In thrombosis and haemostasis, coagulation and platelet activation pathways culminate to form solid fibrin clots, which can become vaso-occlusive or prevent excessive bleeding. We report a novel mechanism describing how developing fibrin clots prolong and modulate the reactivity of thrombin, an enzyme propagating platelet and coagulation activation and forming fibrin from fibrinogen. Using immunological and genetic approaches, we delineate how thrombin bound to the A and Baachains of fibrin E-domains regulates lateral fibrin fibre extension. Our data reveal that fibrin-bound thrombin remains active and is temporarily protected against inactivation by antithrombin-III. Immunological displacement of thrombin from fibrin profoundly lowered its capacity, whereas a peptide mimicking the AA-chain binding-site increased its reactivity. In a cohort of patients with congenital dysfibrinogenemia, carrying FGA, FGB or FGG mutations associated with bleeding or thrombosis phenotypes, we noticed a high thrombin capacity and suppressed thrombinantithrombin-III complex formation, pointing to a prolonged active thrombin lifetime, likely due to abnormal formation of thrombin-containing fibrin. In conclusion, the combination of an impaired clotting and increased thrombogenicity may explain the paradoxical bleeding and thrombotic complications observed in such patients. Development of fibrin-directed agents may offer new therapeutic opportunities to normalize hemostasis or prevent thrombosis.

Membranolytic peptides are potential cancer therapeutics, although targeting cancer cells specifically remains an unmet challenge. We have modified the membranolytic peptide MP1, from Polybia paulista, to direct its action specifically to some cancer cells, thereby improving its cancer therapeutic characteristics and reducing its nonspecific toxicity. MP1 was modified by addition of sequences allowing binding to the cancer biomarker EGFR, with or without sequences directing cleavage by the cancer biomarker MMP-2. Toxicity was assessed in human breast cell lines and was correlated with EGFR expression and MMP-2 activity. Efficacy as an antitumor agent was assessed in MDA-MB-468 xenograft models. C-terminal addition of targeting sequences generally reduced cellular toxicities of peptides relative to wildtype MP1. Cell lines that retained the highest sensitivities to these fusion peptides expressed the highest EGFR and/or MMP-2 levels, supporting specific cytotoxic activity directed to these biomarkers. Treatment with an MMP-2 inhibitor significantly reduced the cell-killing activity of peptides containing MMP-2 cleavage sites, further supporting specific targeting. Fusion peptides significantly induced apoptosis and reduced survival in EGFR/MMP-2 high cancer cells, while sparing EGFR/MMP-2 low cells in standard tissue culture and 3D-spheroids. Systemic treatment with the EGFR-MMP-MP1 fusion significantly reduced tumor size in MDA-MB-468 xenograft models, confirming in vivo efficacy against cancer cells and acceptable systemic toxicity. We conclude that EGFR-MMP-MP1 peptides represent a novel cancer therapeutic for further development.

The aim of this study was to understand the effect of pH and oil subphase when stabilising emulsions by plant protein-based microgels versus non-microgelled counterparts. Potato protein microgels (PoPM) were used to stabilise Pickering oil-in-water (O-W) emulsions at pH 3 and 7 and subphase of varying polarity-tetradecane and octanol and compared against conventional emulsions stabilised by non-microgelled potato protein (PoP). Confocal microscopy and static light scattering were used to monitor changes in droplet size and microstructure during storage, whilst interfacial shear rheology was used to assess the viscoelasticity of the adsorbed interfacial protein layers using various subphases. Despite the larger oil droplet sizes and lower interfacial viscoelasticity observed for octanol emulsions, systems stabilised by PoPM appeared more resilient to environmental variations irrespective of pH or subphase. This is most likely due to the ca. 4 x larger size of the adsorbed entities of PoPM, promoting steric hindrance and the higher mechanical strength of the adsorbed PoPM films. Strikingly PoP unfolding (confirmed via dynamic light scattering and small angle X-ray scattering (SAXS)) facilitated similar aggregation of PoP to thermally cross-linked PoPM, with resultant emulsion stability behaviour of PoP-stabilised emulsions resembling PoPM-stabilised ones at pH 3. Overall, this study highlights the importance of understanding how using plant protein microgels may offer benefits to interfacial stabilisation when the polarity of subphase varies, such as stabilising essential oils versus vegetable oils, which has been relatively underexplored in the literature.

Artificial cells assembled from materials such as hydrogels have emerged as platforms to replicate and understand biological functionalities, processes, and behaviors. However, hydrogels lack a lipid membrane, a vital property of cellular systems. Here we develop a process for the assembly of a fluid and stable lipid membrane which coats the hydrogel mesh network within the particle, through electostatically-mediated fusion of nanoscale lipid vesicles. This confers cell-mimetic and biotechnologically relevant properties upon microscale, cell sized, hydrogel artificial cells generated through microfluidics. We exploit the properties of the created membrane to augment existing hydrogel properties through permeability alteration and protection of the hydrogel from small molecule degraders. Furthermore, we show that the lipid membrane is compatible with organelle substructures within the hydrogels, which enables the exploitation of an enhanced material design space to build hydrogel artificial cells that increasingly mimic the organization of cells. This platform paves the way for producing next generation artificial cells and functional microdevices from interfaced hydrogel-lipid materials. Our technologies may underpin new opportunities for integrating membranes into hydrogel-based systems, inlcuding for drug delivery and tissue engineering.

This study investigates the role of size and deformability of potato protein microgels in influencing their interfacial performance at the air-water interface. Microgels produced via a top-down method were studied across different length scales, focusing on the air-water interface. Techniques included internal structure analysis via small-angle X-ray scattering (SAXS), particle deformability and moduli studies using atomic force microscopy (AFM), and compression and deposition of Langmuir-Blodgett monolayers. It was found that microgels have the capacity to reach a jammed interfacial state similar to that of nongelled potato protein, however, compression may be required to promote their intermolecular interactions. Despite this, thicker microgel-laden interfacial layers may have greater capacity to promote steric hindrance and aid stability within foams and emulsions.

Membranolytic peptides have demonstrated potential as cancer therapeutics, although targeting to cancer cells and reducing toxicity associated with activity in normal cells remain unmet challenges. We have investigated the membranolytic peptide MP1, from Polybia paulista , in order to assess ways to reduce its non-specific toxicity and thereby improve its characteristics as a cancer therapeutic. Using a panel of human breast cell lines and cell survival assays, we show that C-terminal addition of an EGFR binding sequence, with or without a linking MMP-2 cleavage sequence, generally reduced the efficacy of the peptides relative to wildtype MP1, as determined by increases in IC50 values. Critically, cell lines that show the highest sensitivities to these fusion peptides (MDA-MB-468, MDA-MB-231) expressed the highest EGFR and/or MMP-2 levels, demonstrating that these additions direct the cytotoxic activity to cells expressing these biomarkers. MMP-2 inhibition significantly reduced the cell-killing activity of peptides containing MMP-2 cleavage sites, further demonstrating targeting to this biomarker. Fusion peptides significantly induced apoptosis and reduced survival in EGFR/MMP-2 high cancer cells, while sparing EGFR/MMP-2 low cells in the context of standard tissue culture and 3D-spheroids. Finally, systemic treatment with the EGFR and MMP-2 cleavage fusion significantly reduced tumour size in MDA-MB-468 xenograft models, confirming in vivo efficacy against cancer cells and acceptable systemic toxicity. We present this EGFR-MMP-MP1 peptide as a novel cancer therapeutic for further pre-clinical and clinical development. ### Competing Interest Statement The authors have declared no competing interest. Engineering and Physical Sciences Research Council, https://ror.org/0439y7842, EP/R03608X/1

Fungal hyphae have demonstrated their importance in developing environmentally friendly, multiscale, composite assemblies where animal-derived proteins have been predominantly used as binders. Now, an ongoing challenge is to replace those high-performance animal protein binders with ecofriendly, plant-based alternatives. While the majority of studies have focused on the binding implied by rheological observations, relatively little is known about how such animal proteins bind to hyphal surfaces at nanometric length scales, and this knowledge is required to replace animal-derived binders with plant protein alternatives. Here, we decode intermolecular interactions of plant protein-based binders such as potato protein (PoP) to fungal (Fusarium venenatum) hyphae in comparison to a classic animal protein-based binder (egg white protein, EWP) using a suite of theoretical and experimental approaches. Self-consistent field calculations modeling fungal hyphae as weakly hydrophobic, parallel cylinders predicted differences in the interaction potentials between the model protein layers, showing that EWP had an attractive potential across a broad range of conditions, in contrast to PoP that was mainly repulsive. Stimulated emission depletion (STED) microscopy of protein-coated fungal hyphae confirmed that EWP delivers a uniform and complete coverage, while PoP naturally aggregates, resulting in more patchy coverage. Experimental interaction forces were measured using colloidal probe atomic force microscopy, confirming the influence of non-Coulombic forces particularly dominating in PoP, and attractive forces in EWP, further differentiating their respective binding mechanisms. Collectively, this multimethodological study provides a first-hand molecular explanation of the weaker hyphal-binding properties of aggregated plant proteins at the nanoscale, consistent with the previously reported macroscale observations.

The development of biocompatible and safe mucoadhesive materials is critical for improving therapeutic strategies, where cationic proteins such as lactoferrin are emerging as promising alternatives to synthetic polymers. Here, we demonstrate how thermal denaturation of lactoferrin can be used as a viable strategy to enhance mucoadhesion. We identify and study in detail the structural changes in lactoferrin upon thermal denaturation using light scattering, circular dichroism spectroscopy, gel-electrophoresis, and atomic force microscopy. Lactoferrin-mucin binding was evaluated using rheology, confocal microscopy, and quartz crystal microbalance with dissipation monitoring. We find that lactoferrin binds to mucin at its native state, heat-treatment at 95 °C enhances its affinity for mucin, and that the adhesion mechanism relies on hydrophobic interactions with no obvious contributions of disulfide bonds. Lactoferrin and its resulting complexes with mucin present high surface activity, which induces an artificial shear-thinning rheological response. While electrostatic interactions have been considered the dominant mucoadhesive mechanism of native lactoferrin up to now, our findings highlight the role of hydrophobic interactions, providing a design route to alter the structural state of the protein to inspire the development of future natural protein-based mucoadhesive systems.

Almost four decades after the identification of the AKT protein and understanding of its role in cancer, barriers remain in the translation of AKT inhibitors for clinical applications. Here, we provide new molecular insight into the first step of AKT activation where AKT binds to the plasma membrane and its orientation is stabilized in a bilayer with lateral heterogeneity (Lo-Ld phase coexistence). We have applied molecular dynamic simulations and molecular and cell biology approaches, and demonstrate that AKT recruitment to the membrane requires a second binding site in the AKT pleckstrin homology (PH) domain that acts cooperatively with the known canonical binding site. Given the precision with which we have identified the protein-lipid interactions, the study offers new directions for AKT-targeted therapy and for testing small molecules to target these specific amino acid-PIP molecular bonds.

The membrane-disrupting peptide Mastoparan-1 (MP-1), derived from the wasp Polybia paulista is known to possess antimicrobial properties, and exhibits enhanced activity against a number of cancer cell lines relative to healthy cells. Due to the mechanism of action of MP-1 it is likely that differences in plasma membrane lipid composition arising from cancer associated mutations, such as localisation of phosphatidylserine (PS) lipids to the outer leaflet of the plasma membrane, are involved in driving that enhanced activity. Rapid screening of MP-1 mutants in a combined approach using model membrane and cell-based biological assays, has led to the identification of a number of derivative peptides with enhanced selectivity for cancer-like membrane models and breast cancer cell lines and provided insights into the mechanism of membrane disruption and cell death. Notably, the morphology of the membrane perturbations observed by Atomic Force Microscopy (AFM) and activity in cell model systems can change considerably in response to single-point mutations in the MP-1 sequence, indicating a complex structure-activity relationship. ### Competing Interest Statement The authors have declared no competing interest. Engineering and Physical Sciences Research Council, https://ror.org/0439y7842, EP/R03608X/1

ABSTRACT It is important to understand the behaviours of fluorescent molecules because, firstly, they are often utilized as probes in biophysical experiments and, secondly, they are crucial cofactors in biological processes such as photosynthesis. A phenomenon called ‘fluorescence quenching’ occurs when fluorophores are present at high concentrations but the mechanisms for quenching are debated. Here, we used a technique called ‘in-membrane electrophoresis’ to generate concentration gradients of fluorophores within a supported lipid bilayer (SLB), across which quenching was expected to occur. Fluorescence lifetime imaging microscopy (FLIM) provides images where the fluorescence intensity in each pixel is correlated to fluorescence lifetime: the intensity provides information about the location and concentration of fluorophores and the lifetime reveals the occurrence of energy-dissipative processes. FLIM was used to compare the quenching behaviour of three commonly-used fluorophores: Texas Red (TR), nitrobenzoaxadiazole (NBD) and 4,4-difluoro-4-bora-3a,4a-diaza- s -indacene (BODIPY). FLIM images provided evidence of quenching in regions where the fluorophores accumulated but the degree of quenching varied between the different fluorophores. The relationship between quenching and concentration was quantified and the ‘critical radius for trap formation’, representing the relative quenching strength, was calculated as 2.70, 2.02 and 1.14 nm, for BODIPY, TR and NBD, respectively. The experimental data supports the theory that quenching takes place via a ‘transfer-to-trap’ mechanism which proposes, firstly, that excitation energy is transferred between fluorophores and may reach a ‘trap site’ resulting in immediate energy dissipation and, secondly, that trap sites are formed in a concentration-dependent manner. Some previous work suggested that quenching occurs only when fluorophores aggregate, or form long-lived dimers, but our data and this theory argues that traps may be ‘statistical pairs’ of fluorophores that exist only transiently. Our findings should inspire future work to assess whether these traps can be charge-transfer states, excited state dimers or something else.

Waxes comprise a diverse set of materials from lubricants and coatings to biological materials such as the intracuticular wax layers on plant leaves that restrict water loss to inhibit dehydration. Despite the often mixed hydrocarbon chain lengths and functional groups within waxes, they show a propensity for ordering into crystalline phases, albeit with a wealth of solid solution behavior and disorder modes that determine chemical transport and mechanical properties. Here, we reveal the microscopic structure and heterogeneity of replica leaf wax models based on the dominant wax types in the Schefflera elegantissima plant, namely C31H64 and C30H61OH and their binary mixtures. We observe defined grain microstructure in C31H64 crystals and nanoscale domains of chain-ordered lamellae within these grains. Moreover, nematic phases and dynamical disorder co-exist with the domains of ordered lamellae. C30H61OH exhibits more disordered chain packing with no grain structure or lamellar domains. Binary mixtures from 0-50% C30H61OH exhibit a loss of grain structure with increasing alcohol content accompanied by increasingly nematic rather than lamellar chain packing, suggesting a partial but limited solid solution behavior. Together, these results unveil the previously unseen microstructural features governing flexibility and permeability in leaf waxes and outline an approach to microstructure analysis across agrochemicals, pharmaceuticals, and food.

Fibrin has unique biomechanical properties which are essential for its role as a scaffold for blood clots. Fibrin is highly extensible and demonstrates significant strain stiffening behaviour, which is essential for stress-distribution in the network. Yet the exact structures of fibrin at the sub-fibre level that contribute to its unique biomechanical characteristic are unknown. Here we show how truncations of the fibrinogen αC-region impact the biomechanical properties of fibrin fibres. Surprisingly, absence of the complete αC-region did not influence the low strain modulus of fibrin fibres but led to premature fibre rupture and decreased extensibility. Intermediate effects were observed with partial deletion of the αC-region, reflected by intermediate rupture stress and toughness. However, overall strain-stiffening behaviour remained even in absence of the αC-region, indicating that strain stiffening is not due to stress being transferred from the αC-region to the protofibril backbone. Upon stress-relaxation, decay constants and their relative contribution to the total relaxation remained similar at all strains, showing that a distinct relaxation process is present until fibre rupture. However, relative contribution of fast relaxation was maximal only in crosslinked fibres if the flexible αC-connector was present. These data show that the αC-region is not the main load-bearing structure within fibrin fibres and point to a critical role for the protofibril backbone instead. We present a revised structural model based on protofibril branching that fully explains the unique biomechanical behaviour of fibrin fibres, while the αC-region primarily acts as a safety latch at the highest of strains. Statement of significance The findings presented in this paper reveal critically important details about how the molecular structure of fibrin contributes to its unique mechanical properties which are essential to fulfil its function as the scaffold of blood clots. In this work we used engineered proteins with alterations in an important but highly disordered area of the molecule called αC-region and we provide direct evidence for the first time for how the absence of either the globular αC-domain, or the complete αC-region impacts the mechanical behaviour of individual fibrin fibres. Using these results we developed a new structural model of protofibril organisation within fibrin fibres that fully explains their strain stiffening, relatively low modulus and their high, largely variable, extensibility.

Concentrated Fusarium venenatum biomass commonly known as mycoprotein (MYC), naturally rich in high quality proteins, has been commercially used to fabricate vegan meat analogues by incorporating potato protein (PoP). In this work, we studied the effect of multivalent cations, focusing particularly on essential micronutrients such as calcium (0-100 mM) and ferric ions (0-1.0 mM), on the microstructure of the fungal hyphae (MYC)-PoP composites, as characterized via rheology and microscopic techniques as a function of pH (3.0-7.0), Na+ (0-200 mM) with and without added PoP. A clear dependency of storage modulus (G ') on pH, ionic strength and specific concentrations of Ca2+ and Fe3+ was observed, with Fe3+, PoP concentration and acidic pH having the largest impact. Microscopy across various length scales revealed that PoP coats the hyphae's rough surface and dominates the interactions between the hyphae. G ' responsiveness to NaCl and CaCl2 concentrations indicated that electrostatic interactions between the fungal hyphae and PoP coverage mainly govern texture properties. Interestingly, Fe3+ induced protein-protein aggregation which led to a reduction in G ' of MYC without PoP. However, the effect of Fe3+ was modulated by Ca2+, where increasing concentration of both salts seemed to narrow the rheological (G ') differences between MYC with and without PoP at higher pH. Fe3+ alone significantly increased the G ' of MYC-PoP except at pH 5.0. Thus, a subtle balancing of pH and added levels of calcium is needed to enable iron supplementation with minimal textural effects when formulating mycoprotein-based meat analogues with PoP as protein binding agent.

This study aims to understand the bulk and interfacial performance of potato protein microgels. Potato protein (PoP) was used to produce microgels of submicrometer diameter via a top-down approach of thermal cross-linking followed by high-shear homogenization of the bulk gel. Bulk "parent" gels were formed at protein concentrations [PoP] = 5-18 wt %, which subsequently varied in their bulk shear elastic modulus (G') by several orders of magnitude (1-100 kPa), G' increasing with increasing [PoP]. The PoP microgels (PoPM) formed from these parent gels had diameters varying between 100 and 300 nm (size increasing with increasing G' and [PoP]), as observed via dynamic light scattering and atomic force microscopy (AFM) of PoPM adsorbed onto silicon. Interfacial rheology (interfacial shear storage and loss moduli, Gi' and Gi″) and interfacial tension (γ) of adsorbed films of PoP (i.e., nonheated PoP) and PoPM (both at tetradecane-water interfaces) were also studied, as well as the bulk rheology of the PoPM dispersions. The results showed that PoPM dispersions (at 50 vol %) had significantly higher bulk viscosity and shear thinning properties compared to the nonmicrogelled PoP at the same overall [PoP], but the bulk rheological behavior was in sharp contrast to the interfacial rheological performance, where Gi' and Gi″ of PoP were higher than for any of the PoPM. This suggests that the deformability and size of the microgels were key in determining the interfacial rheology of the PoPM. These findings may be attributed to the limited capacity for "unfolding" and lateral interactions of the larger PoPM at the interface, which are presumed to be stiffer due to their production from the strongest PoP gels. Our study further confirmed that heating and cooling the adsorbed films of PoPM after their adsorption showed little change, highlighting that hydrogen bonding was limited between the microgel particles.

Abstract Aqueous multiphasic systems have attracted a great deal of interest recently owing to the growing demands of sustainability for the development of stable “oil‐free” emulsions, often complicated by their limited stability against droplet coarsening. Although particles may provide ultrastability to water‐in‐water (W/W) emulsions formed in phase‐separating polymer systems, the need for lubrication in such W/W emulsions presents an important challenge for their use in diverse applications. Herein, W/W Pickering emulsions were stabilized by sustainable plant protein (pea)‐based microgels (PPM) using starch and xanthan gum as the biopolymers to generate the W/W phase separating droplet structures. The lubricity of these systems was compared with that of parallel systems stabilized by animal (whey) protein microgels (WPM). New results reveal that PPM are more soft and adhesive than WPM and outperform the latter in boundary lubrication performance, in striking contrast to the behavior of the non‐microgelled pea or whey proteins. Furthermore, the PPM tend to stabilize a different, less spherical type of W/W droplet than the WPM that may explain the lower friction observed in PPM‐stabilized systems. The novel approach of fabricating W/W emulsions stabilized by sustainable microgels opens up new solutions in designing aqueous lubricants for future nutritional and biomedical applications.

Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility and interactions of biomolecules. However, fluorophores can undergo “self-quenching” of their fluorescence intensity at high concentrations. A greater understanding of concentration-quenching effects is important for avoiding artefacts in fluorescence images and is relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores within supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of Texas Red (TR) fluorophores were generated within 100 × 100 µm corral regions on glass substrates. Application of an electric field in-plane with the lipid bilayer induced the migration of negatively-charged TR molecules towards the positive electrode and created a lateral concentration gradient across each corral. The self-quenching of TR was directly observed in FLIM images as a correlation of high concentrations of fluorophores to reductions in their fluorescence lifetime. By varying the initial concentration of TR fluorophores incorporated into the SLBs from 0.3% to 0.8% (mole/mole), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7% (mole/mole), leading to maximal quenching of 30% up to 70%. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The shape of the calculated concentration profiles revealed evidence of subtle lipid-lipid interactions at high packing densities. Overall, these findings prove that electrophoresis is effective at producing microscale concentration gradients of a molecule-of-interest and that FLIM is an excellent approach to interrogate dynamic changes to molecular interactions via their photophysical state.

With the resource-intensive meat industry accounting for over 50% of food-linked emissions, plant protein consumption is an inevitable need of the hour. Despite its significance, the key barrier to adoption of plant proteins is their astringent off-sensation, typically associated with high friction and consequently poor lubrication performance. Herein, we demonstrate that by transforming plant proteins into physically cross-linked microgels, it is possible to improve their lubricity remarkably, dependent on their volume fractions, as evidenced by combining tribology using biomimetic tongue-like surface with atomic force microscopy, dynamic light scattering, rheology and adsorption measurements. Experimental findings which are fully supported by numerical modelling reveal that these non-lipidic microgels not only decrease boundary friction by an order of magnitude as compared to native protein but also replicate the lubrication performance of a 20:80 oil/water emulsion. These plant protein microgels offer a much-needed platform to design the next-generation of healthy, palatable and sustainable foods.

PDMS (polydimethylsiloxane) is a cheap, optically clear polymer that is elastic and can be easily and quickly fabricated into a wide array of microscale and nanoscale architectures, making it a versatile substrate for biophysical experiments on cell membranes. It is easy to imagine many new experiments will be devised that require a bilayer to be placed upon a substrate that is flexible or easily cast into a desired geometry, such as in lab-on-a-chip, organ-on-chip, and microfluidic applications, or for building accurate membrane models that replicate the surface structure and elasticity of the cytoskeleton. However, PDMS has its limitations, and the extent to which the behavior of membranes is affected on PDMS has not been fully explored. We use AFM and fluorescence optical microscopy to investigate the use of PDMS as a substrate for the formation and study of supported lipid bilayers (SLBs). Lipid bilayers form on plasma-treated PDMS and show free diffusion and normal phase transitions, confirming its suitability as a model bilayer substrate. However, lipid-phase separation on PDMS is severely restricted due to the pinning of domains to surface roughness, resulting in the cessation of lateral hydrodynamic flow. We show the high-resolution porous structure of PDMS and the extreme smoothing effect of oxygen plasma treatment used to hydrophilize the surface, but this is not flat enough to allow domain formation. We also observe bilayer degradation over hour timescales, which correlates with the known hydrophobic recovery of PDMS, and establish a critical water contact angle of 30°, above which bilayers degrade or not form at all. Care must be taken as incomplete surface oxidation and hydrophobic recovery result in optically invisible membrane disruption, which will also be transparent to fluorescence microscopy and lipid diffusion measurements in the early stages.

This study investigated the microstructure and interactions of heat treated concentrated Fusarium venenatum biomass, commonly referred to as 'mycoprotein' (MYC), as well as the impact of 3 wt % egg white protein (EWP) on those interactions - EWP being frequently added as a 'binding agent'. Confocal laser scanning microscopy (CLSM) and cryo-scanning electron microscopy (Cryo-SEM) combined with energy dispersive spectroscopy analysis (EDS) demonstrated the filamentous nature of the MYC, made up of chitin and protein, with a filament aspect ratio typically between 130 and 140. Protein extraction and analysis via SDS-PAGE electrophoresis suggested that the most abundant native protein in the MYC had a molecular weight of 69.2 kDa, probably belonging to the heat shock 70 (HSP70) group. Frequency sweep dynamic shear rheology measurements were performed at combinations of 3 different values of pH (3, 5 and 7), NaCl concentration ([NaCl] = 0, 100 and 200 mM), CaCl2 concentration ([CaCl2] = 0, 50 and 100 mM) and wt. % MYC solids (5, 10, 15 and 20) without added EWP and with added EWP (MYC-EWP). For MYC the storage modulus G ' was seen to increase strongly with increasing wt. %MYC solids but also with [NaCl] and [CaCl2], the latter having greater impact at pH 5 and 7. The opposite trends were observed at pH 3, where G ' decreased with increasing concentration of ions, the highest G ' values for MYC occurring at pH 3 with no added salts. This suggests that electrostatic interactions between the MYC elements are key to the overall texture. On addition of EWP, there was good evidence that EWP completely coats the filaments so that, the hyphal interactions of MYC-EWP are then dominated by the response of EWP to changes in pH and salt composition. Gaining further understanding of the strength and distribution of the loci of these interactions is therefore key to better control of the texture of such meat analogues.