Jean-Louis Maubois

Lactolation of β-lactoglobulin monitored by electrospray ionisation mass spectrometry

Direct monitoring of the Amadori product formation between lactose and β-lactoglobulin was performed by electrospray ionisation mass spectrometry. As expected, the glycation reaction was faster at low water activity than in aqueous system. Heterogeneous β-lactoglobulin glycoforms were observed, with different number of lactose bound: populations of β-lactoglobulin with 1 to 7 and 2 to 11 linked lactose were detected, respectively, after 10 and 22 h of reaction under 65% relative humidity and 50°C. Trypsinolysis of glycated proteins, followed by reverse-phase HPLC coupled to tandem mass spectrometry in the neutral loss scanning mode, indicated that all potential reactive amino groups, except Lys101, were involved in sugar binding. The resulting order of reactivity, as a function of reaction time, was as follows: Lys47,91>α-amino-group, Lys15,70,100>Lys60,69,75,77,83,135,138>Lys8,141.

Application of Membrane Separation Technology to Cheese Production

It has been said that ‘a revolution in cheesemaking as a result of ultrafiltration is coming soon’.1 This revolution has been in the making for the past 25 years and has encompassed not only ultrafiltration but, more recently, microfiltration as well. Indeed, more than 400 000 tonnes of cheese were made using ultrafiltration technology in 1989.2 The history of cheesemaking using membranes commenced in the late 1960s with the invention of the MMV process.3–5 This process, named after its inventors (Maubois, Macquot and Vassel) opened up new avenues for significant advances in cheesemaking, including improvements in plant efficiencies, increases in cheese yield, development of continuous process, and possibilities of creating new cheese varieties. As a result, numerous plants all over the world, but mainly Europe, now use this process to manufacture a wide range of cheeses.6

Mechanisms of absorption of caseinophosphopeptide bound iron.

Binding iron (Fe) to the 1-25 caseinophosphopeptide obtained from enzyme hydrolysis of beta casein (beta CPP) improves Fe bioavailability in the rat. To assess the mechanisms involved in its absorption, a perfused, vascularized duodenal rat loop model was used in controls and in Fe-deficient (bleeding of 25% blood volume) rats. Inhibitors of oxidative phosphorylation [2-4 dinitrophenol (DNP)] and/or of endocytosis [phenylarsine oxide (PAO)] were added to the perfusion solution containing 50 microM Fe as beta CPP bound Fe (Fe-beta CPP) or gluconate (Fe Gluc). Fe-beta CPP enhanced Fe uptake, reduced mucosal storage, and improved net absorption both in controls and in deficient animals. DNP reduced uptake, mucosal storage, and net absorption by the same percentage in Fe-beta CPP and Fe Gluc perfused rats in both control and Fe-deficient animals. PAO decreased uptake, mucosal storage, and net absorption of Fe-beta CPP but not of Fe Gluc. At the end of the experiment Fe serum levels were increased only in Fe Gluc animals. These results confirm the improved bioavailability of beta CPP bound Fe. They suggest that at least part of its absorption can occur by a different pathway than usual Fe salts. Fe-beta CPP can be taken up by endocytosis and absorbed bound to amino acids or peptides.

New genetic variants identified in donkey's milk whey proteins.

Novel genetic variants for donkey milk lysozyme and β-lactoglobulins I and II have been identified by the combined use of peptide mass mapping and sequencing by tandem mass spectrometry in association with database searching. The novel donkey lysozyme variant designated as lysozyme B (Mr 14,631 Da) differed in three amino acid exchanges, N49 → D, Y52 → S, and S61 → N, from the previously published sequence. Three novel genetic variants for donkey β-lactoglobulins were identified. One of them is a type β-lactoglobulin I with three amino acid exchanges at E36 → S, S97 → T, and V150 → I (β-lactoglobulin I B, Mr 18,510 Da). The two others are type β-lactoglobulins II with two amino acid exchanges at C110 → P and M118 → T (β-lactoglobulin II B, Mr 18,227 Da) and with three amino acid exchanges at D96 → E, C110 → P, and M118 → T (β-lactoglobulin II C, Mr 18,241 Da). All these primary structures are closely related to those of homologous proteins in horse milk (percent identity >96%).

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.

The addition of a cocktail of yeast species to Cantalet cheese changes bacterial survival and enhances aroma compound formation

Indigenous yeasts can be detected at high populations in raw milk Cantal cheese, a French Protected Denomination of Origin (PDO) hard cheese. To investigate their use as adjunct cultures to promote flavour development in Cantalet (small Cantal) cheese, three strains isolated from raw milk Cantal cheese, Kluyveromyces lactis, Yarrowia lipolytica, and Pichia fermentans were added at 3 (E3) and 5 (E5) log(10) colony-forming units (cfu)/mL to microfiltered milk at a ratio of 80/10/10 viable cells, respectively. The global microbial, compositional and biochemical changes induced by the presence of yeasts in cheese were determined. Adjunct yeasts did not grow but stayed at viable populations of approximately 4 and 6 log(10) cfu/g in E3 and E5 cheeses, respectively, throughout the ripening period. They were mainly constituted of K. lactis, while P. fermentans and Y. lipolytica were not detectable after 3 and 45 days of ripening, respectively. Several species of indigenous yeasts were also detected in E3 cheeses at the beginning of ripening only, and in the control cheeses without yeasts added. Lactoccoci survived for longer periods in the presence of yeast adjuncts, while, conversely, the viability of Streptococcus thermophilus decreased more rapidly. The addition of yeasts did not influence cheese composition and total free amino acid content. In contrast, it slightly increased lipolysis in both E3 and E5 cheeses and markedly enhanced the formation of some volatile aroma compounds. The concentrations of ethanol, ethyl esters and some branched-chain alcohols were 6 to 10 fold higher in E5 cheeses than in the control cheeses, and only slightly higher in E3 cheeses. This study shows that K. lactis has a potential as cheese adjunct culture in Cantalet cheese and that, added at populations of 4-5 log(10) cfu/g cheese, it enhances the formation of flavour compounds.

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.

Sensitivity of β-casein phosphopeptide-iron complex to digestive enzymes in ligated segment of rat duodenum

Binding iron to the phosphorylated beta(1-25) peptide derived from beta-casein improves iron bioavailability in the rat. The aim of the present work was to learn how injected beta(1-25) and iron-beta(1-25) complex behave in the duodenum of rats using the technique of intestinal ligation in situ and reversed-phase (RP)-high performance liquid chromatography-electrospray mass spectrometry analysis of the lumen contents. The results demonstrate that beta(1-25) is sensitive to digestive enzymes including proteases/peptidases and phosphatases during duodenal transit. The lumen contents of rats perfused with iron free beta(1-25) contained all peptidic sequences derived from beta(1-25). In contrast, the phosphorylated part of beta(1-25) [i.e., beta(15-24)] was not detected in lumen of rats perfused with iron-beta(1-25) complex.

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.

Influence of thermophilic lactic acid bacteria strains on propionibacteria growth and lactate consumption in an Emmental juice-like medium

Swiss-type cheeses such as Emmental are characterized by the successive development of thermophilic lactic acid bacteria (TLAB) and propionibacteria. The aim of this study was to determine whether the choice of TLAB strain influenced propionibacteria. TLAB and propionibacteria were cultured sequentially under the conditions prevailing in cheese. Firstly, 11 Emmental juice-like media were prepared by fermenting casein-enriched milk with pure or mixed cultures of TLAB (Lactobacillus helveticus, Lb, delbrueckii subsp. lactis and Streptococcus thermophilus), differing in their proteolytic activities. TLAB cells were then removed by microfiltration. Finally, five strains of Propionibacterium freudenreichii were grown on these media at 24 °C under anaerobiosis and their growth characteristics and lactate consumption determined. The media mainly differed in their contents of peptides (1.9-5.3 g/kg) and free amino acids (1.0-5.6 g/kg) and the proportions of lactate isomers (42 92% of the L( +) isomer). Propionibacteria were significantly (P < 0.05) influenced by TLAB strains (differences in doubling times of up to 20 % and differences in lactate consumption after 600 h culture of up to 52%). The influence of TLAB was similar for all the propionibacteria tested, depended on the TLAB strains and could not be generalized to the TLAB species. Propionibacteria were stimulated by high peptide levels, low levels of free amino acids and NaCl, a low proportion of L(+ )-lactate and other undetermined factors. However, variations due to TLAB were less than those between propionibacteria strains.