The scientific evidence for the role of milk protein-derived bioactive peptides in humans: A Review
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 et al, 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 et al, 2014, Panchaud et al, 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 et al, 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 et al, 2010, Geerts et al, 2011, Jauhiainen et al, 2009, Jauhiainen et al, 2010, Morifuji et al, 2010, Usinger et al, 2010, Xu et al, 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 et al, 2015, Li-Chan, 2015, Saadi et al, 2015). In this context, food protein-derived peptides and specifically milk protein-derived BAPs have potential as natural alternatives to drugs for disease management (Carrasco-Castilla et al, 2012, Li-Chan, 2015, Saadi et al, 2015, Zambrowicz et al, 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 et al, 2005, Maeno et al, 2005, Ponstein-Simarro Doorten et al, 2009). Similarly, the antimicrobial (lactoferrin- LF f(1–11)) peptides have been evaluated as being safe for human consumption (Brouwer et al, 2011, van der Velden et al, 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 dairy-based 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, Stuknytė et al, 2015, Walsh et al, 2004) or permeation through Caco-2 cell monolayers (Picariello et al, 2013, Quirós et al, 2008, Shimizu et al, 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, Jauhiainen et al, 2009). This may, in some instances, be related to analytical limitations associated with the detection of peptides and notably short peptides (Le Maux et al, 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 (<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 et al, 2011, Fekete et al, 2013). This may explain the inter-individual 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, EFSA, 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 et al, 2013, Cicero et al, 2011, Fekete et al, 2013, Qin et al, 2013, Turpeinen et al, 2013, Xu et al, 2008) and caseinophosphopeptides (CPPs) (Yengopal & Mickenautsch, 2009). However, these meta-analyses 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 physiologically 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.
Section snippets
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, Chabance et al, 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 et al, 2014) as outlined in Table 1.
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
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,
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
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 et al, 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
Immunomodulatory and antimicrobial peptides
The consumption of milk proteins has been associated with immunostimulatory effects in humans. A reduction in low-grade 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
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
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, Minkiewicz, Darewicz, Protasiewicz, & Mogut, 2015). Despite these scientific advances and increasing knowledge on the
Acknowledgement
The work described herein was supported by Enterprise Ireland under Grant Number TC2013-0001.
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