: −92.97%

T2D: 299 ± 64% Healthy: 132 ± 63%

(Manders et al., 2005)

57–61 y T2D: 11 Healthy: 11 60–64 y 씹 T2D: 13 50–70 y 씹 and 씸 T2D: 36

T2D: 41 and 204% (for (2) and (3), respectively) Healthy: 66 and 221% (for (2) and (3), respectively) n.d.

(Manders et al., 2006)

(1) = Placebo (2) = 0.3 g/kg CNH + 0.1 g/kg Leu

T2D: −15 and −12% (for (2) and (3), respectively) Healthy: −92 and −97% (for (2) and (3), respectively) T2D: −11 ± 3% Healthy: n.s. n.s.

n.d.

(Manders et al., 2007)

T2D: −4.7% for (3) and (4); n.s. for (2)

T2D: 26.1 and 51.8% for (3) and (4), respectively

(Geerts et al., 2011)

57–59 y 씹 T2D: 13

T2D: n.s. effect with (2) and −6% (3)

T2D: n.s. effect with (2) and 26% for (3) and (4), respectively

(Jonker et al., 2011)

44–79 y 씹 T2D : 60

T2D: −22 ± 32 and −23 ± 36% for (2) and (3), respectively

T2D: 99 ± 41 and 110 ± 10% for (2) and (3), respectively

(Manders et al., 2014)

(1) = Placebo (2) = 0.4 g/kg CNH (1) = Placebo (2) = 15 g CN (3) = 15 g CNH (4) = 15 g CNH + 5 g Leu (1) = 50 g CHO (2) = (1) + 6 g CNH (3) = (1) + 12 g CNH (1) = 0.7 g/kg CHO (2) = (1) + 0.3 g/kg CN (3) = (1) + 0.3 g/kg CNH

Reference

(Manders et al., 2006)

CHO: carbohydrates; CN: casein; CNH: casein hydrolysate; n.d.: not determined; n.s.: not significant; T2D: type 2 diabetes; 씹: males; 씸: females. a In each study, the percentage increase or decrease is expressed relatively to the control sample (1).

receptors (Anderson & Moore, 2004; Bendtsen, Lorenzen, Bendsen, Rasmussen, & Astrup, 2013; Luhovyy et al., 2007; McGregor & Poppitt, 2013; Sam, Troke, Tan, & Bewick, 2012; Turgeon & Rioux, 2011). In terms of direct intake of milk protein-derived peptides, particular attention has been given to CMP. The satiating properties of CMP have been considered to involve an increased release of CCK and inhibition of gastric secretions (Guilloteau et al., 2010; Gustafson et al., 2001; Thomä-Worringer, Sørensen, & López-Fandiño, 2006; Yvon, Beucher, Guilloteau, Le Huerou-Luron, & Corring, 1994). However, most human studies conducted to date with CMP appear to have failed to demonstrate a clear satiating effect followed by a reduction in food intake (Chungchunlam, Henare, Ganesh, & Moughan, 2014; Gustafson et al., 2001; Poppitt, Strik, McArdle, McGill, & Hall, 2013; Yvon et al., 1994). This was also the case in the study conducted by Keogh et al. (2010) showing no significant effect of a CMP preload (50 g) on plasma CCK level, food intake and subjective satiety in obese subjects compared to a glucose control. Similarly, CMP alone used as a preload before a meal did not reduce food intake in healthy humans (Chungchunlam et al., 2014). Although a CMP preload failed to increase pre-test meal satiety, a positive role of CMP on compensatory food intake (additional food intake throughout the day) has been shown in females in contrast with male subjects (Burton-Freeman, 2008). This was evidenced by a significant decrease in food intake on the study day. The differences between male and female subjects could be linked to the fact that hormonal changes also differed, with pre-lunch CCK concentration being twice as high in women as in men. Differences in human satiety study out-

comes may arise from various factors including the metabolic/ health status of the individuals participating in the study, their age, sex, the timing of the satiety evaluation and also the quality and amount of protein ingested. The emotional status of the subjects is also a very important parameter in food intake (Raspopow, Abizaid, Matheson, & Anisman, 2014).

7. Immunomodulatory and antimicrobial peptides The consumption of milk proteins has been associated with immunostimulatory effects in humans. A reduction in lowgrade inflammation in healthy adults was correlated with the intake of dairy products in the ATTICA study (18 months, 3042 subjects) (Panagiotakos, Pitsavos, Zampelas, Chrysohoou, & Stefanadis, 2010). It is not clear, however, if the origin of this effect was the milk protein components. CPPs (290 mg mixture of β-CN f(1–28) and αs2-CN f(1–32)), formulated in cakes, were ingested for 30 days by 7 healthy subjects. Immunostimulatory effects were seen, with higher faecal IgA levels compared to the control group (Kitamura & Otani, 2002). Studies with a commercial WPH (MEIN™, Meiji Dairies Co., Tokyo, Japan) have shown a significant reduction in inflammatory markers (highsensitivity C-reactive protein (hsCRP), IL-6, IL-8, and tumour necrosis factor (TNF)-α) after 12 weeks’ consumption in elderly patients participating in a low-exercise programme and presenting chronic obstructive pulmonary disease (Sugawara et al., 2012).

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Antimicrobial and immunostimulatory activities are linked. For instance, specific milk protein-derived BAPs with immunomodulatory action have been shown to increase the resistance to pathogens in the GIT (Gauthier, Pouliot, & Saint-Sauveur, 2006). Various milk proteins and peptides have been reported to have a wide range of antimicrobial properties in vitro (Agyei & Danquah, 2012; Benkerroum, 2010; Hartmann & Meisel, 2007; Morris & FitzGerald, 2009; Wilson, Buchanan, Allan, & Tikoo, 2012). Specific modes of action, involving their buffering capacity and inhibitory activity on microbial enzymes, have been proposed for the antibacterial activity of CPPs (Rose, 2000). A few studies involving in situ experiments with humans wearing dental appliances showed that CPP–ACP could increase the pH (from 5.0 to 5.8) through its buffering capacity, following a carbohydrate challenge (Caruana, Al Mulaify, Moazzez, & Bartlett, 2009). Another human intervention study evaluated the effect of CPP-amorphous calcium phosphate fluoride (ACPF) on bacteria growth in white spot lesions found after debonding of orthodontic appliances. It was shown after 4 weeks of topical treatment with CPP–ACPF that there was a reduction in aciduric bacteria (from 47.4 to 38.1%) and Streptococcus mutans (from 9.6 to 6.6%) counts (Beerens et al., 2010). In contrast, no significant effect of CPP–ACP could be found in humans wearing fixed orthodontic appliances (Marchisio, Esposito, & Genovesi, 2010). Peptides originating from LF have also been shown to have a wide range of antimicrobial and anti-inflammatory activities in vivo, notably human LF f(1–11) (Brouwer et al., 2011). This peptide has also been evaluated for safety in humans. It was reported to be safe (no adverse effects on various health markers or cytotoxicity) in healthy humans and in haematopoietic stem cell transplantation patients in intravenous doses as high as 5000 µg over 5 days (van der Velden et al., 2009). However, its pharmacokinetics are unknown; this has been attributed to analytical limitations where detection of the peptide in the plasma was not possible due to the very low doses employed (≤5000 µg) (van der Velden et al., 2009).

8.

Other bioactivities

8.1.

Opioid peptides

Opioid peptide sequences typically contain Tyr-Gly-Gly-Phe at their N-terminal side, and in the case of atypical opioid peptides, a Tyr residue is found at the N-terminus (Teschemacher, Koch, & Brantl, 1997). These peptides have been reported to behave like morphine in the brain. These have been found within milk proteins, both in CN (caseinomorphins) and WP (lactorphins). Although numerous small animal studies have been carried out with milk protein-derived BAPs with opioid activity, they do not appear to have been extensively evaluated in humans (Artym & Zimecki, 2013; Teschemacher et al., 1997). In addition, in an EFSA opinion report, the clinical evidence to date of the potential role of β-casomorphin 7 (β-CN f(60–66)) on various health conditions has been judged to be inconclusive especially since no dose–effect relationship appears to have been reported in the literature (De Noni et al., 2009). Following ingestion of milk/dairy products, opioid peptides have been detected in different parts of the body including

the GIT of humans (Svedberg, de Haas, Leimenstoll, Paul, & Teschemacher, 1985), the brain (Nyberg et al., 1989; Pasi, Mahler, Lansel, Bernasconi, & Messiha, 1993) and different body fluids of infants and adults (Koch et al., 1988; Kost et al., 2009; Renlund et al., 1993; Righard et al., 2014). Human β-casomorphin-8 ((βCN f(51–58)) could be detected in the milk of a lactating human female suffering from post-partum depression (Renlund et al., 1993). Similarly, fragments originating from human β-casomorphin-8 were identified in the plasma of women during pregnancy and post-parturition (Koch et al., 1988). Other studies have reported high levels of human β-casomorphin-8 in the sera of women suffering from post-partum depression (Righard et al., 2014). In addition, elevated levels of human β-casomorphin-8 both in the milk and human sera of women presenting signs of mastitis have also been detected (Righard et al., 2014). These levels were reduced when the infection was cleared up. Therefore, it has been proposed that there was a relationship between human β-casomorphin-8 concentration in physiological fluids, mastitis, and post-partum depression (Righard et al., 2014). It has been suggested that β-casomorphin-8 originating from human milk is able to reach the circulation and the brain owing to its hydrophobic character (Pasi et al., 1993; Renlund et al., 1993). A connection between β-casomorphin and schizophrenia, autism and apnoea in sudden infant death syndrome has also been proposed (Kamin´ski, Cies´lin´ska, & Kostyra, 2007; Sun et al., 2003). However, to date, the clinical evidence does not appear to be clear in this regard (De Noni et al., 2009; Sun et al., 2003). Similarly, human ((β-CN f(51–57)) and bovine β-casomorphin-7 could be detected in the plasma of infants (<1 y) fed with breast milk or cow’s milk infant formulae (Kost et al., 2009). β-Casomorphin-7 fragments were identified in the plasma both in the pre- and postprandial phases. A positive correlation was reported with the elevated plasma levels of human β-casomorphin-7, normal psychomotor development and muscle tone. Human β-casomorphin-7 was reported to be twice as high in infants with normal functions as compared with infants at risk of developmental delay and increased muscle tone. In contrast, it was the opposite for bovine β-casomorphin-7 (Kost et al., 2009). β-Casomorphin-7 can be generated from β-CN variants A1 and B but not from the A2 variant. Some studies have suggested a link between the onset of type 1 diabetes, ischaemic heart disease, autism, schizophrenia, and β-casomorphin-7 (Elliott, Harris, Hill, Bibby, & Wasmuth, 1999; Laugesen & Elliott, 2003). A role of milk protein-derived peptides in satiety and hypertension, possibly involving opioid receptor agonism effects, has also been proposed (Froetschel, Azain, Edwards, Barb, & Amos, 2001; Nurminen et al., 2000). However, to date, there is no conclusive evidence for these effects in humans (De Noni et al., 2009).

8.2.

Antioxidant peptides

While a large amount of information is available in the literature reporting the antioxidant activity of milk BAPs in vitro, there is limited evidence showing these effects in humans (Power et al., 2013). The link between in vitro and in vivo antioxidant capacities has not been clearly established (Lacroix & Li-Chan, 2014). Furthermore, the anti- or pro-oxidant mechanisms of milk protein-derived BAPs is not fully understood (Pihlanto, 2006).

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The antioxidant properties of milk peptides have mostly been investigated in the context of physical exercise. Reactive oxygen species (ROS) generated by respiratory metabolism may lead to muscle inflammation, fatigue and reduced post-exercise performance. A recent study was conducted with elite Brazilian soccer players who were supplemented with a whey protein concentrate (WPC) hydrolysate (0.5 g protein/kg of body mass/ day) for 12 weeks (Lollo et al., 2014). WPC hydrolysate supplementation led to a decrease in creatine kinase (CK; −42%) and lactate dehydrogenase (LDH; −38%), which are in vivo markers of oxidative stress and tissue damage (Lollo et al., 2014).

8.3. Populations with specific needs: allergy and phenylketonuria (PKU) Hydrolysed milk proteins are used as ingredients in different nutritional formulations for populations with specific nutritional needs. These comprise infants with cow’s milk protein allergy (CMPA) and people suffering from PKU. The aim of milk protein hydrolysis includes the destruction of allergenic epitopes (CMPA formulation), an increased digestibility of the proteins or the generation of ingredients with low Phe levels for PKU sufferers. Oral tolerance is defined as a stimulation of type 1-regulatory T (Tr-1) cells leading to an immune response suppression involving IL-10 secretion (Adel-Patient et al., 2011, 2012; Gauthier et al., 2006; Prioult, Pecquet, & Fliss, 2004). The utilisation of infant formulae containing partially or extensively hydrolysed milk proteins is used in order to reduce allergenic milk protein epitopes (Berg, 2013; Elsayed, Hill, & Do, 2004; Zheng, Shen, Bu, & Luo, 2008). PKU is a genetic metabolic disorder characterised by a phenylalanine hydrolase (the enzyme responsible for the conversion of Phe to Tyr) malfunction or deficiency (Clemente, 2000; Ney et al., 2009). CMP is free from Phe residues. Therefore, it is an interesting protein-based ingredient for use in the formulation of food products for PKU nutrition (Ney et al., 2009; Soltanizadeh & Mirmoghtadaie, 2014). Another alternative is the generation of milk protein hydrolysates with reduced Phe levels. In order to prepare these formulae, the first step consists in enzymatic hydrolysis of milk proteins to release Phe, followed by Phe removal with adsorbent resins. Depending on the processing conditions, Phe removal efficiencies ranging from 69 to 99% may be attained (Cogan, Moshe, & Mokady, 1981; López-Bajonero, Lara-Calderón, Gálvez-Mariscal, Velazques-Arellano, & López-Munguia, 1991; Silva, De Marco, Afonso, Lopes, & Silvestre, 2007; Silvestre, Silva, Silva, Silva, & Amorin, 2011; Silvestre et al., 2013).

9. Conclusions and perspectives on the efficacy of milk BAPs in humans The physiological effects of milk protein-derived BAPs have been studied in humans for many decades. Analysis of human fluids and tissues has been made easier with the development of analytical techniques such as MS, the improvement of detector sensitivity (Panchaud et al., 2012) and the development of software which allow treatment of large amounts of data (Iwaniak,

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Minkiewicz, Darewicz, Protasiewicz, & Mogut, 2015). Despite these scientific advances and increasing knowledge on the structure–function activity relationship of BAPs, there is still a high level of uncertainty on the role of milk BAPs in human health. Many reasons for this have been identified during the compilation of data for this review. In particular, when analysing EFSA opinions on specific BAPs, it appears that the main reason for negative opinion lies in the poor characterisation of the BAPS, the lack of dose–response relationships, the low bioavailability of the peptides and their low-potency (De Noni et al., 2009; EFSA, 2010, 2011). Although the proteome of milk proteins has been well characterised, the peptide composition of the resultant hydrolysates may be difficult to determine. This may be linked to the fact that the identification of short peptides (<5 amino acid residues) within complex milk protein hydrolysates still represents a technical challenge (Le Maux et al., 2015; Panchaud et al., 2012). However, many of these short peptides are thought to play a major role in the bioactive properties associated with milk BAPs. The lack of detailed information on the identity of the active components within hydrolysates may also be due to the fact that these cannot be easily identified and quantified in physiological fluids. In addition, because milk protein hydrolysates are composed of a large number of peptides, it may be quite difficult to attribute a specific physiological effect to a particular peptide. Furthermore, due to their low complexity, short peptides may be found within a wide range of different food proteins and possibly within endogenous proteins. Recently, gut endogenous proteins (originating from saliva, gastric secretions, bile, pancreatic secretions, mucins, epithelial cells, plasma proteins and the microbiota) have been suggested as a potential source of BAPs in humans (Moughan, Rutherfurd, Montoya, & Dave, 2013). These gut endogenous protein-derived peptides may be released in amounts twice as high as those arising from the diet (Dave, Montoya, Rutherfurd, & Moughan, 2014; Moughan et al., 2013). It was suggested using an in silico digestion approach that certain gut endogenous proteins could yield peptides which had the same structure as previously identified BAPs, including ACE inhibitory peptides (Dave et al., 2014). Given that BAPs may also be generated from endogenous proteins, this highlights the difficulty of including relevant human controls in human interventions. Many other confounding factors may negatively affect the outcomes of human intervention studies, including external (e.g. the diet, environment, etc.) and internal (the genotype, health status, microbiota, body composition, psychological and nutritional status, etc.) factors. It is then understandable why elucidating the role of BAPs in humans is more challenging when compared to drug compounds which are normally not present within the human body. In order to measure significant differences between the control and the test subject groups, it appears critical that the intervention with the test compound(s) allows a significant increase in BAP concentration in vivo. In this context, an increase in the bioaccessibility and bioavailability of BAPs may help in enhancing the concentration of the BAP at the target organ/receptor. Intestinal absorption of peptides has been reported to involve a transcellular (endocytosis/transcytosis) or paracellular route (Fernández-Musoles et al., 2013; Gardner, 1983; Jahan-Mihan, Luhovyy, El Khoury, & Anderson, 2011). The BAP

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concentrations identified in the GIT (Boutrou et al., 2013; Ledoux et al., 1999; Meisel et al., 2003), sera and plasma (Table 1; Chabance et al., 1998; Foltz et al., 2007) of humans have been deemed sufficient to mediate bioactivity in vivo. However, a low bioavailability for BAPs has been reported, possibly due to the extensive degradation of the peptides (>90%) occurring in the intestine, notably during permeation (Jahan-Mihan et al., 2011). Further processing of BAPs by intestinal and serum peptidases may be responsible for a low bioavailability, which can constitute a limiting factor, explaining the limited potential of BAPs in humans (Gardner, 1983). Different studies have investigated ways to enhance delivery of bioactive components in humans. Increasing the dose of BAP ingested may help to increase their bioavailability. However, this strategy may induce high costs and cause sensory defects (e.g. bitterness) with low/ limited benefits for the biological outcomes. Another alternative is the utilisation of microencapsulation strategies (Barbosa et al., 2004; Brayden & Baird, 2013; de Vos, Faas, Spasojevic, & Sikkema, 2010; Morais et al., 2004; Onwulata, 2012; Yang et al., 2012). An additional advantage of microencapsulation of milk protein hydrolysates is the bitterness masking effect and potential increased stability during storage (Favaro-Trindade, Santana, Monterrey-Quintero, Trindade, & Netto, 2010; Rocha, Trindade, Netto, & Favaro-Trindade, 2009; Subtil et al., 2014). Paracellular and transcellular enhancers also exist which may improve the bioavailability of peptides. However, due to safety concerns, these are not currently being used in humans (Maher & Brayden, 2012). On assessing the scientific evidence linking the positive role of milk protein BAPs on human health, several studies appear to show a positive effect in certain health conditions. However, in many instances, this effect is lower than that observed with pharmaceutical drugs. To date, two EFSA opinions on the role of the milk BAPs (C12 peptide and LTPs) and the reduction of BP in humans have been published. The C12 peptide claims were rejected on the basis that it was insufficiently characterised in the products which were fed to the subjects during the human trials. In addition, there was no direct relationship between the maintenance of a normal BP and the administration of the C12 peptide (EFSA, 2010). Similarly, a negative opinion was given for the effect of the LTPs on BP reduction in humans (EFSA, 2011). Although the peptides were sufficiently characterised, it was estimated that certain human studies presented methodological limitations (inadequately powered studies, treatment allocation, randomisation, blinding and statistical treatment). Therefore, the outcomes of the meta-analyses provided in support of the dossier to EFSA were not taken into account. In addition, because no clear mechanism of action was proposed to explain the effects seen at the doses tested, a negative opinion was delivered for LTPs. A few recommendations may be proposed to improve the quality of the scientific data generated in human intervention studies with milk BAPs. It appears evident from the contradictory results with BAP-based ingredients that there is a real need to establish universal guidelines for the evaluation of these BAPs in humans. Several studies have highlighted the fact that certain subjects are non-responders to the treatment applied (Usinger, Jensen, Flambard, Linneberg, & Ibsen, 2010), which may in certain instances involve the nature of their gut microbiota (Flint, Scott, Louis, & Duncan, 2012; Norheim

et al., 2012). In addition, interindividual differences exist in the digestion of food proteins in humans (Boutrou et al., 2013; Moughan et al., 2013). Human studies need to take these aspects into consideration in order to draw adequate conclusions on the outcomes of the interventions with milk BAPs. In addition, it appears that certain studies are underpowered from a statistical point of view, which has detrimental consequences on the scientific quality of some of the data published to date. For example, in their meta-analysis on BP reducing LTP, Cicero et al. (2013) calculated that a population of 1000 subjects would be required for a study powered at 80% with a 5% α risk. Although it appears challenging to design studies with such a large number of subjects, meta-analyses (larger population groups taken into account) may help to overcome this limitation. However, the human studies included in meta-analyses are not always conducted under the same conditions (dose, duration, population, etc.). Double-blind randomised clinical trials have been suggested for a better design of human interventions (Qin et al., 2013). Consensus methodologies are also needed in terms of the analytical tools employed to adequately identify and quantify BAPs in biological tissues and fluids. This may help to develop better designed human intervention studies and provide case studies with sufficient scientific evidence to support health claims as this is a requirement with numerous national and international health agencies such as EFSA, which grants positive opinions on the basis of human intervention and structure–activity relationship outcomes (Jäkälä & Vapaatalo, 2010; Lalor & Wall, 2013).

Acknowledgement The work described herein was supported by Enterprise Ireland under Grant Number TC2013-0001.

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