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Spatiotemporal assessment of lipid and protein oxidation is key for understanding quality deterioration in emulsified food products containing polyunsaturated fatty acids. In this work, we first mechanistically validated the use of the lipid oxidation-sensitive fluorophore BODIPY 665/676 as a semi-quantitative marker for local peroxyl radical formation. Next, we assessed the impact of microfluidic and colloid mill emulsification (respectively producing mono- and polydisperse droplets) on local protein and lipid oxidation kinetics in whey protein isolate (WPI)-stabilized emulsions. We further used BODIPY 581/591 C11 and CAMPO-AFDye 647 as colocalisation markers for lipid and protein oxidation. The polydisperse emulsions showed an inverse relation between droplet size and lipid oxidation rate. Further, we observed less protein and lipid oxidation occurring in similar sized droplets in monodisperse emulsions. This observation was linked to more heterogeneous protein packing at the droplet surface during colloid mill emulsification, resulting in larger inter-droplet heterogeneity in both protein and lipid oxidation. Our findings indicate the critical roles of emulsification methods and droplet sizes in understanding and managing lipid oxidation.
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Emulsões , Oxirredução , Tamanho da Partícula , Proteínas do Soro do Leite , Proteínas do Soro do Leite/química , Emulsões/química , Compostos de Boro/química , Cinética , Peróxidos/química , Lipídeos/químicaRESUMO
Models predicting lipid oxidation in oil-in-water (O/W) emulsions are a requirement for developing effective antioxidant solutions. Existing models do, however, not include explicit equations that account for composition and structural features of O/W emulsions. To bridge this gap, a mechanistic kinetic model for lipid oxidation in emulsions is presented, describing the emulsion as a one-dimensional three phase (headspace, water, and oil) system. Variation in oil droplet sizes, overall surface area of oil/water interface, oxidation of emulsifiers, and the presence of catalytic transition metals were accounted for. For adequate predictions, the overall surface area of oil/water interface needs to be determined from the droplet size distribution obtained by dynamic and static light scattering (DLS, SLS). The kinetic model predicted well the formation of oxidation products in both mono- and polydisperse emulsions, with and without presence of catalytic transition metals.
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Emulsões , Lipídeos , Oxirredução , Polissorbatos , Emulsões/química , Cinética , Polissorbatos/química , Lipídeos/química , Água/química , Tamanho da Partícula , Modelos Químicos , Óleos/químicaRESUMO
Lipid oxidation limits the shelf-life of dried microencapsulated oils (DMOs), such as infant formula. However, it is poorly understood how lipid oxidation is affected by different types of emulsifiers. To improve our understanding, we prepared DMOs with different emulsifiers (whey protein isolate (WPI), pea protein isolate (PPI), and non-proteinaceous CITREM) and studied lipid oxidation in both the free and encapsulated fat. Only a small difference in oxidation rate was observed between these fat fractions for all formulations. We ascribed this to a non-discrete distribution of the fractions and the subsequent low fractionation selectivity as shown by Raman microscopy. The DMO with PPI showed hardly any oxidation during a 7-week incubation at 40 °C, whereas the DMOs with WPI and CITREM both reached significantly higher contents of oxidation products (lipid hydroperoxides, aldehydes, and epoxides). The enhanced stability of DMO-PPI could not be ascribed to the presence of phytic acid. In conclusion, we demonstrate the potential of using PPI to produce oxidatively stable DMOs.
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Emulsificantes , Emulsões , Oxirredução , Emulsificantes/química , Emulsões/química , Proteínas do Soro do Leite/química , Proteínas de Ervilha/química , Secagem por Atomização , Composição de Medicamentos , Lipídeos/química , Fórmulas Infantis/químicaRESUMO
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.
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Lipid oxidation constitutes the main source of degradation of lipid-rich foods, including food emulsions. The complexity of the reactions at play combined with the increased demand from consumers for less processed and more natural foods result in additional challenges in controlling this phenomenon. This review provides an overview of the insights acquired over the past two decades on the understanding of lipid oxidation in oil-in-water (O/W) emulsions. After introducing the general structure of O/W emulsions and the classical mechanisms of lipid oxidation, the contribution of less studied oxidation products and the spatiotemporal resolution of these reactions will be discussed. We then highlight the impact of emulsion formulation on the mechanisms, taking into consideration the new trends in terms of emulsifiers as well as their own sensitivity to oxidation. Finally, novel antioxidant strategies that have emerged to meet the recent consumer's demand will be detailed. In an era defined by the pursuit of healthier, more natural, and sustainable food choices, a comprehensive understanding of lipid oxidation in emulsions is not only an academic quest, but also a crucial step towards meeting the evolving expectations of consumers and ensuring the quality and stability of lipid-rich food products.
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Emulsões , Oxirredução , Emulsões/química , Humanos , Lipídeos/química , Antioxidantes/química , Antioxidantes/metabolismo , Metabolismo dos LipídeosRESUMO
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.
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Lipossomos , Polímeros , EmulsõesRESUMO
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 â¼ 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 â¼ 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, â¼8% for FCD, domains of only â¼2 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.
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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.
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Condimentos , Lipoproteínas LDL , Radicais Livres/metabolismo , Oxirredução , Lipoproteínas LDL/metabolismo , Peroxidação de LipídeosRESUMO
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 â¼60 % 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.
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Compostos de Epóxi , Peróxidos Lipídicos , Aldeídos/química , Espectroscopia de Ressonância Magnética , OxirreduçãoRESUMO
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.
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Gema de Ovo , Água , Gema de Ovo/química , Emulsões/química , Imageamento por Ressonância Magnética , Viscosidade , Água/químicaRESUMO
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.
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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.
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Proteínas do Leite , Prótons , Digestão , Cinética , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética , Proteínas do Leite/metabolismo , Proteólise , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
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)'.
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Microscopia , Imagem Individual de Molécula , Emulsões , Imageamento TridimensionalRESUMO
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.
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Imageamento por Ressonância Magnética , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Reologia/métodos , TemperaturaRESUMO
AIMS: Dicarbonyl compounds contribute to the formation of advanced glycation endproducts (AGEs) and the development of insulin resistance and vascular complications. Dicarbonyl stress may already be detrimental in obesity. We evaluated whether diet-induced weight loss can effectively reverse dicarbonyl stress in abdominally obese men. MATERIALS AND METHODS: Plasma samples were collected from lean (n = 25) and abdominally obese men (n = 52) in the fasting state, and during a mixed meal test (MMT). Abdominally obese men were randomized to 8 weeks of dietary weight loss or habitual diet, followed by a second MMT. The α-dicarbonyls methylglyoxal (MGO), glyoxal (GO) and 3-deoxyglucosone (3-DG) and AGEs were measured by UPLC-MS/MS. Skin autofluorescence (SAF) was measured using the AGE reader. T-tests were used for the cross-sectional analysis and ANCOVA to assess the treatment effect. RESULTS: Postprandial glucose, MGO and 3-DG concentrations were higher in obese men as compared to lean men (p < 0.05 for all). Fasting dicarbonyls, AGEs, and SAF were not different between lean and obese men. After the weight loss intervention, fasting MGO levels tended to decrease by 25 nmol/L (95%-CI: -51-0.5; p = 0.054). Postprandial dicarbonyls were decreased after weight loss as compared to the control group: iAUC of MGO decreased by 57% (5280 nmol/Lâmin; 95%-CI: 33-10526; p = 0.049), of GO by 66% (11,329 nmol/Lâmin; 95%-CI: 495-22162; p = 0.041), and of 3-DG by 45% (20,175 nmol/Lâmin; 95%-CI: 5351-35000; p = 0.009). AGEs and SAF did not change significantly after weight loss. CONCLUSION: Abdominal obesity is characterized by increased postprandial dicarbonyl stress, which can be reduced by a weight loss intervention. Registered under ClinicalTrials.gov Identifier no. NCT01675401.
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Dieta Redutora , Obesidade Abdominal/dietoterapia , Período Pós-Prandial , Estresse Fisiológico , Redução de Peso/fisiologia , Adulto , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Estudos Transversais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , MasculinoRESUMO
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.
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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.
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Espectroscopia de Ressonância de Spin Eletrônica/métodos , Análise de Alimentos , Lipídeos/química , Detecção de Spin/métodos , Óxidos N-Cíclicos , Radicais Livres/análise , Radicais Livres/química , Compostos Nitrosos , OxirreduçãoRESUMO
The unambiguous identification of unknown compounds is of utmost importance in the field of metabolomics. However, current identification workflows often suffer from error-sensitive methodologies, which may lead to incorrect structure annotations of small molecules. Therefore, we have developed a comprehensive identification workflow including two highly complementary techniques, i.e. liquid chromatography (LC) combined with mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR), and used it to identify five taste-related retention time and m/z features in soy sauce. An off-line directed two-dimensional separation was performed in order to purify the features prior to the identification. Fractions collected during the first dimension separation (reversed phase low pH) were evaluated for the presence of remaining impurities next to the features of interest. Based on the separation between the feature and impurities, the most orthogonal second dimension chromatography (hydrophilic interaction chromatography or reversed phase high pH) was selected for further purification. Unknown compounds down to tens of micromolar concentrations were tentatively annotated by MS and structurally confirmed by MS and NMR. The mass (0.4-4.2 µg) and purity of the isolated compounds were sufficient for the acquisition of one and two-dimensional NMR spectra. The use of a directed two-dimensional chromatography allowed for a fractionation that was tailored to each feature and remaining impurities. This makes the fractionation more widely applicable to different sample matrices than one-dimensional or fixed two-dimensional chromatography. Five proline-based 2,5-diketopiperazines were successfully identified in soy sauce. These cyclic dipeptides might contribute to taste by giving a bitter flavour or indirectly enhancing umami flavour.
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Fracionamento Químico , Metabolômica , Cromatografia Líquida , Espectroscopia de Ressonância Magnética , Espectrometria de MassasRESUMO
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.