Droplet size dependency and spatial heterogeneity of lipid oxidation in whey protein isolate-stabilized emulsions.

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.

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.

Effect of Nonprotein Components for Lipid Oxidation in Emulsions Stabilized by Plant Protein Extracts.

Plant protein ingredients are rich in non-protein components of which the antioxidant and pro-oxidant effects are expected to be considerable. In this paper, commercial soy and pea protein isolates and concentrates were selected by using their soluble fractions to prepare oil-in-water (O/W) emulsions. Emulsions stabilized with soy protein isolates were more prone to lipid oxidation than those with soy protein concentrate or pea protein isolate. Compositional analysis revealed that the soluble fraction of soy protein isolates contained higher concentrations of phenolic compounds and metals (iron and copper) but lower mineral and ash contents than those of soy protein concentrate and pea protein isolate. Correlating the composition to oxidation in emulsions highlighted the significant role of non-protein components, alongside the protein's oxidative state. These findings are relevant for the use of alternative proteins in food formulation, a practice often promoted as sustainable yet that may come with repercussions for oxidative stability.

Oleofoams: The impact of formulating air-in-oil systems from a lipid oxidation perspective.

Air-in-oil foams, or oleofoams, have a great potential for food applications as they can at least partially replace animal or hydrogenated fats, without compromising on textural properties. Yet, there are some challenges to tackle before they can largely be implemented for real-life applications. One of those is the lack of data regarding their oxidative stability. This is an important point to consider, as although using oils rich in polyunsaturated fatty acids (PUFAs) is highly desirable from a nutritional perspective, these fatty acids are particularly prone to oxidation, which leads to major degradations of food quality. This work thus aimed to investigate the oxidative stability of oleofoams prepared with omega-3 PUFA-rich vegetable oils (rapeseed or flaxseed oil) and various types of high melting point lipid-based oleogelators (stearic acid, glyceryl monostearate and stearyl alcohol) when incubated at room temperature. The physical structure and stability of the oleofoams was monitored by various techniques (visual observations, microscopy, DSC, NMR, SAXS and WAXS). Lipid oxidation was assessed by combined measurements of primary (conjugated diene hydroperoxides) and secondary (thiobarbituric acid reactive substances - TBARS) products. We found that the oxidative stability of oleofoams was higher compared to that of the corresponding bulk oil. This protective effect was also found when the oil was simply mixed with the oleogelator without incorporation of air bubbles (i.e., forming an oleogel), and was somewhat modulated depending on the type of oleogelator. These results suggest that oleogelators and the structural changes that they induce limit the cascaded propagation of lipid oxidation in oil-continuous matrices, which is promising in the perspective of future applications.

Lipid oxidation in emulsions: New insights from the past two decades.

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.

In vitro digestion of high-lipid emulsions: towards a critical interpretation of lipolysis.

Investigating the gastrointestinal fate of food emulsions is critical to unveil their nutritional relevance. To this end, the protocol standardized by COST INFOGEST 2.0 is meaningful for guiding in vitro digestion experiments. In contrast with studies addressing emulsions with low dispersed phase volume fraction (φ 0.05-0.1), we presently raise some points for a proper interpretation of the digestibility of emulsions with high lipid content using the pH-stat method. Oil-in-water high internal phase emulsions (HIPEs) were submitted to gastric pre-lipolysis with the addition of rabbit gastric lipase (RGE). Commercial mayonnaise (φ 0.76) was systematically diluted (φ 0.025, 0.05, 0.1, 0.15, 0.25, 0.4, and 0.76) to cover a wide range of enzyme-to-lipid ratios (8.5-0.3 U per µmol for RGE and 565.1-18.6 U per µmol for pancreatin, in the gastric and intestinal phases, respectively). Lipolysis was tracked either by fatty acid titration (NaOH titration) or completed by analysis of lipid classes and fatty acid composition. Gastric lipase resulted in substantial lipid hydrolysis, reaching 20 wt% at low lipid fractions (φ 0.025 and 0.05). Likewise, the kinetics and extent of lipolysis during intestinal digestion were modulated by the enzyme-to-substrate ratio. A logarithmic relationship between lipid hydrolysis and lipid concentration was observed, with a very limited extent at the highest lipid content (φ 0.76). A holistic interpretation relying on FFA titration and further evaluation of all lipolytic products appears of great relevance to capture the complexity of the effects involved. Overall, this work contributes to rationally and critically evaluating the outcomes of static in vitro experiments of lipid digestion.

Tiny, yet impactful: Detection and oxidative stability of very small oil droplets in surfactant-stabilized emulsions.

HYPOTHESIS: The shelf life of multiphase systems, e.g. oil-in-water (O/W) emulsions, is severely limited by physical and/or chemical instabilities, which degrade their texture, macroscopic appearance, sensory and (for edible systems) nutritional quality. One prominent chemical instability is lipid oxidation, which is notoriously complex. The complexity arises from the involvement of many physical structures present at several scales (1-10,000 nm), of which the smallest ones are often overlooked during characterization. EXPERIMENTS: We used cryogenic transmission electron microscopy (cryo-TEM) to characterize the coexisting colloidal structures at the nanoscale (10-200 nm) in rapeseed oil-based model emulsions stabilized by different concentrations of a nonionic surfactant. We assessed whether the oxidative and physical instabilities of the smallest colloidal structures in such emulsions may be different from those of larger colloidal structures. FINDINGS: By deploying cryo-TEM, we analyzed the size of very small oil droplets and of surfactant micelles, which are typically overlooked by dynamic light scattering when larger structures are concomitantly present. Their size and oil content were shown to be stable over incubation, but lipid oxidation products were overrepresented in these very small droplets. These insights highlight the importance of the fraction of "tiny droplets" for the oxidative stability of O/W emulsions.

Comparison of the effect of various sources of saturated fatty acids on infant follow-on formulas oxidative stability and nutritional profile.

Fortification of infant follow-on formulas (IFF) with docosahexaenoic acid (DHA), which is prone to lipid oxidation, is required by European regulation. This study aimed to identify lipid formulation parameters that improve the nutritional profile and oxidative stability of IFF. Model IFF were formulated using different lipid and emulsifier sources, including refined (POM) or unrefined red palm oil (RPOM), coconut oil (COM), dairy fat (DFOM), soy lecithin, and dairy phospholipids (DPL). After an accelerated storage, RPOM and DFOM with DPL had improved oxidative stability compared to other IFF. Specifically, they had a peroxide value twice lower than POM and 20% less loss of tocopherols for DFOM-DPL. This higher stability was mainly explained by the presence of compounds such as carotenoids in RPOM and sphingomyelin in DFOM-DPL very likely acting synergistically with tocopherols. Incorporation of dairy lipids and carotenoids into DHA-enriched IFF compositions seems promising to enhance their stability and nutritional quality.

Targeting Interfacial Location of Phenolic Antioxidants in Emulsions: Strategies and Benefits.

It is important to have larger proportions of health-beneficial polyunsaturated lipids in foods, but these nutrients are particularly sensitive to oxidation, and dedicated strategies must be developed to prevent this deleterious reaction. In food oil-in-water emulsions, the oil-water interface is a crucial area when it comes to the initiation of lipid oxidation. Unfortunately, most available natural antioxidants, such as phenolic antioxidants, do not spontaneously position at this specific locus. Achieving such a strategic positioning has therefore been an active research area, and various routes have been proposed: lipophilizing phenolic acids to confer them with an amphiphilic character; functionalizing biopolymer emulsifiers through covalent or noncovalent interactions with phenolics; or loading Pickering particles with natural phenolic compounds to yield interfacial antioxidant reservoirs. We herein review the principles and efficiency of these approaches to counteract lipid oxidation in emulsions as well as their advantages and limitations.

Physical and Oxidative Stabilization of Oil-In-Water Emulsions by Roasted Coffee Fractions: Interface- and Continuous Phase-Related Effects.

Emulsions fortified with polyunsaturated fatty acids are highly relevant from a nutritional perspective; however, such products are prone to lipid oxidation. In the current work, this is mitigated by the use of natural antioxidants occurring in coffee. Coffee fractions with different molecular weights were extracted from roasted coffee beans. These components were positioned either at the interface or in the continuous phase of emulsions where they contributed to emulsion stability via different pathways. Coffee brew as a whole, and its high-molecular-weight fraction (HMWF), was able to form emulsions with good physical stability and excellent oxidative stability. When added post-homogenization to the continuous phase of dairy protein-stabilized emulsions, all coffee fractions were able to slow down lipid oxidation considerably without altering the physical stability of emulsions, though HMWF was more effective in retarding lipid oxidation than whole coffee brew or low-molecular-weight fraction. This is caused by various effects, such as the antioxidant properties of coffee extracts, the partitioning of components in the emulsions, and the nature of the phenolic compounds. Our research shows that coffee extracts can be used effectively as multifunctional stabilizers in dispersed systems leading to emulsion products with high chemical and physical stability.

Design insights for upscaling spontaneous microfluidic emulsification devices based on behavior of the Upscaled Partitioned EDGE device.

Microfluidic emulsification has the potential to produce emulsions with very controlled droplet sizes in a subtle manner. To support in unleashing this potential, we provide guidelines regarding upscaling based on the performance of Upscale Partitioned EDGE (UPE) devices, using rapeseed oil as the to-be-dispersed phase and whey proteins as the emulsifier. The UPE5x1 device (11,000 droplet formation units (DFUs) of 5 × 1 µm) produced 3.5-µm droplets (CV 3.2 %) at 0.3 mL/h; UPE10x2 (8,000 DFUs of 10 × 2 µm) produced 7-µm droplets (CV 3.2 %) at 0.5 mL/h, and at higher pressures, 32-µm droplets (CV 3-4 %) at 4 mL/h. These productivities are relatively high compared to those of other devices reported in literature (e.g., Microchannel, Tsukuba and Millipede, Harvard). Based on these results, and on others from literature, we conclude that: (1) the continuous phase channel dimensions need to be chosen such that they allow for gradual filling of this channel with droplets without decreasing the pressure over the droplet formation units significantly; (2) the dispersed phase supply channel design should create a wide stable droplet formation pressure range to increase productivity; and (3) higher productivities can be obtained through the choice of the ingredients used; low viscosity dispersed phase and an emulsifier that increases the interfacial tension without negatively affecting device wettability is preferred (e.g., whey protein outperforms Tween 20). These results and design guidelines are expected to contribute to the first food emulsion products prepared with microfluidics.

Lipid oxidation products in model food emulsions: do they stay in or leave droplets, that's the question.

Lipid oxidation is a major factor limiting the shelf life of food and other emulsion products. In this work, we explore which lipid oxidation products may transfer between oil droplets in model food emulsions stabilized by excess amounts of surfactant, and whether this affects the overall reaction. No significant differences in concentrations of triglyceride-bound hydroperoxides were found before and after mixing 'clean' oil droplets with pre-oxidized ones. Shorter and more hydrophilic lipid oxidation products, such as 4-hydroperoxy-2-nonenal and 2,4-decadienal, were found to equilibrate between oil droplets within 30 min. Adding exogenous 4-hydroperoxy-2-nonenal to an emulsion led to overall higher lipid oxidation values, although this effect was not systematic nor instantaneous. Therefore, it may be questioned whether transfer and subsequent initiation are always relevant for oxidizing emulsion systems. In future research, this question should be addressed for complex emulsions that are closer to real-life food products.

A unifying approach to lipid oxidation in emulsions: Modelling and experimental validation.

Lipid oxidation is a longstanding topic within the field of food technology, and is strongly related to loss of product quality and consumer acceptance. Both for bulk oils and emulsions, the chemical phenomena involved in lipid oxidation have been extensively researched, and various reaction pathways have been identified. They are different in bulk oil compared to oil-in-water (O/W) emulsions in which the oil-water interface plays a prominent role. Most probably because of the complexity of the reaction scheme in combination with mass transfer effects, there is no model that describes lipid oxidation in emulsions in a unified fashion, and that is the aim that we have set ourselves to achieve. We use lipid oxidation data previously obtained in O/W emulsions made with 5 different emulsifiers (2 surfactants, and 3 proteins), in well-mixed systems where the oxygen-to-oxidizable lipid ratio is strictly controlled. We use data pertaining to headspace oxygen concentration, and to primary and secondary lipid oxidation products to develop a model based on reaction kinetics, including not only the classical reaction scheme (starting from an unsaturated lipid, LH) but also radical initiation from hydroperoxides, which is thought to be an effect that is overlooked in the classical description of the initiation step. We were able to describe the course of the reactions in these emulsions using the same reaction rate constants for all emulsions, with the exception of the two related to radical-based initiation. In Tween 20- and Tween 80-stabilized emulsions, initiation stems most probably solely from decomposition of hydroperoxides; this implies that lipid oxidation in these emulsions is co-determined by the initial ("pre-existing") hydroperoxide concentration. In protein-stabilized emulsions, on the other hand, lipid radical initiation is probably linked to reactions involving proteins (co-oxidation reactions), whereas initiation through decomposition of hydroperoxides seems less important, if at all. From this, we can conclude that the difference between both types of emulsions with regard to lipid oxidation mechanisms is related to differences in radical initiation. The developed model can serve as a unified basis for understanding lipid oxidation in emulsions, through which additional effects beyond the bare reaction kinetics, such as mass transfer effects, can be identified and used to e.g., quantify antioxidant effects, which is part of follow-up research.

Interfacial protein-protein displacement at fluid interfaces.

Protein blends are used to stabilise many traditional and emerging emulsion products, resulting in complex, non-equilibrated interfacial structures. The interface composition just after emulsification is dependent on the competitive adsorption between proteins. Over time, non-adsorbed proteins are capable of displacing the initially adsorbed ones. Such rearrangements are important to consider, since the integrity of the interfacial film could be compromised after partial displacement, which may result in the physical destabilisation of emulsions. In the present review, we critically describe various experimental techniques to assess the interfacial composition, properties and mechanisms of protein displacement. The type of information that can be obtained from the different techniques is described, from which we comment on their suitability for displacement studies. Comparative studies between model interfaces and emulsions allow for evaluating the impact of minor components and the different fluid dynamics during interface formation. We extensively discuss available mechanistic physical models that describe interfacial properties and the dynamics of complex mixed systems, with a focus on protein in-plane and bulk-interface interactions. The potential of Brownian dynamic simulations to describe the parameters that govern interfacial displacement is also addressed. This review thus provides ample information for characterising the interfacial properties over time in protein blend-stabilised emulsions, based on both experimental and modelling approaches.

Alkyl chain length modulates antioxidant activity of gallic acid esters in spray-dried emulsions.

Lipid oxidation is a well-recognized issue in dried food emulsions, such as infant milk formula. Antioxidants can be used to mitigate this issue; however, their efficiency in such complex systems is far from understood. In this study, antioxidant polarity is varied through the alkyl chain length of gallic acid esters (0-16 carbon atoms) incorporated to O/W emulsions that are subsequently spray-dried. During processing and subsequent storage of the samples, antioxidants with more than eight carbon atoms are effective. Both for encapsulated fat and surface free fat, we observe a slight cut-off effect, meaning that beyond eight alkyl groups, a more nonpolar antioxidant is slightly less effective. Depending on the antioxidant polarity, lipid oxidation is faster either in the encapsulated or in the surface free fat. The insights obtained contribute to understanding lipid oxidation in low moisture food emulsions, and thus lead to effective antioxidant strategies.

Evaluation of oxygen partial pressure, temperature and stripping of antioxidants for accelerated shelf-life testing of oil blends using 1H NMR.

Lipid oxidation compromises the shelf-life of lipid-containing foods, leading to the generation of unpleasant off-flavours. Monitoring lipid oxidation under normal shelf-life conditions can be time-consuming (i.e. weeks or months) and therefore accelerated shelf-life conditions are often applied. However, little is known on their impact on the lipid oxidation mechanisms. In this study, different oxygen partial pressures (PO2; 10 and 21%), temperatures (20, 30 and 40 °C), and the removal of antioxidants through stripping of the oil were tested to accelerate lipid oxidation. Increasing the incubation temperature of stripped oil blends from 30 to 40 °C reduced the onset of lipid oxidation from 4 to 2 weeks, whereas the PO2 had no impact. Surprisingly, at room temperature, an increase in PO2 resulted in a longer onset time (10 weeks under 10% oxygen, 15 weeks under 21% oxygen). We hypothesize that this is due to a shift in (initiation) mechanism. In non-stripped oil, an increase in PO2 from 10 to 21% decreased the onset time from 16 to 10 weeks (40 °C). Temperature elevations and stripping led to a shift towards more trans-trans diene hydroperoxides, as compared to the cis-trans conformation. Additionally, oil stripping led to an increase in oxidized PUFAs with three or more double bonds in which the hydroperoxide group is located between the double bond pattern, instead of on the edge of it. Lastly, it was shown that small additions of LC-PUFAs (0, 0.3, 0.6, 1.2 and 2.3%, w/w) accelerate lipid oxidation, even in relatively stable stripped oils. In conclusion, increased PO2 and slightly elevated temperatures hold fair potential for accelerated shelf-life testing of non-stripped oils with a limited impact on the lipid oxidation mechanisms, whereas stripping significantly changes propagation mechanisms.

Droplet Microfluidics for Food and Nutrition Applications.

Droplet microfluidics revolutionizes the way experiments and analyses are conducted in many fields of science, based on decades of basic research. Applied sciences are also impacted, opening new perspectives on how we look at complex matter. In particular, food and nutritional sciences still have many research questions unsolved, and conventional laboratory methods are not always suitable to answer them. In this review, we present how microfluidics have been used in these fields to produce and investigate various droplet-based systems, namely simple and double emulsions, microgels, microparticles, and microcapsules with food-grade compositions. We show that droplet microfluidic devices enable unprecedented control over their production and properties, and can be integrated in lab-on-chip platforms for in situ and time-resolved analyses. This approach is illustrated for on-chip measurements of droplet interfacial properties, droplet-droplet coalescence, phase behavior of biopolymer mixtures, and reaction kinetics related to food digestion and nutrient absorption. As a perspective, we present promising developments in the adjacent fields of biochemistry and microbiology, as well as advanced microfluidics-analytical instrument coupling, all of which could be applied to solve research questions at the interface of food and nutritional sciences.

Towards Oxidatively Stable Emulsions Containing Iron-Loaded Liposomes: The Key Role of Phospholipid-to-Iron Ratio.

To encapsulate soluble iron, liposomes were prepared using unsaturated phospholipids (phosphatidylcholine from egg yolk), leading to high encapsulation efficiencies (82-99%). The iron concentration affected their oxidative stability: at 0.2 and 1 mM ferrous sulfate, the liposomes were stable, whereas at higher concentrations (10 and 48 mM), phospholipid oxidation was considerably higher. When applied in oil-in-water (O/W) emulsions, emulsions with liposomes containing low iron concentrations were much more stable to lipid oxidation than those added with liposomes containing higher iron concentrations, even though the overall iron concentration was similar (0.1 M). Iron-loaded liposomes thus have an antioxidant effect at high phospholipid-to-iron ratio, but act as pro-oxidants when this ratio is too low, most likely as a result of oxidation of the phospholipids themselves. This non-monotonic effect can be of crucial importance in the design of iron-fortified foods.

Conformational Changes of Whey and Pea Proteins upon Emulsification Approached by Front-Surface Fluorescence.

Proteins are widely used to stabilize emulsions, and plant proteins have raised increasing interest for this purpose. The interfacial and emulsifying properties of proteins depend largely on their molecular properties. We used fluorescence spectroscopy to characterize the conformation of food proteins from different biological origins (dairy or pea) and transformation processes (commercial or lab-made isolates) in solution and at the oil-water interface. The fourth derivative of fluorescence spectra provided insights in the local environment of tryptophan (Trp) residues and thus in the protein structure. In emulsions, whey proteins adsorbed with their Trp-rich region at the oil-water interface. Proteins in the commercial pea isolate were present as soluble aggregates, and no changes in the local environment of the Trp residues were detected upon emulsification, suggesting that these structures adsorb without conformational changes. The lab-purified pea proteins were less aggregated and a Trp-free region of the vicilin adsorbed at the oil-water interface.

Air-water interfacial behaviour of whey protein and rapeseed oleosome mixtures.

HYPOTHESIS: Plant seeds store lipids in oleosomes, which are storage organelles with a triacylglycerol (TAG) core surrounded by a phospholipid monolayer and proteins. Due to their membrane components, oleosomes have an affinity for the air/oil-water interface. Therefore, it is expected that oleosomes can stabilise interfaces, and also compete with proteins for the air-water interface. EXPERIMENTS: We mixed rapeseed oleosomes with whey protein isolate (WPI), and evaluated their air-water interfacial properties by interfacial rheology and microstructure imaging. To understand the contribution of the oleosome components to the interfacial properties, oleosome membrane components (phospholipids and membrane proteins) or rapeseed lecithin (phospholipids) were also mixed with WPI. FINDINGS: Oleosomes were found to disrupt after adsorption, and formed TAG/phospholipid-rich regions with membrane fragments at the interface, forming a weak and mobile interfacial layer. Mixing oleosomes with WPI resulted in an interface with TAG/phospholipid-rich regions surrounded by whey protein clusters. Membrane components or lecithin mixed with proteins also resulted in an interface where WPI molecules aggregated into small WPI domains, surrounded by a continuous phase of membrane components or phospholipids. We also observed an increase in stiffness of the interfacial layer, due to the presence of oleosome membrane proteins at the interface.