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Glycation of β-lactoglobulin (β-Lg) with either lactose or galactose in a solid-state medium was monitored using gel electrophoresis techniques and liquid chromatography coupled to electrospray ionisation mass spectrometry (LC/ESI-MS). The kinetics of glycation monitored by SDS polyacrylamide gel electrophoresis showed a molecular weight increase over time of the β-Lg bands for both sugars, but no significant amounts of aggregated proteins were observed. The isoelectric point of the protein, observed by isoelectric focusing gel electrophoresis, was dramatically affected by galactosylation. LC/MS measurements of β-Lg variants A and B, over the whole glycation reaction time, showed a larger extent of glycation with galactose (from 4 up to 22 adducts) as compared with lactose (from 0 up to 14 adducts), and confirmed that early Maillard reaction products were the main species observed. Based on the relative abundances obtained from the deconvoluted mass spectra after a 8 h 15 min incubation time at 60°C, the mean values of lactose and galactose molecules bound to the protein species were calculated to be 10.4 and 17.9, and 10.5 and 18.6, for variants A and B, respectively. Furthermore, the charge state distribution data obtained by ESI-MS was studied using different methanol percentages, and indicated that adduct formation with lactose, but more significantly galactose, tends to improve the stability properties of the native protein towards denaturation. Copyright © 2003 John Wiley & Sons, Ltd.

Incorporation of milk fat and milk fat globule membrane fragments in infant formulas could be an interesting way to get closer to the composition and structure of human milk and its physiological impacts.Two adapted formulas were processed containing either vegetable fat (STD) or a combination of milk and vegetable fats stabilized by a mixture of proteins and milk fat globule membrane fragments (EXP). The formulas were distributed with an automatic milk feeder to piglets until slaughter at 28 days. Intestinal contents and tissues (proximal jejunum and ileum) as well as mesenteric lymph nodes (MLN) were collected. The residual immunoreactivity of β-lactoglobulin (β-lg) and caseins (Cns) present in the digestive compartments was determined by ELISA. Mononuclear cells isolated from MLN were cultured in vitro to evaluate their cytokine secretory profiles. Microbiota diversity and composition was assessed using DHPLC and metasequencing analysis. Finally the immunomodulatory capacity of peptide and lipid fractions extracted from the jejunal contents of STD and EXP piglets were tested in vitro on MLN cells from sow’s suckling piglets. The immunoreactivity of β-lg and Cns was higher in EXP than in STD intestinal contents. The secretory activity of MLN cells was modified by the formula composition, the highest IFNγ secretion was obtained in EXP piglets. A major immunosuppressor effect of lipid fractions was evidenced with both formulas. Microbiota clustering successfully discriminated infant formulas according to the presence of exclusive vegetable vs. a combination of milk and vegetable lipids. Differences in proteobacteria and firmicutes phyla were observed. In conclusion we evidenced that lipid composition of formulas had influence on gut physiology through the release of immunomodulatory lipids in contact with the mucosa, modulation of the dynamic of proteolysis and microbiota composition.

National audience; Beside differences in composition, infant formula and human milk exhibit structural differences. Native milk fat globule is a 4–5 μm particle composed by a core of triglycerides surrounded by a tri-layer of phospholipids. Fat in infant formula appears as small droplets stabilized by milk proteins. Similarly, type of proteins and casein micelle size differ between these two foods. Two infant formulas and human milk were submitted to a simulated in vitro dynamic digestion mimicking the gastro-intestinal tract of a 1-month old baby. Digested samples were collected and characterized at different scales. Structure of the native human milk fat globule persisted in the stomach during the first 2 h of digestion. Kinetics of proteolysis and lipolysis presented some differences with casein being digested more rapidly in the formula than in the human milk. It now needs to determine whether these differences have some physiological consequences on the neonate.; Outre des différences de composition, les formules infantiles présentent des différences structurales avec le lait maternel. Le globule gras natif du lait maternel correspond à une particule de 4–5 μm de diamètre constituée d’un cœur de triglycérides entouré d’une tri-couche de phospholipides. La matière grasse des formules infantiles apparaît sous forme de petites gouttelettes stabilisées par des protéines. De même, le type de protéines et la taille des micelles de caséines diffèrent entre ces deux aliments. Deux formules infantiles et un lait maternel ont été soumis à un protocole de digestion in vitro en conditions dynamiques simulant le tractus gastro-intestinal d’un enfant d’un mois. Les digestas ont été collectés et caractérisés à différents niveaux d’échelle. La structure du globule gras natif du lait maternel persiste dans l’estomac lors des deux premières heures de digestion. Les cinétiques de protéolyse et lipolyse sont différentes avec des caséines digérées plus rapidement dans les formules que dans le lait maternel. Il reste cependant à déterminer si ces différences ont des conséquences physiologiques pour le nourrisson.

Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and beta-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between beta-Lg molecules; (2) disulfide bond involving two kappa-casein molecules; and (3) a disulfide bond between two peptides, one from beta-Lg and the other from kappa-casein. The involved sites in this last bond were Cys(160) of beta-Lg and Cys(88) of kappa-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and beta-lactoglobulin derived from goat milk.

Link between goat milk lipolysis and sensorial quality of lactic goat cheeses made from raw or pasteurised milk. There is an important variability of the lipolysis level in goat milk, due to various physiological and breeding factors. The aim of this study was to evaluate the impact of these natural variations on the sensorial quality of fresh and ripened lactic goat cheeses. The effect of milk pasteurisation was also studied. The results show that the variation of the lipolysis level of goat milk do not have any significant effect on the sensorial quality of ripened cheeses. On the contrary, the sensorial quality of fresh cheeses is linked to the lipolysis level of milk. These results allowed us to recommend some means of controlling the sensorial quality of goat cheeses. For ripened cheeses, a satisfying sensorial quality could be obtained via the control of the technological parameters, notably the moisture in non-fat cheese at the demoulding. For fresh cheeses, the lipolysis level of milk should be checked before cheesemaking, and milks with high lipolysis levels should not be used. Pasteurisation of milk induced a decrease in the lipolysis level of cheeses. The use of pasteurisation led to a limitation of the appearance of organoleptic defects in rip- ened cheeses, but the overall flavour and odour of ripened cheeses made from pasteurised milk was also reduced compared to cheeses made from raw milk. goat milk / lipolysis / goat cheese / sensorial quality / pasteurisation

The strong and characteristic “goat” flavour is one of the most important quality components of French cheeses made from goat milk. This study was designed in order to evaluate the impact of different ripening strains on this typical flavour. Goat milk was incubated with selected ripening strains (yeasts and moulds), and two types of cheeses were produced: Sainte-Maure cheese (lactic coagulation technology) and Camembert-type cheese (lactic-rennet coagulation technology). Sensory evaluation of the cheeses was performed by trained panellists, and physico-chemical parameters (including lipolysis) were measured. The results showed that cheeses made with Geotrichum candidum as a ripening agent were characterised by a strong typical flavour, whereas the use of Penicillium candidum was detrimental for the sensorial quality of cheeses. These results were related to the lipolytic and flavouring activities of the different strains, which displayed considerable quantitative and qualitative variations.

Compositional factors involved in the variable acidification capacity of goat milk by lactic starters. This study was designed to define the factors involved in the variable acidification capacity of goat milk by lactic starters. Acidification capacity and compositional parameters of 28 goat milk samples were determined. The influences of the period of production and of some tech- nological treatments (cold storage and heating) were also investigated. The results point out the influ- ence of milk composition, and notably the role of the buffering components such as proteins and minerals. An improved activity of lactic starters is observed in milks with a high level of proteins and minerals. The lactation period, through its effect on the protein concentration, affects the acidification capacity of goat milk. Cold storage does not exert any major influence on the acidification process. However, the results indicate that heating of milk lead to an improved activity of lactic starters at the beginning of the acidification period. goat milk / acidification / lactic starter / composition

Goat milk is characterized by a very low heat stability, which could be related to compositional factors. In this paper, the role of the heat-induced interaction between whey proteins and casein micelles is considered. The effect of the casein micelle structure is evaluated using milk samples with different α SI -casein genotypes, and the protein interactions are studied at various pH values in order to take into account the strong pH-dependence of the heat stability, The results suggest that the heat-induced interaction of β-lactoglobulin with κ-casein is of minor importance at natural pH, but is promoted at more elevated pH.

An in vitro study was conducted to investigate the sensitivity of lactose-β-lactoglobulin conjugates to β-galactosidase from Kluyveromyces lactis. The hydrolysis was monitored by ion exchange chromatography. Compared to free lactose or lactulose, which were rapidly hydrolysed, protein-bound lactose was not hydrolysed by β-galactosidase even after an extended hydrolysis time. Tryptic digestion of the conjugates before addition of β-galactosidase improved the substrate accessibility and led to the release of about 50% of the linked lactose. The results strongly suggest that the resistance of protein bound lactose is linked to the globular and compact structure of lactose-β-lactoglobulin conjugates.

Bovine beta-LG was modified by glycation with lactose in a powdered state or in an aqueous solution. An immunological characterization was performed using monoclonal antibodies with defined epitopes. The results showed that the structural changes were confined to the AB loop region of the molecules when glycation was conducted in a restricted water environment and had little consequences on the association state of glycated beta-LG. The protein conformation was much more extensively modified when glycation was performed in an aqueous solution at 60 degrees C, despite a lower glycation extent. These structural changes were located at the dimer interface (AB loop, GH loop, beta-strand I, and alpha-helix). These results allowed us to establish a relationship between the conformational changes and the modification of the association state of the glycated protein (formation of disulfide bridges between the free thiol groups of two monomers), previously described.

y Since temperature and water activity are among the most important parameters that affect the Maillard reaction, the glycation sites in pure, native bovine β-lactoglobulin were determined after a mild heat treatment (60°C) in an aqueous solution and after a treatment under a restricted water environment (50°C, 65% relative humidity). In both systems, the results obtained underlined the structural heterogeneity of β-lactoglobulin (β-LG) glycoforms with respect to the number of lactose residues linked per protein molecule and to the binding sites involved. Subsequently, the effect of the glycation conditions on both the association behaviour and the conformational changes of the glycated β-LG were characterised by proteolytic susceptibility, binding of the fluorescent probe 8-anilino-1-naphtalene-sulfonic acid, SDS-PAGE and size exclusion chromatography. The results showed that dry-way glycation did not significantly alter the native-like behaviour of the protein while the treatment in solution led to important structural changes. These changes resulted in a specific denatured β-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation to form high molecular weight species, via non-covalent interactions. The use of monoclonal antibodies with defined epitopes, raised against native β-LG. confirmed that the protein conformation was much more modified when glycation was performed in a solution while the structural changes induced during dry-way treatment were limited to the AB loop region of the protein.

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.

Lactose reacts nonenzymatically with beta-lactoglobulin (beta-LG), the major whey protein, under mild heat treatment and the formation of the complex may be monitored by mass spectrometry. Using Reverse Phase HPLC coupled with Electrospray Ionization MS (ESI-MS) we have measured the global extent of glycosylation and examined the distribution of lactose among the beta-LG glycoforms. Identification of lactosylated sites by trypsinolysis and Tandem MS indicate that, although the glycosylation reaction was non-specific and potentially involved all the reactive sites (alpha- and epsilon-amino groups), beta-LG appeared to have at least two populations of lysine with the distinct ability to react with lactose. These results underline the structural heterogeneity of beta-LG glycoforms, with respect to the number of lactose linked per molecule and to the binding sites involved, which could affect the biological function of beta-LG.

The first months of life correspond to a key period in human life where dramatic physiological changes (establishment of microbiota, development of the immune system, etc.) occur. In order to better control these changes it is necessary to understand the behaviour of food in the gastrointestinal tract of the newborn. Infant formula is the only food for the newborn when breast-feeding is impossible. The kinetics of digestion of milk proteins and the nature of the peptides liberated in the small intestine throughout infant formula digestion have never been extensively investigated so far and were therefore studied using the piglet as a model of the newborn child. Piglets were fed infant formula by an automatic delivery system during 28 d, and slaughtered 30, 90 and 210 min after the last meal. Contents of stomach, proximal and median jejunum and ileum were collected and characterised. The extent of β-lactoglobulin (β-lg), α-lactalbumin (α-la) and casein proteolysis was monitored by inhibition ELISA, SDS-PAGE, immunoblotting and MS. At 30 min after the last meal, caseins were shown to be extensively hydrolysed in the stomach. Nevertheless, peptides originating mainly from β-caseins (from 509 to 2510 Da) were identified in the jejunum and ileum of the piglets. β-Lg partially resisted gastric digestion but completely disappeared in the stomach after 210 min. α-La had a similar behaviour to that of β-lg. Two large peptides (4276 and 2674 Da) generated from β-lg were present in the ileum after 30 and 210 min and only one (2674 Da) after 90 min.

Lactose crystallisation and Maillard reaction are two major modifications occurring in milk and whey powders during processing and storage. In this work, the aim was first to monitor the solid-state early Maillard reaction (EMR) in whey protein concentrates (WPC) heated at 60 °C and various water activities, and then, to characterise the physical changes that occur as a consequence of heat treatment in skim milk powder (SMP) and WPCs. After aw adjustment, SMP and WPC were heated at 60 °C in hermetic conditions to induce an amino-sugar reaction. Furosine analysis (% of blocked lysine) was used to monitor the progress of EMR. The results showed that the kinetic of EMR was linked to the initial aw of the powders. Heating of SMP may lead to the crystallisation of lactose in humidified powders, without apparently affecting the progress of EMR. Surprisingly, lactose crystallisation - monitored by micro-Differential Scanning Calorimetry - was easily induced in heated SMP, whereas it was delayed or even inhibited in WPC. The results showed that this effect was dependent on the protein/lactose ratio in WPCs., Cristallisation du lactose et réaction de Maillard précoce dans les poudres de lait écrémé et les concentrés de protéines du lactosérum. La cristallisation du lactose et la réaction de Maillard sont deux modifications importantes survenant au cours de la production et du stockage des poudres de lait et de lactosérum. L'objectif de ce travail était dans un premier temps de suivre la réaction de Maillard précoce (RMP) dans des concentrés de protéines de lactosérum (CPL) chauffés à 60 °C à différentes activités d'eau et, dans un deuxième temps, de caractériser les modifications physiques consécutives au traitement thermique (comparaison poudres de lait écrémé et CPL). Les poudres de lait écrémé et les CPL - équilibrés à différentes activités d'eau - étaient chauffés à 60 °C dans des conditions hermétiques pour induire la RMP (suivie par l'analyse de la furosine). Les résultats indiquaient que la cinétique de la RMP était liée à l'activité d'eau initiale de poudres. Dans les poudres de lait écrémé humidifiées et chauffées, la cristallisation du lactose ne semblait pas avoir d'impact sur l'évolution de la RMP. La microcalorimétrie différentielle à balayage permettait d'observer que la cristallisation du lactose, induite facilement dans les poudres de lait écrémé, était retardée voire inhibée dans les CPL. Les résultats indiquent que cet effet était dépendant du rapport protéine/lactose dans les CPL.

The Maillard reaction is commonly encountered during food processing or storage, and also in human nutrition, hence there is a need for analytical methodologies to identify and characterize the modified proteins. This paper reports specific methods using mass spectrometric techniques to localize protein modifications induced by lactose and galactose on β-lactoglobulin (β-Lg) under solid-state glycation conditions. The extent of glycation was first determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The specific identification of lactose-modified amino acid residues was realized using both NanoESI-MS, NanoESI-MS/MS (neutral loss scanning modes) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) (with and without guanidination of lysine residues) on unfractionated digests. The results indicated that, after 8.25 h of incubation, the lysine residues were the main targets of lactose-induced modification. In addition to the 15 lysine residues, Leu1 (NH2 terminal) and the Arg124 were also found to be modified, thus leading to a total of 17 different modified amino acid residues (versus 15 found by LC/ESI-MS measurement). In a second set of experiments, different strategies consisting of constant neutral loss and precursor ion scanning were compared to characterize galactose-induced modifications. Owing to the high level of β-Lg glycation, the combined use of these different strategies appeared to be necessary for determining the galactose-modified sites after 8.25 h of incubation. Thus, among the 22 galactose adducts deduced from the LC/ESI-MS measurement, apart from the N-terminal and classical lysine residues, we also observed a few arginine residues (Arg40, Arg124 and Arg148) that were modified, and also dialkylations on specific lysine residues (Lys47, Lys75). Copyright © 2003 John Wiley & Sons, Ltd.

The effect of glycation with lactose on the association behavior and conformational state of bovine β-lactoglobulin (β-LG) was studied, using size exclusion chromatography, polyacrylamide gel electrophoresis, proteolytic susceptibility, and binding of a fluorescent probe. Two modification treatments were used, i.e., aqueous solution glycation and dry-way glycation. The results showed that the latter treatment did not significantly alter the nativelike behavior of the protein while the former treatment led to important structural changes. These changes resulted in a specific denatured β-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation into a high molecular weight species, via noncovalent interactions. The association behavior of glycated β-LG is discussed with respect to the known multistep denaturation/aggregation process of nonmodified β-LG. Keywords: β-Lactoglobulin; glycation; denaturation; aggregatio...

The solubility of lactose-beta-lactoglobulin conjugates at pH 4.6, after heating near neutral pH in phosphate buffer/0.116 M NaCI, was investigated by size exclusion chromatography and compared with unmodified protein. Heated conjugates in the temperature range 65-90 degrees C showed greater solubility at pH 4.6. The proportion of soluble protein increased with the number of bound lactose molecules. Total solubility was obtained for conjugates with nine lactose residues attached per monomer of beta-lactoglobulin. The protective effect of bound sugar toward precipitation was associated with the formation of soluble disulfide cross-linked dimers, highly accessible to trypsin digestion. These results suggested that bound lactose, through steric hindrance and high surface hydrophilicity, prevents the thiol-disulfide exchange reactions of the polymerization-aggregation process of lactose-beta-lactoglobulin conjugates.

In food processing as well as in human nutrition and physiology, the increasing importance of the Maillard reaction has brought about the need for analytical means to detect and characterize the protein-bound Amadori products. In this paper, we describe a highly selective and sensitive method for detecting peptides glycated with lactose (lactolated peptides) from a complex hydrolysate using electrospray ionization tandem mass spectrometry. Protonated molecular ions of lactolated peptides gave a product-ion spectrum, with the dominant mode of decomposition including cleavage of the O-glycosidic bond followed by dehydration steps giving a characteristic neutral loss of 216 Da. Optimization of the ion marker [M + H]+− 216, identified as a furylium ion, was investigated. It remained dominant regardless of the nature of the glycated peptides, the collision energy used, or the charge state of the parent ion. An approach for detecting lactolated peptides from protein digest was proposed during reverse-phase high-performance liquid chromatography (HPLC)/electrospray mass spectrometry and reverse-phase HPLC/tandem mass spectrometry using neutral loss scanning. This technique detected picomole amounts of lactolated peptides.