Unravelling the effect of droplet size on lipid oxidation in O/W emulsions by using microfluidics.

Lipid oxidation in emulsions is hypothesised to increase with decreasing droplet size, as this increases the specific oil-water interfacial area, where lipid oxidation is expected to be initiated. In literature, however, contradictory results have been reported, which can be caused by confounding factors such as the oil droplet polydispersity and the distribution of components between the available phases. In this work, monodisperse surfactant-stabilised emulsions with highly controlled droplet sizes of 4.7, 9.1, and 26 µm were produced by microfluidic emulsification. We show that lipid oxidation increases with decreasing droplet size, which we ascribe to the increased contact area between lipids and continuous phase prooxidants. Besides, a significant amount of oxygen was consumed by oxidation of the surfactant itself (Tween 20), an effect that also increased with decreasing droplet size. These insights substantiate the importance of controlling droplet size for improving the oxidative stability of emulsions.

Response to "Liposome vesicle cannot be formed in non-aqueous phase".

In a recent letter to the editor Prof Khosravi-Darani responded to our paper ''Unravelling mechanisms of protein and lipid oxidation in mayonnaise at multiple length scales''. In our work, we observed liposomes in the continuous phase of mayonnaise. In the letter the objection was made that liposomes cannot be formed in a non-aqueous phase which, however, was not argued in our publication. As mayonnaise is an oil-in-water (O/W) emulsion and its continuous phase is aqueous, liposomes may be observed in this phase. Therefore, the objection from Prof Khosravi-Darani does not apply to our work.

Capillary Flow-MRI: Quantifying Micron-Scale Cooperativity in Complex Dispersions.

Strongly confined flow of particulate fluids is encountered in applications ranging from three-dimensional (3D) printing to the spreading of foods and cosmetics into thin layers. When flowing in constrictions with gap sizes, w, within 102 times the mean size of particles or aggregates, d, structured fluids experience enhanced bulk velocities and inhomogeneous viscosities, as a result of so-called cooperative, or nonlocal, particle interactions. Correctly predicting cooperative flow for a wide range of complex fluids requires high-resolution flow imaging modalities applicable in situ to even optically opaque fluids. To this goal, we here developed a pressure-driven high-field magnetic resonance imaging (MRI) velocimetry platform, comprising a pressure controller connected to a capillary. Wall properties and diameter could be modified respectively as hydrophobic/hydrophilic, or within w ∼ 100-540 µm. By achieving a high spatial resolution of 9 µm, flow cooperativity length scales, ξ, down to 15 µm in Carbopol with d ∼ 2 µm could be quantified by means of established physical models with an accuracy of 13%. The same approach was adopted for a heterogeneous fat crystal dispersion (FCD) with d and ξ values up to an order of magnitude higher than those for Carbopol. We found that for strongly confined flow of Carbopol in the 100 µm capillary, ξ is independent of flow conditions. For the FCD, ξ increases with gap size and applied pressures over 0.25-1 bar. In both samples, nonlocal interactions span domains up to about 5-8 particles but, at the highest confinement degree explored, ∼8% for FCD, domains of only ∼2 particles contribute to cooperative flow. The developed flow-MRI platform is easily scalable to ultrahigh field MRI conditions for chemically resolved velocimetric measurements of, e.g., complex fluids with anisotropic particles undergoing alignment. Future potential applications of the platform encompass imaging extrusion under confinement during the 3D printing of complex dispersions or in in vitro vascular and perfusion studies.

Unravelling mechanisms of protein and lipid oxidation in mayonnaise at multiple length scales.

In mayonnaise, lipid and protein oxidation are closely related and the interplay between them is critical for understanding the chemical shelf-life stability of mayonnaise. This is in particular the case for comprehending the role of low-density lipoprotein (LDL) particles acting as a main emulsifier. Here, we monitored oxidation and the concomitant aggregation of LDLs by bright-field light microscopy and cryogenic transmission electron microscopy. We further probed the formation of protein radicals and protein oxidation by imaging the accumulation of a water-soluble fluorescent spin trap and protein autofluorescence. The effect of variation of pH and addition of EDTA on the accumulation of the spin trap validated that protein radicals were induced by lipid radicals. Our data suggests two main pathways of oxidative protein radical formation in LDL particles: (1) at the droplet interface, induced by lipid free radicals formed in oil droplets, and (2) in the continuous phase induced by an independent LDL-specific mechanism.

Non-Invasive Rheo-MRI Study of Egg Yolk-Stabilized Emulsions: Yield Stress Decay and Protein Release.

A comprehensive understanding of the time-dependent flow behavior of concentrated oil-in-water emulsions is of considerable industrial importance. Along with conventional rheology measurements, localized flow and structural information are key to gaining insight into the underlying mechanisms causing time variations upon constant shear. In this work, we study the time-dependent flow behavior of concentrated egg-yolk emulsions with (MEY) or without (EY) enzymatic modification and unravel the effects caused by viscous friction during shear. We observe that prolonged shear leads to irreversible and significant loss of apparent viscosity in both emulsion formulations at a mild shear rate. The latter effect is in fact related to a yield stress decay during constant shearing experiments, as indicated by the local flow curve measurements obtained by rheo-MRI. Concurrently, two-dimensional D-T2 NMR measurements revealed a decrease in the T2 NMR relaxation time of the aqueous phase, indicating the release of surface-active proteins from the droplet interface towards the continuous water phase. The combination of an increase in droplet diameter and the concomitant loss of proteins aggregates from the droplet interface leads to a slow decrease in yield stress.

Quantitative assessment of epoxide formation in oil and mayonnaise by 1H-13C HSQC NMR spectroscopy.

Lipid oxidation is detrimental for the quality of oil-based foods. Historically, lipid oxidation research focussed on hydroperoxides and aldehydes, but a third class, the epoxides, have been proposed to resolve observed mechanistic anomalies. Here, we developed a 2D 1H-13C HSQC NMR spectroscopic method to quantify epoxides in food in a reproducible (relative standard deviation ≤11.6 %) and sensitive (LoQ 0.62 mmol/kg oil) manner. Lipid hydroperoxides, aldehydes, and epoxides generated in rapeseed oil and mayonnaise were quantified over time by NMR. Epoxides accounted at most for 10-40 % of the products. They were formed after hydroperoxide accumulation, most likely primarily via alkoxyl radical intermediates, which limits their potential as an early oxidation marker. As 99 % and ∼60 % of the epoxide signal intensities were assigned in a fatty acid and sub-structure specific manner, respectively, our quantitative HSQC method will enable unravelling and quantitative modelling of lipid oxidation mechanisms.

Quantifying cooperative flow of fat crystal dispersions.

We quantify the cooperative flow behaviour of fat crystal dispersions (FCDs) upon varying crystallization conditions. The latter enabled altering the multiscale microstructure of the FCDs, from the nanometer-sized platelets, and the dispersed fractal aggregates, up to the strength of the mesoscopic weak-link network. To the goal of characterizing strongly-confined flow in these optically-opaque materials, we acquire high-resolution rheo-magnetic-resonance-imaging (rheo-MRI) velocimetry measurements using an in-house developed 500 µm gap Couette cell (CC). We introduce a numerical fitting method based on the fluidity model, which yields the cooperativity length, ξ, in the narrow-gap CC. FCDs with aggregates sizes smaller than the confinement size by an order of magnitude were found to exhibit cooperativity effects. The respective ξ values diverged at the yield stress, in agreement with the Kinetic Elasto-Plastic (KEP) theory. In contrast, the FCD with aggregates sizes in the order of the gap size did not exhibit any cooperativity effect: we attribute this result to the correspondingly decreased mobility of the aggregates. We foresee that our optimized rheo-MRI measurement and fitting analysis approach will propel further similar studies of flow of other multi-scale and optically-opaque materials.

Non-invasive monitoring of in vitro gastric milk protein digestion kinetics by 1H NMR magnetization transfer.

Processing of milk involves heating, which can modify the structure and digestibility of its proteins. In vitro models are useful for studying protein digestion. However, validating these models with in vivo data is challenging. Here, we non-invasively monitor in vitro gastric milk protein digestion by protein-water chemical exchange detected by 1H nuclear magnetic resonance (NMR) magnetization transfer (MT). We obtained either a fitted composite exchange rate (CER) with a relative standard error of ≤10% or the MT ratio (MTR) of the intensity without or with an off-resonance saturation pulse, from just a single spectral acquisition. Both CER and MTR, affected by the variation in the amount of semi-solid protons, decreased during in vitro gastric digestion in agreement with standard protein content analyses. The decrease was slower in heated milk, indicating slower breakdown of the coagulum. Our results open the way to future quantification of protein digestion in vivo by MRI.

Enabling single-molecule localization microscopy in turbid food emulsions.

Turbidity poses a major challenge for the microscopic characterization of food systems. Local mismatches in refractive indices, for example, lead to significant image deterioration along sample depth. To mitigate the issue of turbidity and to increase the accessible optical resolution in food microscopy, we added adaptive optics (AO) and flat-field illumination to our previously published open microscopy framework, the miCube. In the detection path, we implemented AO via a deformable mirror to compensate aberrations and to modulate the emission wavefront enabling the engineering of point spread functions (PSFs) for single-molecule localization microscopy (SMLM) in three dimensions. As a model system for a non-transparent food colloid such as mayonnaise, we designed an oil-in-water emulsion containing the ferric ion binding protein phosvitin commonly present in egg yolk. We targeted phosvitin with fluorescently labelled primary antibodies and used PSF engineering to obtain two- and three-dimensional images of phosvitin covered oil droplets with sub 100 nm resolution. Our data indicated that phosvitin is homogeneously distributed at the interface. With the possibility to obtain super-resolved images in depth, our work paves the way for localizing biomacromolecules at heterogeneous colloidal interfaces in food emulsions. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Validation of temperature-controlled rheo-MRI measurements in a submillimeter-gap Couette geometry.

A temperature-controlled submillimeter-gap (500 µm) rheo-magnetic resonance imaging (MRI) Couette cell has been developed to measure confined flow of soft structured materials under controlled temperature. The proposed setup enables performing rheo-MRI measurements using (i) a spatially uniform temperature control over the range 15°C to 40°C and (ii) a high spatial resolution up to 10 µm, as a consequence of the improved mechanical stability of the in-house developed rotating elements. Here, we demonstrate the performance of the cell for the rheo-MRI velocimetry study of a thixotropic fat crystal dispersion, a complex fluid commonly used in food manufacturing. The submillimeter-gap geometry and variable temperature capability of the cell enable observing the effects of shear- and temperature-induced fat recrystallization on both wall slip and shear banding under strongly confined flow. Our improved rheo-MRI setup opens new perspectives for the fundamental study of strongly confined flow, cooperative effects, and the underlying interparticle interactions and for ultimately aiding optimization of products involved in spreading/extrusion, such as cosmetics and foods.

Quantitative and Predictive Modelling of Lipid Oxidation in Mayonnaise.

Food emulsions with high amounts of unsaturated fats, such as mayonnaise, are prone to lipid oxidation. In the food industry, typically accelerated shelf life tests are applied to assess the oxidative stability of different formulations. Here, the appearance of aldehydes at the so-called onset time, typically weeks, is considered a measure for oxidative stability of food emulsions, such as mayonnaise. To enable earlier assessment of compromised shelf-life, a predictive model for volatile off-flavor generation is developed. The model is based on the formation kinetics of hydroperoxides, which are early oxidation products and precursors of volatile aldehydes, responsible for off-flavor. Under accelerated shelf-life conditions (50 °C), hydroperoxide (LOOH) concentration over time shows a sigmoidal curvature followed by an acceleration phase that occurs at a LOOH-concentration between 38-50 mmol/kg, here interpreted as a critical LOOH concentration (CCLOOH). We hypothesize that the time at which CCLOOH was reached is related to the onset of aldehyde generation and that the characterization of the LOOH-generation curvature could be based on reaction kinetics in the first days. These hypotheses are tested using semi-empirical models to describe the autocatalytic character of hydroperoxide formation in combination with the CCLOOH. The Foubert function is selected as best describing the LOOH-curvature and is hence used to accurately predict onset of aldehyde generation, in most cases within several days of shelf-life. Furthermore, we find that the defining parameters of this model could be used to recognize antioxidant mechanisms at play.

Evaluation of PBN spin-trapped radicals as early markers of lipid oxidation in mayonnaise.

Quality deterioration of mayonnaise is caused by lipid oxidation, mediated by radical reactions. Assessment of radicals would enable early lipid oxidation assessment and generate mechanistic insights. To monitor short-lived lipid-radicals, N-tert-butyl-α-phenylnitrone (PBN), a spin-trap, is commonly used. In this study, the fate of PBN-adducts and their impact on lipid oxidation mechanisms in mayonnaise were investigated. The main signals detected by Electron Spin Resonance (ESR) were attributed to L-radicals attached to 2-methyl-2-nitrosopropane (MNP), one of three degradation products of the PBN-peroxy-adduct. The second degradation product, benzaldehyde, was detected with Nuclear Magnetic Resonance (1H NMR), in line with MNP-L adduct generation. For the third class of degradation products, LO-radicals, their scission products were detected with 1H NMR and indicated that LO-radicals have a major impact on downstream oxidation pathways. This precludes mechanistical studies in presence of PBN. Degradation products of PBN-adducts can, however, be used for early assessment of antioxidants efficacy in oil-in-water emulsions.

Quantitative Spatiotemporal Mapping of Lipid and Protein Oxidation in Mayonnaise.

Lipid oxidation in food emulsions is mediated by emulsifiers in the water phase and at the oil-water interface. To unravel the physico-chemical mechanisms and to obtain local lipid and protein oxidation rates, we used confocal laser scanning microscopy (CLSM), thereby monitoring changes in both the fluorescence emission of a lipophilic dye BODIPY 665/676 and protein auto-fluorescence. Our data show that the removal of lipid-soluble antioxidants from mayonnaises promotes lipid oxidation within oil droplets as well as protein oxidation at the oil-water interface. Furthermore, we demonstrate that ascorbic acid acts as either a lipid antioxidant or pro-oxidant depending on the presence of lipid-soluble antioxidants. The effects of antioxidant formulation on local lipid and protein oxidation rates were all statistically significant (p < 0.0001). The observed protein oxidation at the oil-water interface was spatially heterogeneous, which is in line with the heterogeneous distribution of lipoprotein granules from the egg yolk used for emulsification. The impact of the droplet size on local lipid and protein oxidation rates was significant (p < 0.0001) but minor compared to the effects of ascorbic acid addition and lipid-soluble antioxidant depletion. The presented results demonstrate that CLSM can be applied for unraveling the roles of colloidal structure and transport in mediating lipid oxidation in complex food emulsions.

Advances in our understanding of the structure and functionality of edible fats and fat mimetics.

The reasons for the increased world-wide incidence of obesity, type-2 diabetes, and cardiovascular disease include sedentary lifestyles and poor food choices. Regulatory agencies in several countries now require companies to add unattractive front of package labels to their products where salt, sugar and fat (or saturated fat) levels are prominently displayed. After the demise of partially hydrogenated fats, saturated fat has become the new target. Consumption of saturated fat over polyunsaturated oil has been clearly shown to increase cholesterol levels in humans. However, saturated fats provide the functionality required in many food products. To complicate matters, concerns over sustainability, veganism, genetically modified organisms, animal welfare, as well as religious beliefs, severely limit our sources of saturated fat. In this review we will discuss recent advances in our understanding of the nano and mesoscale structure of fats, responsible for their physical functionality and contrast it to that of fat mimetics. Fat mimetics include polymeric networks of ethylcellulose, emulsion-templated networks of proteins and polysaccharides, colloidal and self-assembled fibrillar networks of polar lipid crystals, as well as solid o/w emulsions of oil trapped within crystallized lamellar mesophases. Clean label and economic considerations will also be touched upon.

Full 1 H and 13 C NMR spectral assignment of conjugated valerolactone metabolites isolated from urine of black tea consumers by means of SPE-prepLC-MS-LC-MS-NMR.

The health benefits of black tea have been linked to polyphenol metabolites that target specific modes of action in the human body. A major bottleneck in unravelling the underlying mechanisms is the preparative isolation of these metabolites, which hampers their structural elucidation and assessment of in vitro bioactivity. A solid phase extraction (SPE)-preparative liquid chromatography (prepLC)-MS-LC-MS-NMR workflow was implemented for preparative isolation of conjugated valerolactone metabolites of catechin-based polyphenols from urine of black tea consumers. First, the urine was cleaned and preconcentrated using an SPE method. Subsequently, the clean urine concentrate was injected on a preparative LC column, and conjugated valerolactones were obtained by MS-guided collection. Reconstituted fractions were further separated on an analytical LC column, and valerolactone fractions were collected in an MS-guided manner. These were reconstituted in methanol-d4 and identified and quantified using 1D and 2D homo- and hetereonuclear NMR experiments (at a field strength of 14.1 T), in combination with mass spectrometry. This resulted in the full spectral 1 H and 13 C NMR assignments of five conjugated valerolactones. These metabolites were collected in quantities of 8-160 µg and purities of 70-91%. The SPE-prepLC-MS-LC-MS-NMR workflow is suitable for isolating metabolites that occur at sub-µM concentrations in a complex biofluid such as urine. The workflow also provides an alternative for cumbersome and expensive de novo synthesis of tea metabolites for testing in bioactivity assays or for use as authentic analytical standards for quantification by mass spectrometry.

31 P NMR assessment of the phosvitin-iron complex in mayonnaise.

Lipid oxidation is the main reason for the limited shelf life of mayonnaise. One of the main catalysts of this process is iron, which is introduced in its ferric (Fe(III)) form via phosvitin, an egg yolk phosphoprotein rich in phosphoserines. The binding of Fe(III) to phosvitin and its ability to establish a redox couple with Fe(II) is believed to determine the oxidation rate of unsaturated lipids. In this work, a 31 P NMR based method was developed to quantify loading of phosvitin with Fe(III) and its reductive release. Both features could be quantified in model phosvitin solutions by exploiting the paramagnetic broadening of 31 P NMR signal of phosphoserine residues by Fe(III). This method was then successfully applied to quantify the phosvitin-Fe(III) loading in mayonnaise water phase by liquid NMR, whereas 31 P NMR MAS could only provide a qualitative measure. The 31 P NMR method showed a direct relation between loading of the Fe(III)-phosvitin complex and lipid oxidation.

Heterogeneity of Network Structures and Water Dynamics in κ-Carrageenan Gels Probed by Nanoparticle Diffusometry.

A set of functionalized nanoparticles (PEGylated dendrimers, d = 2.8-11 nm) was used to probe the structural heterogeneity in Na+/K+ induced κ-carrageenan gels. The self-diffusion behavior of these nanoparticles as observed by 1H pulsed-field gradient NMR, fluorescence recovery after photobleaching, and raster image correlation spectroscopy revealed a fast and a slow component, pointing toward microstructural heterogeneity in the gel network. The self-diffusion behavior of the faster nanoparticles could be modeled with obstruction by a coarse network (average mesh size <100 nm), while the slower-diffusing nanoparticles are trapped in a dense network (lower mesh size limit of 4.6 nm). Overhauser dynamic nuclear polarization-enhanced NMR relaxometry revealed a reduced local solvent water diffusivity near 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)-labeled nanoparticles trapped in the dense network, showing that heterogeneity in the physical network is also reflected in heterogeneous self-diffusivity of water. The observed heterogeneity in mesh sizes and in water self-diffusivity is of interest for understanding and modeling of transport through and release of solutes from heterogeneous biopolymer gels.

Rapid Quantitative Profiling of Lipid Oxidation Products in a Food Emulsion by 1H NMR.

Lipid oxidation is one of the most important reasons for the compromised shelf life of food emulsions. A major bottleneck in unravelling the underlying mechanisms is the lack of methods that provide a rapid, quantitative, and comprehensive molecular view on lipid oxidation in these heterogeneous systems. In this study, the unbiased and quantitative nature of 1H NMR was exploited to assess lipid oxidation products in mayonnaise, a particularly oxidation-prone food emulsion. An efficient and robust procedure was implemented to produce samples where the 1H NMR signals of oxidation products could be observed in a well resolved and reproducible manner. 1H NMR signals of hydroperoxides were assigned in a fatty acid and isomer specific way. Band-selective 1H NMR pulse excitation allowed immediate and precise (RSDR = 5.9%) quantification of both hydroperoxides and aldehydes with high throughput and large dynamic range at levels of 0.03 mmol/kg. Explorative multivariate data modeling of the quantitative 1H NMR profiles revealed that shelf life temperature has a significant impact on lipid oxidation mechanisms.

Quantification of food polysaccharide mixtures by 1H NMR.

Polysaccharides are food ingredients that critically determine rheological properties and shelf life. A qualitative and quantitative assessment on food-specific polysaccharide mixtures by 1H NMR is presented. The method is based on the identification of intact polysaccharides, combined with a quantitative analysis of their monosaccharide constituents. Identification of the polysaccharides is achieved by 1H NMR line shape fitting with pure compound spectra. The monomeric composition was determined using the Saeman hydrolysis procedure, followed by direct monosaccharide quantification by 1H NMR. In the quantification, both the monosaccharide degradation during hydrolysis, as well as a correction for the non-instantaneous polysaccharide dissolution were taken into account. These factors were particularly important for the quantification of pectins. The method showed overall good repeatability (RSDr=4.1±0.9%) and within-laboratory reproducibility (RSDR=6.1±1.4%) for various food polysaccharides. Polysaccharide mixtures were quantitatively resolved by a non-negative least squares estimation, using identified polysaccharides and their molar monosaccharide stoichiometry as prior knowledge. The accuracy and precision of the presented method make it applicable to a wide range of food polysaccharide mixtures with complex and overlapping 1H NMR spectra.

Assessment of dietary exposure and effect in humans: The role of NMR.

In human nutritional science progress has always depended strongly on analytical measurements for establishing relationships between diet and health. This field has undergone significant changes as a result of the development of NMR and mass spectrometry methods for large scale detection, identification and quantification of metabolites in body fluids. This has allowed systematic studies of the metabolic fingerprints that biological processes leave behind, and has become the research field of metabolomics. As a metabolic profiling technique, NMR is at its best when its unbiased nature, linearity and reproducibility are exploited in well-controlled nutritional intervention and cross-sectional population screening studies. Although its sensitivity is less good than that of mass spectrometry, NMR has maintained a strong position in metabolomics through implementation of standardisation protocols, hyphenation with mass spectrometry and chromatographic techniques, accurate quantification and spectral deconvolution approaches, and high-throughput automation. Thus, NMR-based metabolomics has contributed uniquely to new insights into dietary exposure, in particular by unravelling the metabolic fates of phytochemicals and the discovery of dietary intake markers. NMR profiling has also contributed to the understanding of the subtle effects of diet on central metabolism and lipoprotein metabolism. In order to hold its ground in nutritional metabolomics, NMR will need to step up its performance in sensitivity and resolution; the most promising routes forward are the analytical use of dynamic nuclear polarisation and developments in microcoil construction and automated fractionation.