Chocolate is a complex colloidal system of multiple dispersed particle types, including sugar, cocoa solids and often dairy proteins. However, the use of plant proteins in such systems remains unexplored. This study investigates the behaviour of pea protein isolate (PPI), sugar particles and their blends in oil, focussing on their effects on the rheological and crystallisation properties of cocoa butter (CB). Both PPI and sugar particles aggregated in CB, increasing viscosity and forming elastic oleogel-like systems. In blended systems, PPI disrupted sugar particle-particle interactions. Greater sugar particle aggregation was observed in sunflower oil, attributed to surface-active impurities in CB acting as dispersants for hydrophilic particles. Oil type had little influence on PPI aggregation and the modified MPQ-rcp model indicated that PPI and sugar particles exhibit similar interactions in CB. PPI accelerated CB crystallisation, whereas the presence of sugar delayed crystallisation, so that mixed particle systems had similar kinetics to pure CB. Over extended storage, concentrated particle suspensions curb the solid fat content (SFC), attributed to the restricted mobility of oil due to the high dispersed volume fractions-as visualised by Raman microscopy. At low SFC, particles are active in the fat crystal network, increasing elasticity. However, at high SFC, the elasticities were comparable to that of pure CB. This demonstrates the potential of plant protein particles to provide rheology modification benefits to sugar-oil mixtures and to reduce solid fat content, providing a foundation for the development of more sustainable and nutritious confectionery systems.

This study investigated the tribological performance of Pickering water-in-oil emulsions and dual Pickering water-in-oil-in-water double emulsions (DEs) stabilized with particles at both the interfaces. W/O emulsions were stabilized by cocoa butter-based oleogel (CBolg) crystals, while DEs incorporating these emulsions were stabilized by whey protein microgels (WPM). The influence of temperature (21 and 37 degrees C) and surface texture (smooth vs biomimetic tongue-like surface) were investigated in tribology of W/O emulsions (30-60 % v/v water) and DEs (with 20 and 60 wt% W/O phase). In smooth surfaces, CBolg played a critical role in reducing the friction coefficient (mu) primarily via a fat-driven lubrication mechanism that was temperature dependent. While in DEs, smaller oil droplets encapsulating water provided similar lubrication to oil-based systems until starvation occurred. Strikingly, the water content in W/O emulsions exhibited distinct differences between emulsion systems within the biomimetic tongue-like surfaces, demonstrating lower lubricity at higher water concentration. Confocal microscopy images analyzed using Machine Learning (ML)-supported droplet segmentation enabled a more precise evaluation of structural changes within DEs when subjected to tribological stress. We demonstrated that although changes in inner droplet size altered in DEs, their contribution to the overall lubrication performance was minimal, due to their limited entrainment. Of more importance, the tribological performance was governed by the WPM with minimal influence from the droplet-entrained phenomena. These fundamental insights highlight the relevance of structured water in understanding frictional performance in emulsified systems, with structural integrity, composition, and topography of the tribological surface emerging as key factors.

Abstract N-Alkyl-D-galactonamides are amphiphilic galactose derivatives that form supramolecular hydrogels through self-assembly. To enable the processing of these hydrogels at low temperatures in water only, we investigated the reversible complexation of N-heptyl and N-nonyl-D-galactonamides with molybdenum. These molecules readily react with ammonium molybdate to form well-defined dinuclear molybdenum complexes in water. Acidification of the complex solution triggers the formation of transparent, reversible, and thixotropic supramolecular gels, which are markedly distinct from the hydrogels derived from the parent N-alkyl-d-galactonamides. The gelation process is reversible and arises from the self-assembly of N-alkyl-d-galactonamide-molybdenum complexes, driven by the segregation of fatty chains in an aqueous environment and the neutralization of molybdate charges at low pH. In addition, at low pH, polyoxometalates are known to form large aggregates that can also play a role in the self-assembly. The complexes and resulting gels were characterized using 1H, 13C, and 95Mo NMR spectroscopy, mass spectrometry, tensiometry, rheology, small-angle X-ray scattering, transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The N-nonyl-D-galactonamide complex self-organizes into core–shell cylinders that self-assemble further in hexagonal packing, while the N-heptyl-D-galactonamide complex self-organizes in ellipsoidal flat bicelles. In both cases, this organization yields highly ordered molybdate arrays with regular spacings of 3.9 or 2.8 nm, respectively. An alternative gelation method via wet-spinning is also demonstrated. Injection of the complex solution into an acidic bath produces gel membranes and threads. The unique properties of these metallogels, namely, organization of molybdate arrays, in 2D or in 3D, transparency, reversibility, and thixotropy, open promising avenues for shaping composite materials in electrochemistry, electronics, and catalysis.

The aim of this study was to understand how beeswax can be used to stabilize water-in-oil emulsions and examine the impact of droplet volume fraction on emulsion stability. Pickering water-in-oil (W/O) emulsions were successfully designed using beeswax oleogels (4 wt%) as the sole stabilizer via a facile homogenization approach. The effects of beeswax crystals on the formation, stability, and structural organization of W/O emulsions (containing up to 70 % v/v water) were systematically investigated. Oleogelation reduced the interfacial tension between oil and water from 21 +/- 1 mN/m to 14 +/- 1 mN/m, enabling beeswax crystals to stabilize emulsions without the need for any synthetic surfactant. X-ray diffraction analysis revealed that beeswax crystals in oleogels exhibited a stable beta' polymorph with orthorhombic packing, maintaining a reduced long-chain spacing from 7.8 nm in pure beeswax to 6.3 nm, which contributed to the stabilization of Pickering W/O emulsions. The emulsions ranged in size (D32) from 8 to 16 mu m depending upon the droplet volume fraction and showed no significant change over storage period of 6 weeks. Microscopic observations of the interface demonstrated that emulsion stability was achieved through the synergistic effects of both the Pickering stabilization and bulk network stabilization. This dual stabilization mechanism was further confirmed by thermal cycling experiments and in situ crystallization analysis at the interface as well as rheological measurements showing gel formation at higher volume fractions of droplets. Overall, these findings highlight the potential of beeswax to stabilize W/O emulsions for applications in food and allied soft matter industries.

This contribution reviews the recrystallization of high-melting components in PO (PO)-based fat blends monitored by differential scanning calorimetry (DSC) and time- and temperature-resolved small- and wide-angle X-ray scattering (SAXS/WAXS) in four different case studies. Different temperature-time profiles, including annealing, were applied to investigate the crystallization kinetics with focus on mixed crystal formation (co-crystallization) and its potential for being controlled. The main focus was hereby to capture phase segregation events during melting. These segregation effects are caused by true recrystallization from either the melt or melt-mediated polymorphic transition of the tripalmitin (PPP)-rich high-melting fraction. The first case study illustrates the formation of mixed crystals by step-wise crystallization and evaluation of the subsequent melting DSC curve. By applying low heating rates, the evolution of the observed melting points combined with information on lamellar spacings revealed a time-dependent formation of the medium-melting fraction. The second case study discusses the effects and interplay of heating rate, crystallization temperature, and partial melting on the phase segregation in PO crystallization. The third case study focuses on PPP as driver for phase segregation in a palm-based commercial hardstock (PCM). Fourth, the same PCM was subjected to different crystallization routines including annealing to modify the mixed crystals formed. Deduced from SAXS/WAXS observations, annealing during the cooling step proved greatly aiding the formation of a mixed crystal, which would withstand phase segregation of PPP during the melting.

Alpha olefin sulfonate (AOS) is a widely used anionic surfactant, yet its phase behaviour has not previously been systematically mapped. Here, a comprehensive phase diagram is constructed over a concentration range of 2-70 wt% and temperatures from 25 to 80 °C using cross-polarized optical microscopy and small-angle X-ray scattering (SAXS). Polarized microscopy was used to delineate isotropic and anisotropic regions and identify phase boundaries, while SAXS provided quantitative structural characterisation, including lattice parameters, electron density reconstructions, and micellar size and shape analysis. Measurements were performed in sealed capillaries under controlled thermal conditions, with refined temperature increments used to resolve phase transitions. Five distinct phases were identified with decreasing water content: a micellar dispersion; a 2D hexagonal phase; a 2D ribbon phase; a previously unreported 3D hexagonal phase; and two lamellar phases. This temperature- and concentration-dependent sequence is structurally analogous to that of sodium dodecyl sulfate (SDS), though key distinctions arise from the molecular architecture of AOS, which promotes unique intermediate mesophases through differences in molecular packing and constrained hydration. Structural analysis reveals a systematic evolution of spatial water organisation, progressing from excess bulk water in the micellar phase, to tubular confinement within hexagonal and ribbon phases, to discrete water pockets embedded within surfactant bilayers in the 3D hexagonal phase, and finally to residual interfacial hydration in the lamellar phases. This work establishes the relationship between hydration state and mesophase structure, and provides the first complete structural phase diagram for this important surfactant system, enabling prediction of phase behaviour under the complex conditions encountered in industrial processing.

Particles with some degree of hydrophilicity are known to aggregate when directly dispersed in non-aqueous media. Proteins are generally insoluble in oil and have complex surface properties, but they may form networks in oil like more simple colloidal particles, depending on particle size and surface hydrophilicity. Here, the particle size of pea protein isolate (PPI) particles in oil was reduced to submicron sizes by stirred media milling. The rheology of milled PPI oil suspensions was compared to dispersions prepared with two types of colloidal silica particles - hydrophobic and hydrophilic. PPI particles form structured aggregates in oil which break down under shear that, similarly to silica, can form an elastic network like an oleogel system. As PPI size decreased, aggregation increased, shown by higher apparent viscosities and gel strength. PPI particles with an average size of 1 μm exhibited elastic behaviour (G' > G'') at 11.2 wt%. Rheological scaling models obtained two fractal dimensions: a higher intra-floc dimension and a lower network backbone dimension, suggesting that colloidal PPI and silica particles have an inhomogeneous microstructure with denser particle flocs compared to a relatively sparse backbone. For smaller PPI particles the inter- and intra-floc fractal dimensions become like that of hydrophobic silica, suggesting that the average 'surface' character of the PPI may be close to that of the silica. Therefore despite the complexity of the protein surface, parallels can be drawn with simpler colloidal systems. Pre-wetting the particles with ethanol tuned this behaviour, highlighting the role of surface chemistry in gel formation.

This contribution provides a comprehensive review of nanostructural aspects in pure triglyceride compared with more complex, real fat systems. Alongside this journey on understanding the crystallization of triglycerides, we provide a practical guide on using X-ray scattering to its fullest extent. Data from public-domain literature of monoacid and mixed-acid saturated and mono-unsaturated triglycerides are reviewed in great depth. On the nanoscale, lamellar stacking and hydrocarbon tilt angles are discussed, and on the molecular scale, trends in the chain packing density and concomitant geometry changes are explained. Useful tools to evaluate lab-scale and synchrotron X-ray scattering data are presented, including (i) electron density profile calculations, decomposing the lamellar repeat distance into the bilayer and monolayer contributions, (ii) refined estimations of the chain tilt angle in the bilayer region, (iii) crystallite size and strain investigations, and (iv) the determination of the area per hydrocarbon chain. Further, the review summarizes representative crystallographic data over the last decades, including extensive lists of long- and short-spacing data. Generalizing the introduced model-free methods to real fat systems makes it possible to also analyze complex phase transitions and their solid fat content as derived from the wide-angle scattering. The main aim of this contribution is to provide a robust set of X-ray scattering methods for a detailed evaluation of polymorphic states and phase transitions supporting research efforts at the interface of academia and industry.

Hybrid membranes, consisting of phospholipids and amphiphilic block polymers, offer enhanced stability compared to liposomes and greater biocompatibility than polymersomes. These qualities make them a versatile platform for a wide range of applications across various fields. In this study, we have investigated the ability of solid-supported polymer-lipid hybrid membranes (SSHM) to act as a platform for bioelectrochemistry of membrane proteins. The redox enzyme, cytochrome bo 3 (cyt bo 3 ), a terminal oxidase in Escherichia coli, was reconstituted into hybrid vesicles (HVs), which were subsequently tested for their ability to form SSHMs on different self-assembled monolayers (SAMs) on gold electrodes. SSHM formation was monitored with electrochemical impedance spectroscopy (EIS), quartz crystal microbalance with dissipation (QCM-D), and atomic force microscopy (AFM). SSHMs were successfully formed on gold electrodes with mixed SAMs of 6-mercapto-1-hexanol and 1-hexanethiol at a 1 : 1 ratio. The activity of cyt bo 3 was confirmed using cyclic voltammetry (CV), with electron transfer to cyt bo 3 mediated by a lipophilic substrate-analogue decylubiquinone (DQ). SSHMs formed with HVs-cyt bo 3 samples, stored for more than one year before use, remain bioelectrocatalytically active, confirming our previously established longevity and stability of HV systems.

In this contribution, the crystallization kinetics of model fat blends containing trisaturated and mono-unsaturated TAGs of different saturated fatty acid composition are investigated via differential scanning calorimetry (DSC) and time-resolved small and wide angle X-ray scattering (SAXS/WAXS). The effect of inhomogeneity in saturated fatty acid composition was studied by varying between palmitic and stearic sources for trisaturated (POSt - palm stearin and FHRO - fully hydrogenated rapeseed oil) and using palm mid fraction (PMF) and shea stearin (ShSt) sources for mimicking the mono-unsaturated TAGs (PMF - palm mid fraction and ShSt - shea stearin). The kinetics were followed via SAXS/WAXS upon cooling at different rates (1 and 10 °C min-1) and during the isothermal hold at 0 °C (273 K) for 60 min. The blends revealed a multiple step crystallization with co-existing polymorphic forms and a slow restructuring process. Generally, mono-unsaturated triglycerides tended to impede the polymorphic transition of the trisaturated fraction. The trisaturated triglyceride fraction formed an α phase at the beginning of the crystallization. Quickly after, co-existing β' and β phases were recorded. In each case, the subsequent crystallization events were dictated by the mono-unsaturated fraction with ShSt affecting the crystallization more drastically than PMF. Differences were diminished when a low cooling rate was applied. Further, the low cooling rate allowed the formation of a β phase in FHRO-containing samples before an additional β' phase was recorded. The differences in explicit fatty acid composition, TAG diversity and minor components are discussed as primary potential sources of differences in crystallization behavior. Calorimetric experiments capturing the crystallization and melting behavior of the industrial grade binary blends were compared to that of blends based on pure TAGs diluted in rapeseed oil, namely the combinations PPP-POP, SSS-SOS, PPP-SOS, and SSS-POP.

The effects of pea protein isolate (PPI) particles on the crystallisation and rheological properties of cocoa butter (CB) were evaluated. PPI particles were milled to produce two size classes, coarse and fine. Milled PPI particles were found to aggregate in molten CB, increasing the viscosity of the system and forming an elastic network. Greater aggregation was observed as the size of PPI particles was reduced. PPI particles had no effect on the crystal structure of CB, or the polymorphic transition pathway. However, the induction time of crystallisation was shorter, and the crystalline domain size in the alpha-phase was smaller for CB crystals + PPI, indicating heterogeneous nucleation effects. PPI particles increased the elasticity of the system by an order of magnitude only during the early stages of CB crystallisation. This study demonstrates that PPI particles can be incorporated into fat-based systems without disrupting fat crystal structure, with the potential to enhance mechanical strength. This highlights their potential as functional rheological modifiers upon milling in confectionery applications.

Cationic ionizable lipids (CILs) are fundamental components of inverse hexagonal (HII) lipid assemblies, which mediate the encapsulation and release of negatively charged mRNA through a pH-dependent mechanism. Since variations in the structure and composition of the HII phases can significantly impact the biological efficacy of the mRNA-carrying lipid nanoparticles (LNP), a comprehensive understanding of the ionizable lipid HII phases is necessary. We present an integrated approach combining small-angle X-ray scattering (SAXS) experiments, molecular dynamics (MD) simulations and a continuum model to elucidate lipid distribution and water content within HII phases. Our results indicate strong agreement between structures derived from MD simulations and SAXS data. To this end, we introduce a method to correct for periodic boundary artifacts when computing scattering profiles from MD simulations. This enables direct, model-free comparisons between experimental and simulated data, enhancing the reliability of structural interpretations, specifically the water content of the HII phases. Next, we developed a continuum model to extend structural analysis to CIL HII phases for which MD data is unavailable. This integrative framework not only provides molecular-level insights into the ionizable lipid HII mesophase but also enables the prediction of hydration properties across different CIL compositions. The different approaches consistently yield water contents that seem to correlate with the lipids' transfection efficiencies. By bridging experimental and simulation data, our approach offers a powerful tool for the rational design and optimization of lipid nanoparticles, potentially linking a lower water content with an increased therapeutic performance.

The aim of this study was to create stable, dual Pickering -stabilized double emulsions (DEs) where both the internal and external droplets are stabilized by food grade particles. Water -in -oil (W/O) emulsions were stabilized by using cocoa butter -high oleic sunflower oil based oleogels (CBolg) and then W/O emulsion was used as the dispersed phase in water -in -oil -in -water (W/O/W) emulsions, latter stabilized by whey protein microgels (WPM). The capability of the CBolg (10 v/v% cocoa butter) to form W/O and subsequently a W/O/W emulsion was characterized using cross -polarized light microscopy, cryogenic scanning electron microscopy (cryo-SEM), confocal light microscopy (CLSM) and static light scattering. In addition, the characterization of the WPM 's internal structure was evaluated by small angle X-ray scattering (SAXS). Results revealed dual Pickering stabilized W/O/W emulsions with a droplet size of 10 mu m could be formed using whey protein microgel (WPM, 3 -5 v/ v%) as the Pickering stabilizer. Dynamic light scattering (DLS) analysis revealed WPM particles size averaging approximately 100 nm, with internal structures consisting of partially stretched protein strands with smaller compact subunits along the strand (partially folded proteins) was confirmed by SAXS analysis. The dual Pickering -stabilized DE droplets were stable over a month by formation a protective layer of WPM around the oil droplets, as observed in CLSM images, unlike the systems where the external phase was stabilized by nonmicrogelled whey protein, latter destabilized within hours. Such dual Pickering -stabilized DEs may offer new templates for designing healthy low -fat foods and for co -delivery of nutrients and active in multiple compartments.

Periodontal disease is triggered by surface bacterial biofilms where bacteria are less susceptible to antibiotic treatment. The development of liposome-based delivery mechanisms for the therapeutic use of antimicrobial peptides is an attractive alternative in this regard. The cationic antimicrobial peptide LL-37 (human cathelicidin) is well-known to exert antibacterial activity against Porphyromonas gingivalis, a keystone oral pathogen. However, the antibacterial activity of the 16-amino acid fragment (LL17-32) of LL-37, is unknown. In addition, there are still gaps in studies using liposomal formulations as delivery vehicles of antibacterial peptides against this pathogen. This study was designed to examine the influence of the different types of liposomal formulations to associate and deliver LL17-32 to act against P. gingivalis. Chitosans of varying Mw and degree of acetylation (DA) were adsorbed at the surface of soya lecithin (SL) liposomes. Their bulk (average hydrodynamic size, ζ-potential and membrane fluidity) and ultrastructural (d-spacing, half-bilayer thickness and the water layer thickness) biophysical properties were investigated by a panel of techniques (DLS, SAXS, M3-PALS, fluorescence spectroscopy and TEM imaging). Their association efficiency, in vitro release, stability, and efficacy in killing the periodontal pathogen P. gingivalis were also investigated. All liposomal systems possessed spherical morphologies and good shelf-life stabilities. Under physiological conditions, chitosan formulations with a high DA demonstrated enhanced stability in comparison to low DA-chitosan formulations. Chitosans and LL17-32 both decreased SL-liposomal membrane fluidity. LL17-32 exhibited a high degree of association with SL-liposomes without in vitro release. In biological studies, free LL17-32 or chitosans alone, demonstrated microbicidal activity against P. gingivalis, however this was attenuated when LL17-32 was loaded onto the SL-liposome delivery system, presumably due to the restrained release of the peptide. A property that could be harnessed in future studies (e.g., oral mucoadhesive slow-release formulations).

Time-resolved small- and wide-angle X-ray scattering (SAXS/WAXS), differential scanning calorimetry (DSC), and small deformation oscillation were employed to investigate the crystallization kinetics of fat blends containing monoacid saturated triglycerides (H3) and a mixture of H3 and mixed acid saturated triglycerides (H2M, where H denotes long-chain saturated fatty acid and M denotes a medium-chain saturated fatty acid). For the H3 system, the time-resolved DSC signal revealed a two-step crystallization process aligned with the kinetic pathway identified via SAXS/WAXS. H3 first crystallizes in alpha form and quickly transitions into the beta polymorphic form. The rheological data on the complex modulus, due to the speed of the polymorphic transition, do not allow us to distinguish the two crystallization steps clearly. However, the alpha-beta transition complies well with literature data on monoacid saturated TAGs. The H3 + H2M system showed a two-step process in DSC and complex modulus, which could be associated with the polymorphic transition from the alpha to beta ' crystals. No further transition into the beta polymorph or segregation of the H3 fraction was detected, indicating the dominant role of H2M triglycerides. The SAXS data on the system suggest that H3 and H2M triglycerides formed a single solid phase, which is not supported by the DSC melting profile. The variation of cooling rate (5 vs 10 degree celsius/min) established minor differences in crystallization kinetics with their cause yet to be explored in greater detail. This study has generated valuable new insights concerning the polymorphic transition (alpha-beta ' and alpha-beta) in systems forming mixed crystals of monoacid and saturated mixed-acid TAGs using established methods and correlating them to blends of defined TAG group composition for the first time.

Lipid nanoparticles have important applications as biomedical delivery platforms and broader engineering biology applications in artificial cell technologies. These emerging technologies often require changes in the shape and topology of biological or biomimetic membranes. Here we show that topologically-active lyotropic liquid crystal nanoparticles (LCNPs) can trigger such transformations in the membranes of giant unilamellar vesicles (GUVs). Monoolein (MO) LCNPs, cubosomes with an internal nanostructure of space group I m 3 m ${Im3m}$ incorporate into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) GUVs creating excess membrane area with stored curvature stress. Using time-resolved fluorescence confocal and lattice light sheet microscopy, we observe and characterise various life-like dynamic events in these GUVs, including growth, division, tubulation, membrane budding and fusion. Our results shed new light on the interactions of LCNPs with bilayer lipid membranes, providing insights relevant to how these nanoparticles might interact with cellular membranes during drug delivery and highlighting their potential as minimal triggers of topological transitions in artificial cells.

The effect of composition on the polymorphic crystallization in blends of fully saturated and monounsaturated triglycerides was investigated (H3 denotes fully saturated, and H2U denotes monounsaturated triglycerides). Fully hydrogenated rapeseed oil (FHRO) and palm oil stearin (POSt) were used as H3 sources. Palm oil (PO) served as an H2U source. Different H3/H2U ratios and cooling rates (1, 5, and 10 degrees C min-1) were investigated. Differential scanning calorimetry, time-resolved small- and wide-angle X-ray scattering, and oscillatory shear experiments were employed to investigate the melting behavior as well as the nano- and microstructural development. The data reveal different kinetic pathways, even selective cocrystallization, depending on the molecular makeup of the H3 fraction. The POSt-PO blends first crystallize in an alpha phase that transitions into coexisting beta ' and beta phases. The FHRO-PO blends showed a longer persisting alpha phase that transitions into beta even before a beta ' phase could be identified. This indicated separate crystallization of H3 triglycerides. The polymorphic pathways are compared to the melting behavior and microstructural development during crystallization. Further, the question of mixed crystal formation in the H3 fraction of the FHRO-PO blends was addressed by studying H3 blend replicates in the absence of H2U triglycerides. This paper is the second part of a series investigating the interplay of polymorphic transition and mixed crystal formation.

Packing stress in the lipidic inverse hexagonal HII phase arises from the necessity of the ideally cylinder-shaped micelles to fill out the hexagonally-shaped Wigner-Seitz unit cell. Thus, hydrocarbon chains stretch towards the corners and compress in the direction of the flat side of the hexagonal unit cell. Additionally, the lipid/water interface deviates from being perfectly circular. To study this packing frustration in greater detail, we have doped 1-palmitoyl-2-oleoyl-sn-phosphatidylethanolamine (POPE) with increasing molar concentrations of 1,2-palmitoyl-sn-phosphatidylethanolamine (DPPE: 0 to 15 mol%). Due to its effectively longer hydrophobic tails, DPPE tends to aggregate in the corner regions of the unit cell, and thus, increases the circularity of the lipid/water interface. From small angle X-ray diffraction (SAXD) we determined electron density maps. Using those, we analysed the size, shape and homogeneity of the lipid/water interface as well as that of the methyl trough region. At 6 and 9 mol% DPPE the nanotubular water core most closely resembles a circle; further to this, in comparison to its neighbouring concentrations, the 9 mol% DPPE sample has the smallest water core area and smallest number of lipids per circumference, best alleviating the packing stress. Finally, a three-water layer model was applied, discerning headgroup, perturbed and free water, demonstrating that the hexagonal phase is most stable in the direction of the flat faces (compression zones) and least stable towards the vertices of the unit cell (decompression zones).

cocoa butter, crystallization, polymorphism, small angle X-ray scattering Chocolate is a popular product and its supply is increasing, with sales in the UK estimated to be £6.7 billion in 2020, a £200 million increase from 2019.In recent years, confectionary companies have put increasing effort in developing novel recipes to improve the nutritional profile of chocolate and to counteract the increasing price of cocoa butter and address sustainability issues related to some chocolate ingredients.One of this these strategies is the use of cocoa butter equivalents (CBE), which are mixtures of triglycerides from multiple sources (e.g., sunflower oil, mango kernel, sal) that resemble cocoa butter in both physical and chemical properties.Despite being widely used, the crystallization behaviour of many CBEs is still poorly understood.The aim of this work was to develop a fundamental understanding, at the molecular level, of the crystallization behaviour of selected CBEs, and compare it with that of cocoa butter.In order to do so, chromatography was used to determine the composition of CBEs, in terms of fatty acids and triglycerides while the thermodynamic and kinetics of crystallization were studied using polarized microscopy, differential calorimetry and several, unique synchrotron X-ray scattering setups (the multi-capillary holder at Diamond Light Source in the United Kingdom, the combined DSC/SAXS at Elettra Sincrotrone Trieste in Italy and the combined SAXS/rheometry at ESRF in France).The combination of these techniques enabled the determination of crystal properties that affect the sensorial perception of chocolate: namely the type of crystals formed (e.g., polymorphism), their thermal stability and their size and shape distributions.Furthermore, the kinetics of crystallization as a function of CBE composition and the effect of shear were evaluated.The presented multi-technique investigation is the key for a rational design of new chocolate recipes and manufacturing processes.

Cocoa butter equivalents (CBE) are mixtures of triglycerides from multiple sources (e.g., sunflower oil, mango kernel and sal), which resemble cocoa butter (CB) in both physical and chemical properties. Despite being widely used to replace CB in chocolate products, the crystallization behavior of many CBEs is still poorly understood. The aim of this work was to develop a fundamental understanding, at the molecular level, of the crystallization behavior of selected CBEs, and compare it with that of CB. Chromatography was used to determine the composition of CBEs, in terms of fatty acids and triacylglycerides (TAGs), while their thermodynamic behavior and crystallization kinetics were studied using polarized microscopy, differential calorimetry and three different synchrotron X-ray scattering setups. CBEs of different origin and chemical composition (e.g., different ratios of the main CB TAGs, namely POP, SOS and POS) crystallized in different polymorphs and with different kinetics of nucleation, growth and polymorphic transformation. SOS rich CBEs presented showed more polymorphs than CB and POP rich samples; whereas, CBEs with high concentration of POP showed slow kinetic of polymorphic transformation towards the stable β(3L) form. Additionally, it was observed that the presence of small amounts (<1% w/w) of specific TAGs, such as OOO, PPP or SSS, could significantly affect the crystallization behavior of CBEs and CBs in terms of kinetics of polymorphic transformation and number of phases detected (multiple high melting β(2L) polymorphs were identified in all samples studied). Finally, it was found that, regardless of the CBE composition, the presence of shear could promote the formation of stable β polymorphs over metastable β' and γ forms, and reduced the size of the crystal agglomerates formed due to increased secondary nucleation.