Saïd Bouhallab

Identification of C-terminal peptides of bovine β-casein that enhance proliferation of rat lymphocytes

A casein polypeptidic fraction, obtained from a pepsin-chymosin digestion of caseins, showed a mitogenic effect on primed lymph node (LN) cells and unprimed spleen cells of rats. A biologically active C-terminal sequence of bovine beta-casein (residues 192-209) was characterized. The corresponding synthetic peptide had a stimulatory effect on primed LN cells but failed to enhance proliferation of spleen cells. We prepared two chymosin digests (PA and PB) of bovine beta-casein which contained, respectively, 80% and 95% of the sequence including residues 193-209. They induced a significant proliferative response in both LN and spleen cells. It is therefore possible that other active peptides in the PA preparation may be involved in mitogenic activity.

Milk proteins and iron absorption: contrasting effects of different caseinophosphopeptides.

Clusters of phosphoserine residues in cow milk caseins bind iron (Fe) with high affinity. Casein inhibits Fe absorption in humans, but protein hydrolysis lessens this effect. Phosphopeptides from different caseins gave conflicting results on Fe absorption; release of phosphate residues by intestinal alkaline phosphatase could be a key point of that metabolism. The objectives of this study were to compare the absorption of Fe complexed to caseinophosphopeptides (CPP) of the main cow milk caseins β-casein (β-CPP) and αs-caseins (αs1-CPP) and to assess the role of alkaline phosphatase on this absorption. Two experimental models were used: an in vivo perfused rat intestinal loop and an in vitro Caco-2 cell culture model. In addition, we determined the effect of an intestinal phosphatase inhibitor on these various forms of Fe. Gluconate Fe was used as control. In both models, uptake and net absorption of Fe complexed to CPP from αS1-caseins were significantly lower than from Fe complexed to β-CPP. Inhibition of the intestinal phosphatase significantly increased the uptake and the absorption of Fe complexed to β-CPP without effect on the other forms of Fe. These results confirm the enhancing effect of β-casein and its CPP on Fe absorption. The differences between CPP could be explained by their structural and/or conformational features: binding Fe to αS1-CPP could impair access to digestive enzymes, whereas β-CPP–bound Fe is better absorbed than its free form. The differences in protein composition between cow and breast milk, which does not contain α-casein, could explain some of their differences in Fe bioavailability.

The structure of infant formulas impacts their lipolysis, proteolysis and disintegration during in vitro gastric digestion

Milk lipids supply most of the calories necessary for newborn growth in maternal milk or infant formulas. The chemical composition of infant formulas has been optimized but not the structure of the emulsion. There is still a major difference between the native emulsions of milk fat globules and processed submicronic emulsions in infant formulas. This difference may modify the kinetics of digestion of emulsions in newborns and influence lipid metabolism. To check this, semi-dynamic gastric in vitro digestions were conducted on three matrices: a standardized milk emulsion containing native milk fat globules referred to as minimally-processed emulsion and two processed model infant formulas (homogenized or homogenized/pasteurized). Gastric conditions mimicked those reported in newborns. The minimally-processed emulsion was lipolyzed and proteolyzed slower than processed formulas. The difference in initial structure persisted during digestion. The surface of the droplets was the key parameter to control gastric lipolysis kinetics, the pattern of released fatty acids and proteolysis by faster hydrolysis of adsorbed proteins.

Bovine β-lactoglobulin/fatty acid complexes: binding, structural, and biological properties

Ligand-binding properties of β-lactoglobulin (β-lg) are well documented, but the subsequent biological functions are still unclear. Focusing on fatty acids/β-lg complexes, the structure-function relationships are reviewed in the light of the structural state of the protein (native versus non-native aggregated proteins). After a brief description of β-lg native structure, the review takes an interest in the binding properties of native β-lg (localization of binding sites, stoichiometry, and affinity) and the way the interaction affects the biological properties of the protein and the ligand. The binding properties of non-native aggregated forms of β-lg that are classically generated during industrial processing are also related. Structural changes modify the stoichiometry and the affinity of β-lg for fatty acids and consequently the biological functions of the complex. Finally, the fatty acid-binding properties of other whey proteins (α-lactalbumin, bovine serum albumin) and some biological properties of the complexes are also addressed. These proteins affect β-lg/fatty acids complex in whey given their competition with β-lg for fatty acids.

Bioavailability of caseinophosphopeptide bound iron in the young rat

Abstract Iron forms strong and soluble complexes with 1–25 caseinophosphopeptide (CPP) issued from the enzymatic hydrolysis of β- casein . That could prevent iron from insolubilization and low digestive absorption. Young iron deficient rats (5 mg iron/kg diet for 4 weeks) were repleted (200 mg iron/kg diet, 2 weeks) using either FeSO 4 or iron bound to CPP of whole hydrolyzed β- casein (β- cas hydr group) or purified molecule (β- cas (1–25) group). Two other groups were fed a control diet (200 mg/kg as FeSO 4 ) for the 6 weeks, either and libitum (control) or pair fed (PF) to experimental groups. A metabolic balance was performed during the 2nd week of repletion. Experimental groups displayed a higher iron absorption than control groups, but did not differ among themselves. At the end of the 2 weeks repletion period, FeSO 4 and β- cas hydr groups showed similar values of Hb, Hct, and RBC count, which were lower than control and PF groups. Hct and Hb values of the β- cas (1–25) group were higher than β- cas hydr and FeSO 4 groups, but did not differ from control and PF animals. MCV values of control and PF groups did not differ from FeSO 4 group but were lower than the β- cas (1–25) and β- cas hydr groups. The Fe liver content was significantly higher in peptide-bound Fe groups ((1–25) β- cas and β- cas hydr) than the three other groups; the FeSO 4 group showed the lowest levels. Binding iron to phosphocaseinophosphopeptide seems to improve its bioavailability and to hasten the cure of iron deficiency in the young animal.

Separation of small cationic bioactive peptides by strong ion-exchange chromatography

Abstract The high resolving power of a Hyper D cation-exchange column was evaluated to achieve the separation of small cationic bioactive peptides derived from tryptic digest of κ-casein: MAIPPKK (pI = 9.9), MAIPPKK (pI = 10.5), KNQDK (pI = 9.6) and NQDK (pI = 6.35). The influence of pH (1.5–6), gradient slope (2–10 mM sodium chloride/min) and elution of the mixture under isocratic conditions was investigated. Although their physico-chemical properties are very similar, these four peptides were readily resolved with an excellent selectivity and recovery. The selectivity of the exchanger was also expressed toward peptides of the same net positive charge; the most hydrophilic peptide always eluted last. It was also shown that the elution order of these molecules depends on pH. From the observed retention times and the elution order, we have established a simple approach to linearization of peptide retention behaviour on the S-Hyper D support.

Dietary bioactive peptides: Human studies

ABSTRACT Current opinion strongly links nutrition and health. Among nutrients, proteins, and peptides which are encrypted in their sequences and released during digestion could play a key role in improving health. These peptides have been claimed to be active on a wide spectrum of biological functions or diseases, including blood pressure and metabolic risk factors (coagulation, obesity, lipoprotein metabolism, and peroxidation), gut and neurological functions, immunity, cancer, dental health, and mineral metabolism. A majority of studies involved dairy peptides, but the properties of vegetal, animal, and sea products were also assessed. However, these allegations are mainly based on in vitro and experimental studies which are seldom confirmed in humans. This review focused on molecules which were tested in humans, and on the mechanisms explaining discrepancies between experimental and human studies.

In vitro digestion of caseinophosphopeptide-iron complex.

Caseins bind strongly to Fe by their phosphoseryl residues (Hegenauer et al . 1979; Brule & Fauquant, 1982; Bouhallab et al . 1991; Emery, 1992) and keep it soluble at the alkaline pH of the duodenum (Manson & Annan, 1971; Bouhallab et al . 1991). It has been suggested that this strong binding prevents the release of free Fe during digestion and impairs its absorption (West, 1986; Hurrell, 1997), but in human studies hydrolysis by digestive enzymes to give low molecular mass peptides improves Fe absorption (Hurrell et al . 1988, 1989). β-Casein peptide 1–25 (β-CN(1–25)) is the phosphorylated N-terminal fragment of β-CN. Its molecular mass is 3124 Da and it contains four phosphoseryl residues that bind four Fe atoms and keep them soluble (Bouhallab et al . 1991). Preliminary results showed that binding Fe to β-CN(1–25) enhances its bioavailability in the rat (Ait-Oukhatar et al . 1997) and its absorption by the duodenal rat loop model (Peres et al . 1997). We recently showed that the β- CN(1–25)–Fe complex is hydrolysed to a lesser extent than free β-CN(1–25) during duodenal digestion, and that the phosphorylated region of the peptide to which Fe is bound, β-CN(15–24), cannot be detected in the digestive lumen (our unpublished results). These previous studies did not examine the effect of luminal digestion that could release free inorganic Fe. We considered the possibility that Fe bound to β-CN(1–25) is not released in the digestive lumen, and that it reaches the absorbing surface of the small intestine brush border membrane as a complex. To test this hypothesis we examined in vitro the influence of pH and digestive enzymes on the dialysability of the β-CN(1–25)–Fe complex.

Spontaneous Assembly and Induced Aggregation of Food Proteins

Beyond their nutritional value, food proteins are a versatile group of biopolymers with a considerable number of functionalities throughout their extensive structures, conformations and interaction–aggregation behaviour in solution. In the present paper, we give an overview of the induced aggregation and spontaneous reversible assembly of food proteins that lead to a diversity of supramolecular structures. After a brief description of the properties of some food proteins, the first part summarises the aggregation processes that lead to supramolecular structures with a variety of morphologies and sizes. The second part reports on the requirements that drive spontaneous assembly of oppositely charged proteins into reversible supramolecular structures. The promising new applications of these structures in food and non-food sectors are also mentioned.

Reduction of iron/zinc interactions using metal bound to the caseinophosphopeptide 1–25 of β-casein

Abstract We used the isolated, perfused rat duodenal loop system to assess the influence of binding iron (Fe) to soluble 1–25 caseinophosphopeptide (β-CN (1–25)), produced by the hydrolysis of β casein, against the inhibition of its absorption by Zinc (Zn). Fe (100 μM) was perfused as Fe gluconate (Fe Gluc) or bound to the β-CN (1–25) (FeCN), alone (controls) or in presence of Zn as Zn sulfate (Zn SO 4 ) or as (ZnCN) at Fe:Zn ratios ranging from 2:1 to 1:5. Zn SO 4 reduced significantly disappearance from the lumen (Q1) and net Fe absorption (FeAbs) at Fe:Zn ratios from 1:1.5 to 1:5 (p 4 and ZnCN did not reduce significantly Q1, Q2 nor FeAbs for FeCN whatever ratios considered. Binding Fe to β-CN (1–25) prevented Zn from inhibiting its absorption by Zn and could have therapeutic applications in dietary supplementation of trace-elements.