Mixtures of proteins and protein-stabilised droplets: rheology of emulsions and emulsion gels

The soft materials formed from emulsions stabilised by proteins, like yogurt, are referred to as emulsion gels. This designation is however not precise enough to reflect the variety of composition of these materials. Indeed, during emulsification not all the proteins in solution adsorb at the interface and the emulsion is thus a mixture of protein-stabilised droplets and unadsorbed proteins. The composition of this mixture affects the viscosity of the emulsions and the texture of the emulsion gels. The objective of this thesis is to study the rheological properties of protein-stabilised emulsions and the gels they form considering the full range of their composition. A first step has been to characterise separately the purified suspensions of protein-stabilised droplets, and of suspensions of pure proteins and their gelation. These components have then been combined, resulting in emulsions and emulsion gels of well-characterised compositions, thus allowing a rigorous approach to the rheology of these systems. The viscosity of purified suspensions of proteins and of protein-stabilised droplets has been studied. It was found that these systems are conveniently studied in the framework developed for soft colloidal suspensions, for which the viscosity scales with the volume fraction. The properties of the droplet and protein suspensions have then been used to model the behaviour of their mixtures. The viscosity models for the two types of pure suspensions have facilitated the development of a semi-empirical model that relates the viscosity of protein-stabilised emulsions to their composition. The gelation of the pure suspensions has then been characterised. Indeed, at low pH, proteins can aggregate and also form gels, either of protein molecules in solution or of protein-covered droplets. The rheological properties of these fractal networks were found to depend on their volume fraction, in good correspondence with previous studies on colloidal gels. Protein gels and droplet gels display very similar mechanical properties when the scaling by the volume fraction is used to describe their concentration. These results have then been used to characterise the rheological properties of emulsion gels over a wide range of compositions. The choice of parameters is important and it was shown that using the total volume fraction and the ratio of volume fractions of the components, rather than the individual volume fractions, makes it possible to change paradigm for these systems, from droplet- filled protein gels to composite gels. Using this approach it was demonstrated that the rheological properties of pure protein gels, emulsion gels and pure droplet gels vary continuously with their composition. Finally, the influence of the size of droplets has been briefly studied. Larger droplets were produced and the rheological properties of the droplet suspensions and droplet gels were compared with the results for smaller droplets and for proteins. It appeared that the increase in size only causes minor changes in the rheological behaviour of the emulsion and emulsion gel, and the variation with the volume fraction is consistent with the other types of samples

Rheology of protein-stabilised emulsion gels envisioned as composite networks. 1 - Comparison of pure droplet gels and protein gels

Protein-stabilised emulsion gels can be studied in the theoretical framework of colloidal gels, because both protein assemblies and droplets may be considered as soft colloids. These particles differ in their nature, size and softness, and these differences may have an influence on the rheological properties of the gels they form. Pure gels made of milk proteins (sodium caseinate), or of sub-micron protein-stabilised droplets, were prepared by slow acidification of suspensions at various concentrations. Their microstructure was characterised, their viscoelasticity, both in the linear and non-linear regime, and their frequency dependence were measured, and the behaviour of the two types of gels was compared. Protein gels and droplet gels were found to have broadly similar microstructure and rheological properties when compared at fixed volume fraction, a parameter derived from the study of the viscosity of the suspensions formed by proteins and by droplets. The viscoelasticity displayed a power law behaviour in concentration, as did the storage modulus in frequency. Additionally, strain hardening was found to occur at low concentration. These behaviours differed slightly between protein gels and droplet gels, showing that some specific properties of the primary colloidal particles play a role in the development of the rheological properties of the gels.Comment: 27 pages, 6 figure

Rheology of protein-stabilised emulsion gels envisioned as composite networks. 2 - Framework for the study of emulsion gels

The aggregation of protein-stabilised emulsions leads to the formation of emulsion gels. These soft solids are classically envisioned as droplet-filled matrices. Here however, it is assumed that protein-coated sub-micron droplets contribute to the network formation in a similar way to proteins. Emulsion gels are thus envisioned as composite networks made of proteins and droplets. Emulsion gels with a wide range of composition are prepared and their viscoelasticity and frequency dependence are measured. Their rheological behaviours are then analysed and compared with the properties of pure gels presented in the first part of this study. The rheological behaviour of emulsion gels is shown to depend mostly on the total volume fraction, while the composition of the gel indicates its level of similarity with either pure droplet gels or pure protein gels. These results converge to form an emerging picture of protein-stabilised emulsion gel as intermediate between droplet and protein gels. This justifies a posteriori the hypothesis of composite networks, and opens the road for the formulation of emulsion gels with fine-tuned rheology.Comment: 22 pages, 5 figure

Viscosity of protein-stabilised emulsions:contributions of components and development of a semi-predictive model

Protein-stabilised emulsions can be seen as mixtures of unadsorbed proteins and of protein-stabilised droplets. To identify the contributions of these two components to the overall viscosity of sodium caseinate o/w emulsions, the rheological behaviour of pure suspensions of proteins and droplets were characterised, and their properties used to model the behaviour of their mixtures. These materials are conveniently studied in the framework developed for soft colloids. Here, the use of viscosity models for the two types of pure suspensions facilitates the development of a semi-empirical model that relates the viscosity of protein-stabilised emulsions to their composition.Comment: 28 pages, 11 figure

Viscoelastometric Testing to Assess Hemostasis of COVID-19: A Systematic Review

Infection by SARS-CoV-2 is associated with a high risk of thrombosis. The laboratory documentation of hypercoagulability and impaired fibrinolysis remains a challenge. Our aim was to assess the potential usefulness of viscoelastometric testing (VET) to predict thrombotic events in COVID-19 patients according to the literature. We also (i) analyzed the impact of anticoagulation and the methods used to neutralize heparin, (ii) analyzed whether maximal clot mechanical strength brings more information than Clauss fibrinogen, and (iii) critically scrutinized the diagnosis of hypofibrinolysis. We performed a systematic search in PubMed and Scopus databases until 31st December 2020. VET methods and parameters, and patients' features and outcomes were extracted. VET was performed for 1063 patients (893 intensive care unit (ICU) and 170 non-ICU, 44 studies). There was extensive heterogeneity concerning study design, VET device used (ROTEM, TEG, Quantra and ClotPro) and reagents (with non-systematic use of heparin neutralization), timing of assay, and definition of hypercoagulable state. Notably, only 4 out of 25 studies using ROTEM reported data with heparinase (HEPTEM). The common findings were increased clot mechanical strength mainly due to excessive fibrinogen component and impaired to absent fibrinolysis, more conspicuous in the presence of an added plasminogen activator. Only 4 studies out of the 16 that addressed the point found an association of VETs with thrombotic events. So-called functional fibrinogen assessed by VETs showed a variable correlation with Clauss fibrinogen. Abnormal VET pattern, often evidenced despite standard prophylactic anticoagulation, tended to normalize after increased dosing. VET studies reported heterogeneity, and small sample sizes do not support an association between the poorly defined prothrombotic phenotype of COVID-19 and thrombotic events

Are Viscoelastometric Assays of Old Generation Ready for Disposal? Comment on Volod et al. Viscoelastic Hemostatic Assays: A Primer on Legacy and New Generation Devices. <i>J. Clin. Med.</i> 2022, <i>11</i>, 860

With the advent of new viscoelastometric hemostatic assay (VHA) devices, with ready-to-use cartridge reagents allowing for their use by people without special laboratory skills, the appreciation of the actual clinical value of VHAs in settings such as severe trauma, post-partum hemorrhage, cardiac surgery and liver transplantation still needs to be fully validated. While two of the newest versions remain based on a ‘cup and pin’ system (ROTEM® sigma, ClotPro®), two other new devices (TEG® 6s, Quantra®) rely on very different technologies: clotting blood is no longer in contact with the probe and challenged by oscillation of one of the components but explored with ultrasound exposure. A systematic literature search (including Sonoclot®) retrieved 20 observational studies (19 prospective). Most studies pointed to imperfect agreements, highlighting the non-interchangeability of devices. Only a few studies, often with a limited number of patients enrolled, used a clinical outcome. No study compared VHA results with conventional laboratory assays obtained through a rapid tests panel. Clinical evidence of the utility of the new VHAs largely remains to be proven through randomized clinical trials, with clinically relevant outcomes, and compared to rapid panel hemostasis testing. The availability of new, improved VHA devices provides an impetus and an opportunity to do so