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The use of egg white in food products for various functional properties was studied along with the influence of salts on the gelling properties of hen egg white. High concentration of sodium chloride decreased the water-holding capacity and microstructure of egg white gels.

Abstract: The conformational changes of egg-white proteins, in a ternary-protein system, at the air–water interface have been studied. Three of the major egg-white proteins, ovalbumin, ovotransferrin and lysozyme, were studied with concentration ratios reflecting those in egg-white. Results were compared to those obtained in a previous work on protein denaturation at the air–water interface in single-protein systems (Lechevalier, V., Croguennec, T., Pezennec, S., Guérin-Dubiard, C., Pasco, M., & Nau, F. (2003). Ovalbumin, ovotransferrin, lysozyme: Three model proteins for structural modifications at the air–water interface. Journal of Agricultural and Food Chemistry 51, 6354–6361). Foaming altered the protein structure more profoundly in the mixture than in single-protein systems. Strong electrostatic interactions were observed between the three proteins. Their existence at the air–water interface could ease intermolecular sulfhydryl–disulfide exchange reactions between ovalbumin and both ovotransferrin and lysozyme. This study highlighted the fact that results obtained on single-protein systems were not easily extrapolable to complex systems, such as egg-white. [Copyright &y& Elsevier]

Combination of plant and animal protein diet is becoming a valuable source of nutrition in the modern diet due to the synergistic functional properties inherent in these protein complexes. Moreover, the synergy between animal and plant proteins can contribute to the high stability and improved solubility of the encapsulated bioactive ingredients (e.g., essential oils). Therefore, the study was designed to evaluate the plant (pea protein (PP) and lupine protein (LP)) and animal protein (whey protein, WP) mixed systems as a wall material for microencapsulation of mānuka essential oil, as an example of bioactive compound. Moreover, physicochemical properties and in vitro release profile of encapsulated mānuka essential oil were studied. Mānuka essential oil microcapsules exhibited low moisture content (5.3-7.1 %) and low water activity (0.33-0.37) with a solubility of 53.7-68.1 %. Change in wall material ratio significantly affected the color of microcapsules, while microcapsules prepared with 1:1 protein/oil ratio demonstrated a high encapsulation efficiency (90.4 % and 89.4 %) for protein mixed systems (PP + WP and LP + WP), respectively. Microcapsules further showed low values for lipid oxidation with a high oxidative stability and antioxidant activity (62.1-87.0 %). The zero order and Korsmeyer-Peppas models clearly explained the release mechanism of encapsulated oil, which was dependent on the type and concentration of the protein mixed used. The findings demonstrated that the protein mixed systems successfully encapsulated the mānuka essential oil with controlled release and high oxidative stability, indicating the suitability of the protein mixed systems as a carrier in encapsulation and application potential in development of encapsulated functional foods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Infant formula (IF) is a complex matrix requiring numerous ingredients and processing steps. The objective was to understand how the quality of protein ingredients impacts IF structure and, in turn, their kinetics of digestion. Four powdered IFs (A/B/C/D), based on commercial whey protein (WP) ingredients, with different protein denaturation levels and composition (A/B/C), and on caseins with different supramolecular organisations (C/D), were produced at a semi-industrial level after homogenization and spray-drying. Once reconstituted in water (13 %, wt/wt), the IF microstructure was analysed with asymmetrical flow field-flow fractionation coupled with multi-angle light scattering and differential refractometer, transmission electron microscopy and electrophoresis. The rehydrated IFs were subjected to simulated infant in vitro dynamic digestion (DIDGI®). Digesta were regularly sampled to follow structural changes (confocal microscopy, laser-light scattering) and proteolysis (OPA, SDS-PAGE, LC-MS/MS, cation-exchange chromatography). Before digestion, different microstructures were observed among IFs. IF-A, characterized by more denatured WPs, presented star-shaped mixed aggregates, with protein aggregates bounded to casein micelles, themselves adsorbed at the fat droplet interface. Non-micellar caseins, brought by non-micellar casein powder (IF-D) underwent rearrangement and aggregation at the interface of flocculated fat droplets, leading to a largely different microstructure of IF emulsion, with large aggregates of lipids and proteins. During digestion, IF-A more digested (degree of proteolysis + 16 %) at 180 min of intestinal phase than IF-C/D. The modification of the supramolecular organisation of caseins implied different kinetics of peptide release derived from caseins during the gastric phase (more abundant at G80 for IF-D). Bioactive peptide release kinetics were also different during digestion with IF-C presenting a maximal abundance for a large proportion of them. Overall, the present study highlights the importance of the structure and composition of the protein ingredients (WPs and caseins) selected for IF formulation on the final IF structure and, in turn, on proteolysis. Whether it has some physiological consequences remains to be investigated., Competing Interests: Declaration of Competing Interest This work was supported by SODIAAL International, a French dairy cooperative, as a CIFRE (Agreement of Training through Research) PhD. Lucile CHAUVET, Marieke Van Audenhaege and Marion Lemaire are employed by SODIAAL International. Other authors declare no conflict of interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

One of the first steps of cheese making is to suppress the colloidal stability of casein micelles by enzymatic hydrolysis and initiate milk gelation. Afterwards, the enzymatic milk gel is cut to promote syneresis and expulsion of the soluble phase of milk. Many studies have reported on the rheological properties of enzymatic milk gels at small strain, but they provide limited information on the ability of the gel to be cut and handled. In this study, we aim to characterize the non-linear properties and the yielding behavior of enzymatic milk gels during creep, fatigue and stress sweep tests. We evidence by both continuous and oscillatory shear tests that enzymatic milk gel displays irreversible and brittle-like failure, as reported for acid caseinate gels, but with additional dissipation during fracture opening. Before yielding, acid caseinate gels display strain-hardening only, while enzymatic milk gels also display strain-softening. By varying the gel aging time and the volume fraction of casein micelles, we are able to attribute the hardening to the network structure and the softening to local interactions between casein micelles. Our study highlights the crucial importance of the nanoscale organization of the casein micelles - or more generally of the building block of a gel - to retain the macroscopic nonlinear mechanical properties of the gel.

In recent years, various types of plant-based meat, dairy, and seafood alternatives merged in the health-conscious consumer market. However, plant-based alternatives present complexity in terms of nutritional profile and absorption of nutrients after food ingestion. Thus, this review summarizes current strategies of plant-based alternatives and their nutritional analysis along with gastrointestinal digestion and bioavailability. Additionally, regulatory frameworks, labeling claims, and consumer perception of plant-based alternatives are discussed thoroughly with a focus on status and future prospects. Plant-based alternatives become a mainstream of many food-processing industries with increasing alternative plant-based food manufacturing industries around the world. Novel food processing technologies could enable the improving of the taste of plant-based foods. However, it is still a technical challenge in production of plant-based alternatives with authentic meaty flavor. In vitro gastrointestinal digestion studies revealed differences in the digestion and absorption of plant-based alternatives and animal-based foods due to their protein type, structure, composition, anti-nutritional factors, fibers, and polysaccharides. Overall, plant-based alternatives may facilitate the replacement of animal-based foods; however, improvements in nutritional profile and in vitro digestion should be addressed by application of novel processing technologies and food fortification. The specific legislation standards should be necessary to avoid consumer misleading of plant-based alternatives.

Eggs are a whole food which affordably support human nutritional requirements worldwide. Eggs strongly resist bacterial infection due to an arsenal of defensive systems, many of which reside in the egg white. However, despite improved control of egg production and distribution, eggs remain a vehicle for foodborne transmission of Salmonella enterica serovar Enteritidis, which continues to represent a major public health challenge. It is generally accepted that iron deficiency, mediated by the iron-chelating properties of the egg-white protein ovotransferrin, has a key role in inhibiting infection of eggs by Salmonella . Ovotransferrin has an additional antibacterial activity beyond iron-chelation, which appears to depend on direct interaction with the bacterial cell surface, resulting in membrane perturbation. Current understanding of the antibacterial role of ovotransferrin is limited by a failure to fully consider its activity within the natural context of the egg white, where a series relevant environmental factors (such as alkalinity, high viscosity, ionic composition, and egg white protein interactions) may exert significant influence on ovotransferrin activity. This review provides an overview of what is known and what remains to be determined regarding the antimicrobial activity of ovotransferrin in egg white, and thus enhances understanding of egg safety through improved insight of this key antimicrobial component of eggs.

Fouling of plate heat exchangers (PHEs) is a recurring problem when pasteurizing whey protein solutions. As Ca 2+ is involved in denaturation/aggregation mechanisms of whey proteins, the use of calcium chelators seems to be a way to reduce the fouling of PHEs. Unfortunately, in depth studies investigating the changes of the whey protein fouling mechanism in the presence of calcium chelators are scarce. To improve our knowledge, reconstituted whey protein isolate (WPI) solutions were prepared with increasing amounts of phosphate, expressed in phosphorus (P). The fouling experiments were performed on a pilot-scale PHE, while monitoring the evolution of the pressure drop and heat transfer coefficient. The final deposit mass distribution and structure of the fouling layers were investigated, as well as the whey protein denaturation kinetics. Results suggest the existence of two different fouling mechanisms taking place, depending on the added P concentration in WPI solutions. For added P concentrations lower or equal to 20 mg/L, a spongy fouling layer consists of unfolded protein strands bound by available Ca 2+ . When the added P concentration is higher than 20 mg/L, a heterogeneously distributed fouling layer formed of calcium phosphate clusters covered by proteins in an arborescence structure is observed.

Understanding the food protein binding to bioactive compounds is of utmost importance for the development of efficient protein-based delivery systems. The binding of lutein to sodium caseinate (NaCas) or native casein micelle (PPCN) was investigated at pH 7 to evaluate the effect of casein supramolecular structures on the interaction. Fluorescence quenching, UV-vis spectroscopy, and dynamic light scattering were carried out. Under the medium conditions of interaction analysis (DMSO-water and ethanol-water), lutein exists as H-type aggregates. The investigation of lutein/casein interaction showed a predominantly static mechanism of fluorescence quenching and the presence of two fluorophore populations on NaCas and PPCN, but only one accessible to lutein. Moreover, the Scatchard plot indicated that lutein interacted with both caseins in one binding site. The interaction of lutein with caseins occurred with binding constant K b of 10 5 M -1 , regardless of casein supramolecular structure., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Heat treatments induce changes in the protein structure in infant milk formulas (IMFs). The present study aims to investigate whether these structural modifications affect protein digestion. Model IMFs (1.3% proteins), with a bovine or a human whey protein profile, were unheated or heated at 67.5 °C or 80 °C to reach 65% of denaturation, resulting in six protein structures. IMFs were submitted to in vitro static gastrointestinal digestion simulating infant conditions. During digestion, laser light scattering was performed to analyze IMF destabilization and SDS-PAGE, OPA assay and cation exchange chromatography were used to monitor proteolysis. Results showed that, during gastric digestion, α-lactalbumin and β-lactoglobulin were resistant to hydrolysis in a similar manner for all protein structures within IMFs (p > 0.05), while the heat-induced denaturation of lactoferrin significantly increased its susceptibility to hydrolysis. Casein hydrolysis was enhanced when the native casein micelle structure was modified, i.e. partially disintegrated in the presence of lactoferrin or covered by heat-denatured whey proteins. The IMF destabilization at the end of the gastric digestion varied with protein structures, with larger particle size for IMF containing native casein micelles. During intestinal digestion, the kinetics of protein hydrolysis varied with the IMF protein structures, particularly for IMFs containing denatured lactoferrin, exhibiting higher proteolysis degree (67.5 °C and 80 °C vs. unheated) and essential amino acid bioaccessibility (67.5 °C vs. unheated). Overall, the protein structures, generated by modulating the whey protein profile and the heating conditions, impacted the IMF destabilization during the gastric phase and the proteolysis during the entire simulated infant digestion.

The process of manufacturing infant milk formulas (IMFs) involves heat treatments that can lead to whey protein denaturation. The objective of the study was to determine how protein composition affects the denaturation kinetics of the whey proteins within IMFs. Three model IMFs (1.3% of cow's milk protein) were produced with a caseins: whey proteins ratio of 40:60, differing only by the whey protein composition. The kinetics of heat-induced denaturation of α-lactalbumin, β-lactoglobulin and lactoferrin were investigated between 67.5 °C and 80 °C by chromatographic quantification of the residual native proteins. Results showed that the heat-denaturation of α-lactalbumin was reduced when β-lactoglobulin was absent. The heat-denaturation of lactoferrin was not affected by the composition of the IMFs but its presence enhanced the heat-denaturation of β-lactoglobulin. This study revealed that, for higher heat treatments (90 °C/15 s, 75 °C/15 min), IMF containing α-lactalbumin and lactoferrin preserved a higher proportion of native whey proteins than IMFs containing β-lactoglobulin., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Animal- and plant-based proteins are present in a wide variety of raw and processed foods. They play an important role in determining the final structure of food matrices. Food proteins are diverse in terms of their biological origin, molecular structure, and supramolecular assembly. This diversity has led to segmented experimental studies that typically focus on one or two proteins but hinder a more general understanding of food protein structuring as a whole. In this review, we propose a unified view of how soft-matter physics can be used to control food protein assembly. We discuss physical models from polymer and colloidal science that best describe and predict the phase behavior of proteins. We explore the occurrence of phase transitions along two axes: increasing protein concentration and increasing molecular attraction. This review provides new perspectives on the link between the interactions, phase transitions, and assembly of proteins that can help in designing new food products and innovative food processing operations.

Understanding the mechanism of interaction between food proteins and bioactives constitutes the preliminary step to design food grade nanocarriers. We investigated the interaction between cyanidin-3-O-glucoside (C3G), and 20nm-sized sodium caseinate nanoparticles (NaCas) at pH 7 and pH 2 by fluorescence spectroscopy and dynamic light scattering. The characterization of the C3G-NaCas interaction indicated that the fluorescence quenching mechanism was predominantly static. C3G interacted with two sets of binding sites with association constants Ka of 10 6 and 10 5 M -1 . Electrostatic interactions dominated at pH 7, while hydrophobic effects were the main force at pH 2. Interestingly, the two sets of binding sites were discriminated by ionic strength at pH 7. The binding of C3G slightly modified the average diameter of NaCas nanoparticles without alteration of its surface charge suggesting a complexation of C3G molecules in the internal casein structure. Thus, NaCas constitutes a putative nanocarrier for anthocyanins in new functional foods., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

During storage, a series of changes occur for dairy powders, such as protein lactosylation and the formation of Maillard reaction products (MRPs), leading to powder browning and an increase of insoluble matter. The kinetics of protein lactosylation and MRP formation are influenced by the lactose content of the dairy powder. However, the influence of lactose in the formation of insoluble matter and its role in the underlying mechanisms is still a subject of speculation. In this study, we aim to investigate the role of lactose in the formation of insoluble matter in a more comprehensive way than the existing literature. For that, two casein powders with radically different lactose contents, standard micellar casein (MC) powder (MC1) and a lactose-free (less than 10 ppm) MC powder (MC2), were prepared and stored under controlled conditions for different periods of time. Powder browning index measurements and solubility tests on reconstituted powders were performed to study the evolution of the functional properties of MC powders during aging. Proteomic approaches [one-dimensional electrophoresis and liquid chromatography-mass spectrometry (LC-MS)] and innovative label-free quantification methods were used to track and quantify the chemical modifications occurring during the storage of the powders. Reducing the amount of lactose limited the browning of MC powders but had no effect on the loss of solubility of proteins after storage, suggesting that the action of lactose, leading to the production of MRC, does not promotes the formation of insoluble matter. Electrophoresis analysis did not reveal any links between the formation of covalent bonds between caseins and loss in solubility, regardless of the lactose content. However, LC-MS analyses have shown that different levels of chemical modifications occur during the MC powder storage, depending upon the presence of lactose. An increase of protein lactosylation and acetylation was observed for the powder with a higher lactose content, while an increase of protein deamidation and dephosphorylation was observed for that containing lower lactose. The decrease of pH in the presence of lactose as a result of Maillard reaction (MR) may explain the difference in the chemical modifications of the two powders. In view of the present results, it is clear that lactose is not a key factor promoting insolubility and for the formation of cross-links between caseins during storage. This suggests that lactosylation is not the core reaction giving rise to loss in solubility.

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 <25 against amorphous aggregates for higher ANS/LF molar ratios. A maximum loading capacity of around 40mg of ANS per gram of LF in the formed heteroprotein coacervates was reached., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Proteins exhibit a rich diversity of functional, physico-chemical and biodegradable properties which makes them appealing for various applications in the food and non-food sectors. Such properties are attributed to their ability to interact and assemble into a diversity of supramolecular structures. The present review addresses the updated research progress in the recent field of complex coacervation made from mixtures of oppositely charged proteins (i.e. heteroprotein systems). First, we describe briefly the main proteins used for heteroprotein coacervation. Then, through some selected examples, we illustrate the particularity and specificity of each heteroprotein system and the requirements that drive optimal assembly into coacervates. Finally, possible and promising applications of heteroprotein coacervates are mentioned., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Under specific conditions, mixing two oppositely charged proteins induces liquid-liquid phase separation. The denser phase, or coacervate phase, can be potentially applied as a system to protect or encapsulate different bioactive molecules with a broad range of food and/or medical applications. The optimization of the design and efficiency of such systems requires a precise understanding of the structure and the equilibrium of the nanocomplexes formed within the coacervate. Here, we report on the nanocomplexes and the dynamics of the coacervates formed by two well-known, oppositely charged proteins β-lactoglobulin (β-LG, pI ≈ 5.2) and lactoferrin (LF, pI ≈ 8.5). Fluorescence recovery after photobleaching (FRAP) and solid-state nuclear magnetic resonance (NMR) experiments indicate the coexistence of several nanocomplexes as the primary units for the coacervation. To our knowledge, this is the first evidence of the occurrence of an equilibrium between quite unstable nanocomplexes in the coacervate phase. Combined with in silico docking experiments, these data support the fact that coacervation in the present heteroprotein system depends not only on the structural composition of the coacervates but also on the association rates of the proteins forming the nanocomplexes.

In the study presented here, we investigated the interaction at pH 5.5 between folic acid (FA) and lactoferrin (LF), a positively charged protein. We found a binding constant Ka of 10(5) M(-1) and a high stoichiometry of 10 mol of FA/mol of LF. The size and charge of the complexes formed evolved during titration experiments. Increasing the ionic strength to 50 mM completely abolished the isothermal titration calorimetry (ITC) signal, suggesting the predominance of electrostatic interactions in the exothermic binding obtained. We developed a theoretical model that explains the complex triphasic ITC profile. Our results revealed a two-step mechanism: FA/LF interaction followed by self-association of the complexes thus formed. We suggest that 10 FA molecules bind to LF to form saturated reactive complexes (FA10/LF) that further self-associate into aggregates with a finite size of around 15 nm. There is thus a critical saturation degree of the protein, above which the self-association can take place. We present here the first results that provide comprehensive details of the thermodynamics of FA/LF complexation-association. Given the high stoichiometry, allowing a load of 55 mg of FA/g of LF, we suggest that FA/LF aggregates would be an effective vehicle for FA in fortified drinks.

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.

The dairy protein β-lactoglobulin (βlg) is known to form a complex with fatty acids (FA). Due to industrial processing, βlg is often in its non-native form in food products, which can modify the FA/βlg complex properties. We investigated the interaction of bovine βlg, in selected structural forms (native βlg, a covalent dimer and as nanoparticles), with linoleate (C18:2). Using fluorescence and Isothermal Titration Calorimetry, linoleate was found to bind βlg at two different binding sites. Regardless of the structural state of βlg, association constants remained in the same order of magnitude. However, the stoichiometry increased up to 6-fold for nanoparticles, compared to that of native βlg. The impact of these structural changes on linoleate uptake in vitro was measured by cytotoxicity assays on Caco-2 cells. The order of cytotoxicity of linoleate was as follows: free>complexed to dimers>complexed to nanoparticles>complexed to native βlg. Therefore, the in vitro cytotoxicity of linoleate could be modulated by altering the state of βlg aggregation, which in turn affects its binding capacity to the FA., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The dairy protein β-lactoglobulin (BLG) is known to bind fatty acids such as the salt of the essential longchain fatty acid linoleic acid (cis,cis-9,12-octadecadienoic acid, n-6, 18:2). The aim of the current study was to investigate how bovine BLG-linoleate complexes, of various stoichiometry, affect the enzymatic digestion of BLG and the intracellular transport of linoleate into enterocyte-like monolayers. Duodenal and gastric digestions of the complexes indicated that BLG was hydrolyzed more rapidly when complexed with linoleate. Digested as well as undigested BLG-linoleate complexes reduced intracellular linoleate transport as compared with free linoleate. To investigate whether enteroendocrine cells perceive linoleate differently when part of a complex, the ability of linoleate to increase production or secretion of the enteroendocrine satiety hormone, cholecystokinin, was measured. Cholecystokinin mRNA levels were different when linoleate was presented to the cells alone or as part of a protein complex. In conclusion, understanding interactions between linoleate and BLG could help to formulate foods with targeted fatty acid bioaccessibility and, therefore, aid in the development of food matrices with optimal bioactive efficacy., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The dairy protein β-lactoglobulin (βlg) is known to bind hydrophobic ligands such as fatty acids. In the present work, we investigated the biological activity in vitro of linoleate once complexed to bovine βlg. Binding of linoleate (C18:2) to bovine βlg was achieved by heating at 60 °C for 30 min at pH 7.4, resulting in a linoleate/βlg molar binding stoichiometry of 1.1, 2.1, and 3.4. Two types of binding sites were determined by ITC titrations. Binding of linoleate induced the formation of covalent dimers and trimers of βlg. The LD(50) on Caco-2 cells after 24 h was 58 μM linoleate. However, cell viability was unaffected when 200 μM linoleate was presented to the Caco-2 cells as part of the βlg complex. The Caco-2 cells did not increase mRNA transcript levels of long chain fatty acid transport genes, FATP4 and FABPpm, or increase levels of the cAMP signal, in response to the presence of 50 μM linoleate alone or as part of the βlg complex. Therefore, it is proposed that βlg can act as a molecular carrier and alter the bioaccessibility of linoleate/linoleic acid.

Understanding the driving forces governing protein assembly requires the characterization of interactions at molecular level. We focus on two homologous oppositely charged proteins, lysozyme and α-lactalbumin, which can assemble into microspheres. The assembly early steps were characterized through the identification of interacting surfaces monitored at residue level by NMR chemical shift perturbations by titrating one (15)N-labeled protein with its unlabeled partner. While α-lactalbumin has a narrow interacting site, lysozyme has interacting sites scattered on a broad surface. The further assembly of these rather unspecific heterodimers into tetramers leads to the establishment of well-defined interaction sites. Within the tetramers, most of the electrostatic charge patches on the protein surfaces are shielded. Then, hydrophobic interactions, which are possible because α-lactalbumin is in a partially folded state, become preponderant, leading to the formation of larger oligomers. This approach will be particularly useful for rationalizing the design of protein assemblies as nanoscale devices.

Self-assembly in aqueous solution of two oppositely charged globular proteins, hen egg white lysozyme (LYS) and bovine calcium-depleted α-lactalbumin (apo α-LA), was investigated at pH 7.5. The aggregation rate of equimolar mixtures of the two proteins was determined using static and dynamic light scattering as a function of the ionic strength (15-70 mM) and protein concentration (0.28-2.8 g/L) at 25 and 45 °C. The morphology of formed supramolecular structures was observed by confocal laser scanning microscopy. When the two proteins are mixed, small aggregates were formed rapidly that subsequently grew by collision and fusion. The aggregation process led on larger length scales to irregularly shaped flocs at 25 °C, but to monodisperse homogeneous spheres at 45 °C. Both the initial rate of aggregation and the fraction of proteins that associated decreased strongly with decreasing protein concentration or increasing ionic strength but was independent of the temperature.

Controlled interactions and assembly of proteins with one another promise to be a powerful approach for generating novel supramolecular architectures. In this study, we report on the ability of oppositely charged globular proteins to self-assemble into well-defined micrometer-sized spherical particles under specific physicochemical conditions. We show that microspheres were spontaneously formed in all binary protein mixtures tested once the physicochemical conditions were optimized. The optimal pH value, initial protein concentrations needed to form microspheres, and protein stoichiometry in these microspheres varied and depended on the structural features of the mixed proteins. We show that charge compensation is required but not sufficient to guide optimal protein assembly and organization into microspheres. Size difference between protein couples (acidic and basic) is a key element that defines optimal pH value for microsphere formation and the protein molar ratio in the formed microspheres. Our findings give new elements that can help to predict the assembly behavior of various proteins in mixed systems.

Apo alpha-lactalbumin (apo alpha-LA) and lysozyme (LYS), two homologous globular proteins have been shown to be able to interact and self-assemble to form microspheres. We report on the organisation and the mechanism of such protein assembly process using a variety of microscopic techniques. We demonstrated that proteins involved into apo alpha-LA/LYS microspheres exchange with those free in solution. The exchange process takes place from the periphery to the centre of the microspheres. The formed spherical particles observed after fixed incubation time were found to be either individual or aggregated according to the total protein concentration leading to structures with different size and morphology. It appears that protein assembly occurs throughout successive steps of aggregated spherical particles that reorganise into biggest isolated microspheres. Direct microscopic observations over time confirm that microspheres resulted from a reorganisation of aggregated, clustered nanospheres. We propose that the formation of apo alpha-LA/LYS microspheres follows an "aggregation-reorganisation" mechanism.

In a previous work, we reported that contrary to native calcium-loaded alpha-lactalbumin (holo alpha-LA), calcium-depleted form (apo alpha-LA) has the ability to self-assemble with lysozyme (LYS) to form different supramolecular structures in temperature-dependent manner. In this study, we examine what happens at molecular scale using fluorescence techniques. Fluorescence anisotropy coupled with fluorescence lifetime measurements provides a means to measure intermolecular interactions. We showed that LYS interacts with both apo alpha-LA and holo alpha-LA to form oligomers, assumed to be heterodimers, at 10 degrees C and 45 degrees C. The dissociation constants for dimerization were found to be in the muM range and increased significantly with increasing ionic strength from 39 to 124 mM. Although the binding constants of holo alpha-LA-LYS and apo alpha-LA-LYS complexes were of the same order of magnitude, the shape or conformation of formed heterodimers differed as assessed by fluorescence parameters in particular correlation time calculations. Such conformation differences could explain why holo alpha-LA-LYS complexes are trapped as heterodimers while the apo alpha-LA-LYS complexes have the ability to further self-assemble into various supramolecular structures.

We have reported previously that the calcium-depleted form of bovine alpha-lactalbumin (apo alpha-LA) interacts with hen egg-white lysozyme (LYS) to form spherical supramolecular structures. These supramolecular structures contain an equimolar ratio of the two proteins. We further explore here the organization of these structures. The spherical morphology and size of the assembled LYS/apo alpha-LA supramolecular structures were demonstrated using confocal scanning laser microscopy and scanning electron microscopy. From confocal scanning laser microscopy experiments with labelled proteins, it was found that LYS and apo alpha-LA were perfectly colocalized and homogeneously distributed throughout the entire three-dimensional structure of the microspheres formed. The spatial colocalization of the two proteins was also confirmed by the occurrence of a fluorescence resonance energy transfer phenomenon between labelled apo alpha-LA and labelled LYS. Polarized light microscopy analysis revealed that the microspheres formed differ from spherulites, a higher order semicrystalline structure. As the molecular mechanism initiating the formation of these microspheres is still unknown, we discuss the potential involvement of a LYS/apo alpha-LA heterodimer as a starting block for such a supramolecular assembly.

The interfacial properties (kinetics of adsorption at the air/water interface, rheology of the interfacial layer) of ovalbumin molecules, unheated or previously heat-denatured in solution (10 g L(-1), pH 7, NaCl 50 mM) under controlled conditions (up to 40 min at 80 degrees C), were investigated. Heat treatments induced the formation of covalent aggregates which surface exhibits a higher hydrophobicity and an increased exposition of sulfhydryl groups when compared to native ovalbumin (unheated). Although they have a larger hydrodynamic size, aggregates adsorb as fast as native ovalbumin at the air/water interface. However, aggregates are able to established rapid contacts in the interfacial layer as shown by the fast increase of both surface pressure and shear elastic constant. In contrast, native ovalbumin needs longer time to developed intermolecular contacts and exhibits lower foam stability even if the shear elastic constant on aging reached higher value than for ovalbumin aggregates.