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Oppositely charged proteins can form soluble assemblies that under specific physical chemical conditions lead to liquid-liquid phase separation, also called heteroprotein coacervation. Increasing evidence suggests that surface charge anisotropy plays a key role in heteroprotein complexation, and coacervation. Here, we investigated complexation of an acidic protein, β-lactoglobulin (BLG), with two basic proteins, rapeseed napin (NAP) and lysozyme (LYS), of similar net charge and size but differing in surface charge distribution. Using turbidity measurements and isothermal titration calorimetry, we confirmed that LYS binds BLG as expected from previous studies. This interaction leads to two types of phase separation phenomena, depending on pH: liquid-solid phase separation in the case of strong electrostatic attraction and liquid-liquid phase separation for weaker attraction. More interestingly, we showed using dynamic light scattering that NAP interacts with BLG, resulting in formation of assemblies in the nanometer size range. The formation of assemblies was also evident when modeling the interactions using Brownian dynamics for both BLG + NAP and BLG + LYS. Similarly, to DLS, BLG and NAP formed smaller assemblies than BLG with LYS. The molecular details rather than the net charge of BLG and NAP may therefore play a role in their assembly. Furthermore, simulated BLG + NAP assemblies were larger than those experimentally detected by DLS. We discuss the discrepancy between experiments and simulations in relation to the limitations of modelling precisely the molecular characteristics of proteins.

The Maillard conjugation of proteins and reducing saccharides is used to modify the technological functionality of whey proteins. In this study, whey protein isolate (WPI) was conjugated with maltodextrin (at 1:1 ratio and two total solid contents of 100 and 200 mg mL−1) through the Maillard reaction and used to form cold-set gels. The glycation reaction increased the strength of hydrogen bonding of whey proteins and preferentially modified α-lactalbumin, in comparison with β-lactoglobulin. It also increased the reducing power of binary protein-saccharide solution and allowed formation of self-standing cold-set WPI gel at a low protein content (i.e., ≈50 mg mL−1). Microscopic imaging showed micro-phase separated maltodextrin domains, interrupting the protein network, in gels made of protein-maltodextrin physical mixtures, whereas Maillard conjugation resulted in more homogenous microstructures at both total solid contents. The Maillard reaction increased gel firmness and water-holding capacity and caused a reduction in the extent of gel swelling.

Whey protein microparticles are of great potential in diverse applications such as fat replacement, emulsification, cargo delivery and formulation of high-protein beverages. We developed a method by which the major whey protein, β-lactoglobulin, (β-lg) is at first encapsulated through a facile procedure within CaCO3 micro-templates, followed by enzymatic crosslinking of the protein and succeeding removal of the template to form protein microparticles. Protein encapsulation increased the colloidal stability and influenced the micro-morphology of CaCO3 microparticles. Dynamic light scattering and ζ-potential measurements, supported by gel electrophoresis indicated that β-lg was entrapped in monomeric form. Heating of the protein-loaded CaCO3 microparticles to gel protein as an alternative to enzymatic crosslinking, was not feasible because pH reduction to 7.0 before heating caused considerable agglomeration and deformation of the carbonate particles. In subsequent to enzymatic crosslinking and template dissolution, confocal laser scanning microscopy illustrated that the resulting protein particles were spherical, micron sized (≈3μ) and structurally homogenous. It was found based on Fourier transform infra-red spectroscopy that enzymatic crosslinking twisted the β-sheet structures of β-lg. Gel electrophoresis and intrinsic fluorescence measurement showed that in addition to the enzymatic polymerization, thiol-disulfide exchange reactions contributed in protein particles formation. It was also concluded based on intrinsic fluorescence measurement that tryptophan microenvironment was not influenced by the enzymatic crosslinking. The fabricated β-lg particles did not undergo significant denaturation by subsequent heating. This may open a route to deliver near-native whey protein microparticles to final consumers.

Whatever the dairy derived fluids that are heat processed, many studies have demonstrated the key role of beta-lactoglobulin (β-lg) denaturation in the initiation of a dairy fouling and mentioned that interactions between β-lg, phosphate and calcium ions are essential in fouling growth. However only few authors have reported the influence of the phosphate/calcium molar ratio on fouling mechanisms. To partly fill this gap, various whey protein solutions (composed mainly of β-lg) having different phosphate:calcium molar ratios were prepared by rehydrating a whey protein isolate powder and by adding calcium chloride and various amounts of soluble inorganic phosphate (noted Pi). Fouling experiments were carried out at pilot scale using a plate heat exchanger (PHE) at a fixed temperature profile and the deposited mass per channel was measured. Finally, the chemical composition and structure of the proteinaceous fouling layer were observed using an Electron Probe Micro Analyser. Results clearly demonstrated that the phosphate:calcium molar ratio modified the deposit structure and influenced substantially the amount of fouling. These results suggest that the relative proportion of calcium phosphate and calcium ions governs the mechanism of fouling build-up and also sets, to a certain extent, the fouling ability of the whey protein solutions.

La lactoferrine et la beta-lactoglobuline sont capables de s’assembler et former des coacervatshétéroprotéiques. Dans cette étude nous avons étudié leur capacité à encapsuler et protégerdes molécules bioactifs et d’intérêt nutritionnel, telle que la vitamines B9. Nous avons dansun premier temps établi la preuve de concept de la formation des coacervats de ces protéinesen présence de B9. Des hauts rendements de coacervation et d’encapsulation ont pu êtreobtenus. Ainsi, en conditions optimales, nous obtenions l’encapsulation de 4 mg de B9/g deprotéines. Comparativement à la B9 seule en solution, la B9 encapsulée est plus stable vis-àvisde traitements tels que la lumière, la lyophilisation ou encore de l’oxydation. L’étudepharmacocinétique conduite chez le rat met en évidence une concentration plasmatiquesupérieure en B9 lorsque celle-ci est administrée sous forme de coacervats, en comparaisonà une administration de B9 seule ou simplement complexée à une des deux protéines. Labiodisponibilité améliorée par les coacervats peut s'explique par l'effet protecteur descoacervats améliorant ainsi la solubilité de la vitamine lors du transit gastro-intestinal.

International audience; The main destabilisation processes in aqueous foams are liquid drainage, coalescence and disproportionation. Due to their amphiphilic nature, proteins can reduce surface tension ϒ, increase liquid phase viscosity and form a visco-elastic interfacial film surrounding the gas bubbles. A correlation is believed to exist between surface rheology of the protein interfacial film and foam stability. Indeed, surface rheology on protein films helps the understanding of foam stability. Two kinds of surface deformation may be applied on a single planar protein film, shearing and expansion/compression (or dilatation). The dilatational elasticity modulus must be larger than 2ϒ to prevent disproportionation. A higher surface shear viscosity is correlated with a better stability against drainage. More generally, higher surface shearing modulus improved stability. However, full understanding still is a challenge because of foam complexity. Foams have confined interstices and spontaneous dynamic rearrangements happen, which cannot be reproduced using only single planar interfaces. Thus, the goal of this work is to bring a multi-scale approach by combining dynamical T1 topological rearrangements of protein films to interfacial rheology measurements.T1 rearrangements occur spontaneously during a foam lifetime or as it flows inside a pipe for example. For small-molecular-weight surfactants, their kinetics have been shown to be an important parameter for the foam stability. In the present work, we aimed at studying the relationship between the rheology of protein interfacial films and the kinetics of T1 rearrangements in protein foams.Two dairy commercial products were studied, sodium caseinate and whey protein isolate (WPI). They differ by their molecular structure and surface rheological properties. Purified β-Lactoglobulin, the main protein in WPI, has also been evaluated. Links between T1, surface rheology and foaming properties will be discussed.

Understanding the mechanism of whey protein fouling along a plate heat exchanger is a challenging issue in the dairy industry. The influence of the calcium/protein molar ratio on β-lactoglobulin (β-LG) denaturation and its consequence both on fouling mass along the plate heat exchanger and the structure of the fouling layer were studied. The experimental design was defined by varying the calcium/protein molar ratio from 2.3 to 22.9 and fixing the total concentration of calcium at 100 ppm. Results established that both the β-LG denaturation rate constants and the distribution of fouling in the plate heat exchanger were strongly impacted by the calcium/protein molar ratio. Structural analysis provided evidence that the fouling layer evolved from a thin and dense structure at low calcium/protein molar ratio towards a thicker and more open structure at higher calcium/protein molar ratio.

The production of natural biocarriers for the protection and controlled delivery of bioactives is of great interest to develop natural functional foods, with enhanced health benefits. The feasibility of heteroprotein complex coacervates formed between two whey proteins (WP), Beta-Lactoglobulin (BLG) and Lactoferrin (LF) to efficiently entrap vitamin B9 has been demonstrated. In the present communication, we investigated the bench scale production of such B9 loaded BLG-LF coacervates (B9-WP coacervates) and their protective ability against B9 degradations during storage (freeze-drying, UV light radiations and accelerated H2O2 induced oxidation). Complex coacervation was performed at bench scale, using commercial protein solutions. The efficiency of two mixing systems, batch versus continuous, were compared. In the continuous mixing system, the formation of B9-WP coacervates was found to be flow-dependant (Figure 1). The highest WP coacervation yield was obtained for a minimum flow rate of 200 mL/min. Under optimized conditions, bench scale efficiency similar to that found for laboratory scale was reached with a coacervation yield of 65 % and a B9 entrapment of 98 %, demonstrating an efficient scaling-up. When entrapped into WP coacervates B9 showed higher stability during freeze-drying with a loss of about 3%, compared to 12% obtained for free unentrapped B9. Additionally, WP coacervates showed good protective effect of B9 against UV irradiation and H2O2 induced oxidation. Hence, heteroprotein complex coacervates constitute a promising biocarriers for the protection of bioactives against chemical and physical degradation. For controlled delivery purpose, in vitro and in vivo studies are in progress to evaluate the effectiveness and optimal release of entrapped B9 molecules. The production of natural biocarriers for the protection and controlled delivery of bioactives is of great interest to develop natural functional foods, with enhanced health benefits. The feasibility of heteroprotein complex coacervates formed between two whey proteins (WP), Beta-Lactoglobulin (BLG) and Lactoferrin (LF) to efficiently entrap vitamin B9 has been demonstrated. In the present communication, we investigated the bench scale production of such B9 loaded BLG-LF coacervates (B9-WP coacervates) and their protective ability against B9 degradations during storage (freeze-drying, UV light radiations and accelerated H2O2 induced oxidation). Complex coacervation was performed at bench scale, using commercial protein solutions. The efficiency of two mixing systems, batch versus continuous, were compared. In the continuous mixing system, the formation of B9-WP coacervates was found to be flow-dependant (Figure 1). The highest WP coacervation yield was obtained for a minimum flow rate of 200 mL/min. Under optimized conditions, bench scale efficiency similar to that found for laboratory scale was reached with a coacervation yield of 65 % and a B9 entrapment of 98 %, demonstrating an efficient scaling-up. When entrapped into WP coacervates B9 showed higher stability during freeze-drying with a loss of about 3%, compared to 12% obtained for free unentrapped B9. Additionally, WP coacervates showed good protective effect of B9 against UV irradiation and H2O2 induced oxidation. Hence, heteroprotein complex coacervates constitute a promising biocarriers for the protection of bioactives against chemical and physical degradation. For controlled delivery purpose, in vitro and in vivo studies are in progress to evaluate the effectiveness and optimal release of entrapped B9 molecules.

Heteroprotein complex coacervation corresponds to the formation of two liquid phases in equilibrium induced by the interaction of two oppositely charged proteins. The more concentrated phase known as coacervate phase, has attracted interest from several fields of science due to its potential applications for example for encapsulation and delivery of bioactives. Prior such application, it is necessary to understand how the presence of small ligands affects the complex coacervation. In this work, we report on the interaction of small ligand with individual proteins β-lactoglobulin (β-LG) and lactoferrin (LF) and consequences on their complex coacervation. ANS (8-Anilinonaphthalene-1-sulfonic acid), a fluorescent probe, was used as model ligand. While ANS did not interact with β-LG, it presented two sets of binding sites with LF inducing its self-aggregation. Depending on its concentration, ANS modulated the shape of β-LG-LF macromolecular assembly. Coacervates were observed for ANS/LF molar ratio

Heat-induced protein gelled deposits, occurring during dairy processing, are industrially cleaned withcaustic-based solutions. In order to develop general dissolution mechanisms of protein hydrogels, inanalogy to general gelation mechanisms for globular proteins, different protein systems must be characterized.We report the first characterization of the dissolution behaviour of heat-set pure bovine serumalbumin (BSA) hydrogels. At low alkali concentrations (0.2 M) the dissolution rate of BSA gels was fairly constant, unlike seen before. Activation energyestimations of the rate yield very low value in certain conditions, again unlike seen before. It is suggestedthat mass transfer limitations occur during dissolution due to the large BSA size.

Mozzarella cheese is a classical dairy product but most research to date has focused on low moisture products. In this study, the microstructure and physicochemical properties of both laboratory and commercially produced high moisture buffalo Mozzarella cheeses were investigated and compared to high moisture bovine products. Buffalo and bovine Mozzarella cheeses were found to significantly differ in their microstructure, chemical composition, organic acid and proteolytic profiles but had similar hardness and meltability. The buffalo cheeses exhibited a significantly higher ratio of fat to protein and a microstructure containing larger fat patches and a less dense protein network. Liquid chromatography mass spectrometry detected the presence of only β-casein variant A2 and a single β-lactoglobulin variant in buffalo products compared to the presence of both β-casein variants A1 and A2 and β-lactoglobulin variants A and B in bovine cheese. These differences arise from the different milk composition and processing conditions. The differences in microstructure and physicochemical properties observed here offer a new approach to identify the sources of milk used in commercial cheese products.

The aim of this work was to examine foaming and interfacial behavior of three milk protein mixtures, bovine α-lactalbumin-β-casein (M1), camel α-lactalbumin-β-casein (M2) and β-lactoglobulin-β-casein (M3), alone and in binary mixtures, at the air/water interface in order to better understand the foaming properties of bovine and camel milks. Different mixture ratios (100:0; 75:25; 50:50; 25:75; 0:100) were used during foaming tests and interfacial protein interactions were studied with a pendant drop tensiometer. Experimental results evidenced that the greatest foam was obtained with a higher β-casein amount in all camel and bovine mixtures. Good correlation was observed with the adsorption and the interfacial rheological properties of camel and bovine protein mixtures. The proteins adsorbed layers are mainly affected by the presence of β-casein molecules, which are probably the most abundant protein at interface and the most efficient in reducing the interfacial properties. In contrast of, the globular proteins, α-lactalbumin and β-lactoglobulin that are involved in the protein layer composition, but could not compact well at the interface to ensure foams creation and stabilization because of their rigid molecular structure.

The production of biocarriers for the protection and controlled delivery of bioactives is of great interest to develop functional foods. The ability of two whey proteins (WP), Beta-Lactoglobulin (BLG) and Lactoferrin (LF), to efficiently entrap vitamin B9 by complex coacervation has been reported at laboratory scale. In the present work, we report on the scaling-up production of B9 loaded BLG-LF coacervates (B9-WP coacervates). Complex coacervation was performed at bench scale, using commercial protein solutions. Under optimized conditions, B9-WP coacervates were produced by static mixing at a flow rate of 300 mL/min. Bench scale efficiency similar to that found for laboratory scale was reached with a coacervation yield of 65% and the B9 entrapment of 98%, demonstrating an efficient scaling-up. B9-WP coacervates showed good protection property for B9 during storage treatments, confirming the efficiency of this type of biocarrier for the development of natural functional foods.

Control of molecular assemblies provides possibility of constructing functional materials in a non-covalent bottom-up manner. Adequate mixing of oppositely charged proteins leads to liquid-liquid phase separation with the formation of reversible heteroprotein complex coacervates having great potential for application as functional biomaterials. For instance, the use of complex coacervates as carrier for nutraceuticals and bioactive components is a blooming research field. Here, we investigated the ability of complex coacervates formed between two whey proteins, Beta-lactoglobulin (BLG) and Lactoferrin (LF), to encapsulate an essential vitamin, the vitamin B9.Across a range of B9:Protein ratios, complex coacervation conditions were determined using turbidity and phase contrast microscopy. Biocarrier efficiency of protein coacervates for B9 was quantified. Stability of B9-Proteins coacervates was monitored after storage at 4°C and 20 °C, and freeze drying. Increasing volumes of B9-Proteins coacervates solutions were tested to evaluate the impact of process conditions on the coacervation yield.By plotting screening maps of B9:Protein ratios and B9 carrying efficiencies (Fig. 1A), we found an optimal biocarrier efficiency of ≈ 10 mg B9/g Proteins. This efficiency found for protein coacervates (Fig. 1B) is similar to that reported for other encapsulation technology like spray-drying [1]. B9-Proteins coacervates after centrifugation (Fig. 1C) were stable over a month storage at 4°C and 20°C, and after freeze drying, without any detectable degradation of B9. Working volumes showed an important impact of the homogenization and centrifugation conditions on the recovered coacervated phase.These heteroprotein systems display useful potentialities as carrying biomaterial [2]. Their formation requires minimal energy input and involves co-products of the dairy industry. Few mg of coacervates can cover the recommended daily intake of bioactives and enhance protein intake. They constitute new potential structured biopolymer-based delivery system. They use directly components of the food product as carrying biomaterial, thus limiting the addition of extraneous components to develop natural functional foods or ingredients.

The design of biocarriers for protection and controlled delivery of bioactives represents a challenge for developing functional foods. We investigated the potentiality of heteroprotein Beta-lactoglobulin (BLG) and Lactoferrin (LF) co-assemblies as biocarriers for vitamin B9 (B9). Using different B9:protein mixing ratios, B9-LF-BLG co-assemblies were obtained and assessed by turbidity and phase contrast microscopy. Kinetics of their formation and stability were monitored. B9 entrapment efficiency was evaluated. Two types of B9-LF-BLG co-assembly were identified: aggregates (at low and high protein concentrations), and heteroprotein coacervates (at intermediate protein concentrations), both exhibiting different kinetics and stability over time. Compiling screening maps of B9-LF-BLG co-assemblies and B9 entrapments, we evidenced that B9-LF-BLG coacervates exhibited higher performance as B9 biocarrier, with an optimal entrapment of ≈10 mg B9/g protein. Therefore, such co-assembly displays useful potentialities as vitamin biocarriers for the design of natural functional foods, offering enhanced health benefits, yet without resorting to non-food additives.

The mechanism of interaction of two oppositely charged macromolecules leading to a liquid-liquid phase separation is called complex coacervation. Examples of complex coacervation between oppositely charged polyelectrolytes are numerous but complex coacervation involving two oppositely charged proteins was only described recently and it results from precise balance of repulsive and attractive interactions between the two proteins1. A better knowledge of the mechanisms of formation and stabilization of such system is essential to design biomaterial with optimized properties such as the encapsulation of bioactives. Here, we investigated the potentiality of two whey proteins, Beta-lactoglobulin (BLG) and Lactoferrin (LF), known for their ability to form complex coacervation2, to spontaneously co-assemble with vitamin B9. Under conditions of LF-BLG coacervation, in contrast to BLG, LF is able to interact with vitamin B9, forming specific nanoparticles3. The ability of such nanoparticles to form coacervates with BLG (B9-LF-BLG coacervates) was further investigated. B9-LF-BLG coacervation was assessed across a range of B9:Proteins mixing ratios and pH conditions by turbidity and phase contrast microscopy. Kinetics of their formation were monitored by light scattering technics. B9 entrapment was evaluated. Storage and time stability of the coacervates were assessed at various temperatures. B9-LF-BLG coacervates were formed in a narrow range of pH, protein concentration and stoichiometry. We evidenced that B9-LF-BLG coacervates exhibited an optimal entrapment of ≈ 10 mg B9/g of proteins. The B9-LF-BLG coacervates were stable over a two weeks storage time. The entrapment efficiency of B9 by the coacervates was found to be similar to that of other encapsulation techniques. Hence the B9-LF-BLG coacervates display useful potentialities as biocarriers for bioactives. They constitute new structured biomaterial based delivery systems with enhanced health benefit to formulate natural and functional ingredient.

The optimisation of dairy unit operations involving heat transfer requires the control of fouling and aggregation phenomena following the denaturation of thermosensitive proteins, in particular β-lactoglobulin (β-lg). This study intends to give a better view of the influence of whey components (whey proteins, lactose, and minerals such as calcium) on β-lg denaturation through a combined kinetic and thermodynamic approach. β-lg denaturation in model solutions of increasing complexity (pure β-lg solution, whey protein solution, and two model wheys differing in mineral content) was characterised at temperatures ranging from 64.5 to 98 °C by following the evolution of soluble β-lg concentration with HPLC. It was demonstrated that whatever the model solution composition, a two-step mechanism (unfolding followed by aggregation) of 1.5-order kinetics could be adopted to describe β-lg denaturation reaction, as the temperature dependence of the denaturation reaction rate was properly fitted by Arrhenius equation. The dependency of kinetic and thermodynamic parameters on solution composition indicated that the presence of whey proteins enhanced β-lg aggregation, whereas lactose showed a small protective effect against β-lg unfolding. Additionally, minerals, especially calcium, tended to stabilise β-lg native state while increasing β-lg aggregation rates. However, at high mineral content, calcium influence could be hindered or even reversed, presumably owing to a lower bioavailability due to complexation with anions such as inorganic phosphates.

The aim of this study is to identify the chemical composition and the microstructure of the deposits obtained after heating camel and cow milks at 80 °C for 60 min using a laboratory-scale device. Like cow milk, camel milk was affected by heat treatment with the reduction of the non-casein nitrogen content reflecting the denaturation of camel whey proteins. The composition of the deposits generated during heating camel and cow milks at 80 °C for 60 min revealed that while camel deposit contained 57 % w/w protein, cow deposit showed a higher protein content of 69 % w/w. The mineral content was 35 % w/w for camel deposit which was higher than that of cow sample, which was 28 % w/w. SEM of both deposits showed a familiar structure of a protein deposit with large clumps composed of smaller aggregates. Camel deposit showed an amorphous structure due to its deficiency in β-lactoglobulin.

International audience; From their amphiphilic nature, proteins in solution adsorb spontaneously at hydrophobic interfaces. Proteins are thus able to change their conformation, which modifies their surface properties. In the confined space near the interface, these modifications favor the formation of many interactions of weak energy (hydrophobic interactions, hydrogen bonds, Van der Waals interactions), and thus the creation, in the interface plane, of a network of intermolecular interactions. Adsorbed proteins can thus, in favorable conditions, constitute at the interface a continuous film with visco-elastic properties. These mechanical properties contribute to the stabilization of the interface created in multiphasic systems such as foams. Our work focuses on the behavior at the air-water interface and, if needed the foam properties, of some model globular proteins: ovalbumin, lysozyme, ovotransferrin, β-lactoglobulin… The aim of our work is to understand, using these models, how protein properties (structure, biochemistry, physico-chemistry), potentially adjusted by the physico-chemical environment, chemical changes or thermo-mechanical treatments, take part in the interfacial behavior, film characteristics and macroscopic proprieties of foams (overrun, stability, texture). Besides, in systems where several proteins with different properties (size, hydrophobicity, charge, conformation flexibility,…) coexist, we look to understand how the interactions between proteins enhance behaviors that cannot be considered as the sum of the single behaviors. The issue over time is to control technological parameters to improve texture properties in relation to foaming capacity.

Expression at the air{water interface of protein assembly potentiality, and interactions at high concentration. OMM Anual meeting

Heating milk at temperatures above 70 °C leads to the exposure of the free thiol group of β-lactoglobulin (β-lg). Free thiol group can then initiate thioldisulphide exchange reactions with other whey proteins or caseins (especially κ-casein, κ-cn) that contain disulphide bonds. The intermolecular aggregation reactions can play a significant role in determining the functional properties of milk. In this study, skim milk was treated with low levels of disulphide reducing agent (β-mercaptoethanol, β-ME) prior to heating. Then the treated milk was made to acid gels. • The functionality of the acid gels were examined using rheology. • The proteins interactions and distributions between the serum and colloidal phase were investigated using electrophoresis.

During the heating of milk (> 70°C), the free thiol group of unfolded βlactoglobulin (β-lg) can interact with the disulphide bonds of other milk proteins. This can lead to the formation of intermolecular disulphide bonds, especially between β-lg and κ-casein (κ-cn)1. The addition of low levels of a disulphide reducing agent will change the ratio of free thiol groups to disulphide bonds in milk proteins and therefore the interactions between the proteins. This study investigated the effect of adding low levels of a reducing agent (β-mercaptoethanol,β-ME) on the involvement of β-lg and κ-cn in disulphide bonding in unheated skim milk, heated skim milk and skim milk that was reduced before heating.

β-lactoglobulin (β-lg) is a major whey protein in bovine milk, which has the ability to bind hydrophobic ligands such as fatty acids. It is well known that the whey protein alpha-lactalbumin also forms a stable complexe with fatty acid, which is cytotoxic to a wide variety of tumour cells. The aim of this project was to investigate the binding stoichiometry of β-lg and linoleic acid (LA; cis,cis-9,12-octadecadienoic acid) and investigate the impact of this complex on viability of intestinal cancer cells in vitro. The interaction between β-lg and fatty acids was studied by HPLC and GC. Complexes with a binding stoichiometry of 1, 2 and 3 LA:β-lg were produced. The effect of the complexes on cell viability was measured by exposing the human colorectal adenocarcinoma epithelial cell line, Caco-2, to various concentrations of complexes from 0 to 72 hours. LA on its own exhibited a cell viability effect at 58 M as measured by MTS assays. A cytotoxic effect at 35 M was measured by an impedance-based biosensing microelectronic real-time cell analyzer system (XCELLigence RTCA system, Roche Diagnostics). Neither the β-lg (0 to 75 μM) nor β-lg/LA complexes (LA concentration from 0 to 200 μM) had an adverse effect on Caco-2 cells. In vivo it is assumed that β-lg gradually releases LA during gut transit which, in turn, may modulate fatty acid uptake by the gut and/or have a satiating effect. Future work will investigate fatty acid uptake and satiety signaling in the gut in response to β-lg/LA complexes.

During the manufacture of dairy products, heat treatments of milk result in the conversion of whey proteins into protein aggregates. These heat-induced aggregates provide new functional properties to dairy products. A protein tailoring approach is developed at UMR-STLO to produce heat-induced aggregates with targeted physicochemical and structural properties, and then to introduce them in a recombined milk to test their functional properties for the formation of dairy acid gels, used as models of yoghurts. It is also well known that denaturation and heat treatments improve interfacial and foaming properties of proteins. The BIA research unit works on understanding the role of heat-induced aggregates in the formation and stability of foams. While non-aggregated proteins adsorb rapidly at the interface, the heat-induced aggregates present in the continuous phase build a protein network that stabilizes the foam. The objective of theses studies is to determine the properties of aggregates that promote the building of new interactions in products that will result in the improvement of custom properties of dairy products and to produce new functional ingredients.; formation de nouvelles entités protéiques ou agrégats thermo-induits. Ces agrégats confèrent des propriétés fonctionnelles aux produits laitiers. Une approche d’ingénierie des agrégats thermo-induits a été développée à l’UMR-STLO qui vise à produire par des moyens variés des agrégats thermo-induits de propriétés physico-chimiques et structurales ciblées, puis à les introduire dans un lait recombiné afin d’évaluer leur propriétés fonctionnelles pour la formation des gels acides, pris comme un modèle du yaourt. Il est connu que la dénaturation et les traitements thermiques améliorent également les propriétés interfaciales et moussantes des protéines. Le laboratoire BIA mène des recherches pour comprendre le rôle des agrégats thermo-induits dans la formation et la stabilisation des mousses. Alors que les protéines non agrégées s’adsorbent rapidement à l’interface, la présence d’agrégats thermoinduits stabilise la mousse par formation d’un réseau de type gel dans la phase continue. L’objectif de ces travaux est de déterminer les propriétés des agrégats qui sont favorables à l’établissement de nouvelles interactions dans les produits, ces dernières étant à l’origine de l’amélioration de leurs propriétés d’usage et de créer de nouveaux ingrédients techno-fonctionnels.

La réactivité des agrégats de b-lactoglobuline peut-elle s’expliquer par l’exposition privilégiée de certains thiols ?. 3ème Rencontres Biologie Physique du Grand Ouest

Previous studies have shown that two altered monomeric species were formed in the early steps of thermal denaturation of bovine beta-lactoglobulin (beta-lg), the well-known Cys121-exposed intermediate (Mcys121), and a new, stable monomer with exposed nonnative Cys119 (Mcys119). In this study, circular dichroism and fluorescence spectroscopies were used to characterize the structural features of these molecules. The structural characteristics of MCys121 after heating and cooling cycles are similar to those of native beta-lg. In contrast, Mcys119 monomer exhibits some characteristics of the well-known molten-globule state. Combined with other published data, these results indicate that heating induces at least two molten globule-like states of beta-lg, a highly reactive Mcys121 that returns to native state after cooling, and a less-reactive Mcys119 that is trapped and stabilized in a molten globule-like state by nonnative disulfide bond.

The role of the free sulfhydryl group of beta-lactoglobulin in the formation of a stable non-native monomer during heat-treatment of beta-lactoglobulin solutions was investigated. Two concomitant events occurred at the earlier stage of heating: unfolding of native globular monomer and intramolecular sulfhydryl/disulfide exchange reaction. Thus, two denatured monomeric species were formed: a non-native monomer with exposed Cys-121 (Mcys121) which became reversible after cooling, and a stable non-native monomer with exposed Cys-119 (Mcys119) which exhibited both a larger hydrodynamic conformation than native monomer and low solubility at pH 4.7. The results also show that the formation of these monomeric species throughout heat-induced denaturation of native beta-lg monomers is faster than their subsequent aggregation. A mechanism describing the behavior of beta-lg denaturation/aggregation during heat-treatment under selected conditions (5.8 mg/ml, low ionic strength, pH 6.6, 85 degrees C) is presented.

Gelation of heat-treated milk protein solutions by acid fermentation was performed using a model system of micellar casein alone (systR), micellar casein and β -lactoglobulin (systB) or micellar casein and egg ovalbumin (systO) dissolved in a milk ultrafiltrate and heat-treated at 90°C for 24 min. Solubility of globular proteins at given pH values and their interactions with casein were determined. Particle size and ζ -potential of the protein systems were measured before and after heat treatment. Acid gelation of the heated systems was monitored using dynamic low-amplitude strain oscillation. Ovalbumin alone or with casein produced larger particles than casein alone or with β -lactoglobulin upon heat treatment. The systems gelled at different pH values, i.e. 4.88, 5.47 and 5.88 in systR, systB and systO, respectively. The latter two pH values were clearly related to the pH of the loss of solubility of the heated globular proteins. While the gelation pattern for systR resembled unheated milk, the pattern for systB and systO showed the usual maximum for tan δ of heated milk.

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...

An in vitro study was conducted to investigate the sensitivity of lactose-b-lactoglobulin conjugates to b-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 b-galactosidase even after an extended hydrolysis time. Tryptic digestion of the conjugates before addition of b-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 compactstructure of lactose-b-lactoglobulin conjugates.

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.

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.

Variously lactosylated species of β-lactoglobulin AB were analysed by capillary electrophoresis at low pH and high pH with and without buffer additives. By simple capillary zone electrophoresis at either low or high pH the lactosylated forms had longer migration times than the native β-lactoglobulin, most likely due to their larger molecular size and lower pI. By micellar electrokinetic chromatography using SDS as detergent the lactosylated forms of β-lactoglobulin had shorter migration times than the native species, due to less interaction with the micelles. The methods used were also able to distinguish between more or less lactosylated forms of β-lactoglobulin. By all methods used, partial separation of at least five forms of β-lactoglobulin were obtained. However, baseline separation of β-lactoglobulin with varying number of lactose units attached was not obtained, probably due to the presence of both variants of β-lactoglobulin with various numbers of lactosyl groups at different sites.