Fatty acid/monoglyceride type and amount modulate fat-soluble vitamin absorption from mixed assemblies in mice.

We investigated the effects of fatty acid/ monoglyceride type and amount on the absorption of fat-soluble vitamins. Micelles or vesicles made with either caprylic acid (CA) + monocaprylin (MC) or oleic acid (OA) + monoolein (MO) at low or high concentrations were infused in bile duct-ligated mice. Retinol + retinyl ester and γ-tocopherol intestinal mucosa contents were higher in mice infused with CA + MC than with OA + MO (up to + 350 % for vitamin A and up to + 62 %, for vitamin E; p < 0.05). Cholecalciferol intestinal mucosa content was the highest in mice infused with micelles with CA + MC at 5 mg/mL (up to + 105 %, p < 0.05). Retinyl ester plasma response was higher with mixed assemblies formed at low concentration of FA + MG compared to high concentration (up to + 1212 %, p < 0.05), while no difference in cholecalciferol and γ-tocopherol plasma responses were measured. No correlation between size or zeta potential and vitamin absorption was found. The impact of FA and MG on fat-soluble vitamin absorption thus differs from one vitamin to another and should be considered to formulate adequate vitamin oral or enteral supplements.

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.

Compositional, Structural, and Kinetic Aspects of Lipid Digestion and Bioavailability: In Vitro, In Vivo, and Modeling Approaches.

Lipid digestion and bioavailability are usually investigated separately, using different approaches (in vitro, modeling, in vivo). However, a few inclusive studies show that their kinetics are closely linked. Lipid bioavailability kinetics is likely involved in the development and evolution of several diseases, so lipid digestion kinetics could be involved as well and can be modulated by food design or combination. To illustrate this possibility, the compositional and structural aspects of lipid digestion kinetics, as investigated using in vitro and modeling approaches, are presented first. Then, in vivo and mixed approaches enabling the study of both kinetics are reviewed and discussed. Finally, disparate modeling approaches are introduced, and a unifying modeling scheme is proposed, opening new perspectives for understanding the role and interactions of various factors (chemical, physical, and biological) involved in lipid metabolism.

Enhancement of the Anti-Angiogenic Effects of Delphinidin When Encapsulated within Small Extracellular Vesicles.

(1) Background: The anthocyanin delphinidin exhibits anti-angiogenic properties both in in vitro and in vivo angiogenesis models. However, in vivo delphinidin is poorly absorbed, thus its modest bioavailability and stability reduce its anti-angiogenic effects. The present work takes advantage of small extracellular vesicle (sEV) properties to enhance both the stability and efficacy of delphinidin. When encapsulated in sEVs, delphinidin inhibits the different stages of angiogenesis on human aortic endothelial cells (HAoECs). (2) Methods: sEVs from immature dendritic cells were produced and loaded with delphinidin. A method based on UHPLC-HRMS was implemented to assess delphinidin metabolites within sEVs. Proliferation assay, nitric oxide (NO) production and Matrigel assay were evaluated in HAoECs. (3) Results: Delphinidine, 3-O-ß-rutinoside and Peonidin-3-galactoside were found both in delphinidin and delphinidin-loaded sEVs. sEV-loaded delphinidin increased the potency of free delphinidin 2-fold for endothelial proliferation, 10-fold for endothelial NO production and 100-fold for capillary-like formation. Thus, sEV-loaded delphinidin exerts effects on the different steps of angiogenesis. (4) Conclusions: sEVs may be considered as a promising approach to deliver delphinidin to target angiogenesis-related diseases, including cancer and pathologies associated with excess vascularization.

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.

In vitro solubilization of fat-soluble vitamins in structurally defined mixed intestinal assemblies.

The structures of fed state intestinal assemblies containing bile components, dietary fat, and fat-soluble vitamins are not well known, although they are involved in lipid transport. In this study, several methods were used to investigate structural transitions upon various dietary lipids or various fat-soluble vitamins incorporation in bile intestinal assemblies. In particular, DLS and turbidimetry were used to study transition points as a function of component concentration, and cryo-TEM and SAXS were used to resolve assembly structures at microscopic and supramolecular scales, respectively. Results showed that increasing the concentration of dietary lipids in bile assembly induced a transition from core-shell micelles to unilamellar vesicles (except with caprylate lipids, always yielding micelles). In these specific assemblies, increasing the concentration of a fat-soluble vitamin either induced a systematic structural transition, defining a solubilization capacity (α-tocopherol or phylloquinone), or induced a structural transition only in micelles (retinol), or did not induce any structural transition up to very high concentrations (cholecalciferol). Using SAXS data, ideal molecular organizations are proposed for assemblies in the absence or presence of α-tocopherol.

A standardised semi-dynamic in vitro digestion method suitable for food - an international consensus.

The link between food and human health is increasingly a topic of interest. One avenue of study has been to assess food disintegration and interactions within the gastrointestinal tract. In vitro digestion models have been widely used to overcome the constrictions associated with in vivo methodology. The COST Action INFOGEST developed an international, harmonised protocol for static simulation of digestion in the upper gastrointestinal tract of adults. This protocol is widely used; however, it is restricted to providing end-point assessment without considering the possible structural changes. On the other hand, there are dynamic models that provide more physiologically relevant data but are expensive and difficult to access. There is a gap between these models. The method outlined in this article provides an intermediate model; it builds upon the harmonised static model and now includes crucial kinetic aspects associated with the gastric phase of digestion, including gradual acidification, fluid and enzyme secretion and emptying. This paper provides guidance and standardised recommendations of a physiologically relevant semi-dynamic in vitro simulation of upper gastrointestinal tract digestion, with particular focus on the gastric phase. Adaptations of this model have already been used to provide kinetic data on nutrient digestion and structural changes during the gastric phase that impact on nutrient absorption. Moreover, it provides a simple tool that can be used in a wide range of laboratories.

Manipulating and studying triglyceride droplets in microfluidic devices.

Triglyceride is the main lipid class in nature, found as droplets in both living systems and man-made products (such as manufactured foods and drugs). Characterizing triglyceride droplets in situ in these systems is complex due to many environmental interactions. To answer basic research questions about droplet formation, structuration, stability, or degradation, microfluidic strategies were developed, allowing well-controlled droplets to be formed, manipulated, and studied. In this review, these strategies are described, starting with the presentation of droplet production devices, with applications essentially related to microencapsulation and delivery, then detailing methods to monitor droplet degradation in situ and in real time, finishing with microfluidic platforms allowing the investigation of many aspects of biological lipid droplets simultaneously.

INFOGEST static in vitro simulation of gastrointestinal food digestion.

Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.

Studying the real-time interplay between triglyceride digestion and lipophilic micronutrient bioaccessibility using droplet microfluidics. 1 lab on a chip method.

This article is the first part of a series reporting on real-time digestion kinetics of triglyceride droplets containing different lipophilic micronutrients. This part focuses on the design, fabrication, and operation of a polydimethylsiloxane microfluidic device which enables the generation and digestion of oil droplets. The micro-channels were made hydrophilic to obtain oil droplets in an aqueous continuous phase. Optimized chip design and outlet control were implemented to provide efficient oil droplet generation, manipulation, and immobilization on a single chip. Highly monodisperse oil droplets were generated, immobilized in an array of traps and monitored in real time by fluorescence using a confocal microscopy method. The device was used to study the kinetics of beta-carotene release during tricaprylin digestion (intestinal lipolysis and micellar solubilization). The effect of the gastric phase on beta-carotene degradation was also investigated using the same method.

Studying the real-time interplay between triglyceride digestion and lipophilic micronutrient bioaccessibility using droplet microfluidics. 2 application to various oils and (pro)vitamins.

The kinetics of micellar solubilization of lipophilic micronutrients (bioaccessibility) in relation with triglyceride digestion remains poorly known. To study this interplay in real-time, a droplet microfluidic method was designed and used as reported in the first part of this article series. In this second part, the interplay between the micellar solubilization of (pro)vitamins (beta-carotene or retinyl palmitate) and the digestion of triglyceride oils (tricaprylin TC, or high-oleic sunflower seed oil HOSO, or fish oil FO) during simulated gastrointestinal digestion was investigated. The relation between the release of both micronutrients and of triglyceride lipolytic products was found to be non-linear. The kinetics of beta-carotene was found to follow the kinetics of lipolytic products, depending on the oil type (TC > HOSO > FO). The effect of the gastric phase on the intestinal phase was also found to follow this order, mostly due to partial lipolysis during the gastric phase.

Bioavailability of Nutrients and Micronutrients: Advances in Modeling and In Vitro Approaches.

The bioavailability of food nutrients and microconstituents is recognized as a determinant factor for optimal health status. However, human and animal studies are expensive and limited by the large amount of potential food bioactive compounds. The search for alternatives is very active and raises many questions. On one hand, in vitro digestion systems are good candidates, but to date only bioaccessibility has been correctly assessed. To go further, to what degree should natural processes be reproduced? What techniques can be used to measure the changes in food properties and structures in situ in a noninvasive way? On the other hand, modeling approaches have good potential, but their development is time-consuming. What compromises should be done between food and physiology realism and computational ease? This review addresses these questions by identifying highly resolved analytical methods, detailed computer models and simulations, and the most promising dynamic in vitro systems.

Bioaccessibility of lipophilic micro-constituents from a lipid emulsion.

Digestion is an important process, the first one in the conversion of food to energy. From this angle, digestion of nutrients was extensively studied, and this process was found to be very efficient. Nevertheless, many molecules contained in food do not bring energy but are essential as they allow maintaining normal body functions. These are the micro-nutrients, including vitamins and minerals. On top of that, recent nutrition research identified many other bioactive molecules (termed micro-constituents as they only represent a small part of the food) playing a role in the health status, e.g. contributing to the prevention of chronic diseases. However, it was shown that their digestion is much less efficient, especially that of lipophilic micro-constituents (such as lipophilic vitamins, carotenoids, cholesterol and other steroids) depending on food structure and composition. Enhancing their health effects through optimal absorption and bioavailability thus requires a comprehensive knowledge of their release from food within the gastrointestinal tract. To study this step, of which the endpoint is termed bioaccessibility, in vitro digestion methods proved to be well adapted to fundamental research. This review reports the effects of the physicochemical parameters controlling the bioaccessibility of various lipophilic micro-constituents from emulsion. Notably, it appears that this bioaccessibility is related to the bioaccessibility of lipid nutrients, as their kinetics are interrelated. This knowledge will enable the formulation of food in terms of structure and composition to obtain optimal bioaccessibility. As the latter likely controls bioavailability, prevention of some metabolic disorders could be targeted in the long term.

In vitro digestion of emulsions: high spatiotemporal resolution using synchrotron SAXS.

Although the biochemical processes of lipid digestion are well-known, the biophysical ones, responsible for the assembly of molecules into functional structures, lack studies resolving both time and space scales. About 35 years ago, the seminal microscopy study of Patton and Carey constituted a major advance to reach this goal. Nowadays, new perspectives arise from the availability of large facilities scattering techniques, able to monitor the dynamics of multi-scale assemblies with unprecedented resolutions. The present small angle X-ray scattering (SAXS) study focused on the roles of the emulsifier and triglyceride in the formation of lipid assemblies during emulsion digestion in vitro. By developing several interpretations of the data in the whole space range (qualitative, shape-dependent and shape-independent models), the characteristic size of the assemblies and their transition times were obtained, which depended on the triglyceride, but not on the emulsifier. The major assembly formed was found to be a spherical mixed micelle, but vesicle was also found to coexist throughout the digestion, although in a lower proportion. The quantitative determination of the sizes and proportions of these assemblies, as well as the evolution of these characteristics during digestion are precious information for nutritional sciences, as these assemblies are the vehicles of lipophilic nutrients and micronutrients towards their absorption site.

Refining in silico simulation to study digestion parameters affecting the bioaccessibility of lipophilic nutrients and micronutrients.

Despite the considerable number of in vivo and in vitro studies on the digestive fate of lipophilic nutrients, micronutrients, and bioactives, the effects of the structure and composition of foods on the physicochemical mechanisms of luminal digestion are still poorly understood. Studying them is indeed complex because the number of parameters is high and many of them are interdependent. To solve this problem, an in silico simulation based on a multi-agent system was recently proposed to study the intestinal bioaccessibility of lipophilic nutrients and micronutrients from a single oil droplet. The roles of lipolysis and solubilization in bile salt were included. The effects of several food and digestion parameters were in line with those reported in the experimental literature. The goal of the research reported in this new article was to include more digestion parameters in the simulation in order to make it more realistic against complex cases. This was done in one specific digestion condition reflecting in vitro experiments, using droplets of tricaprylin or triolein containing vitamin A. The structure and principles of the original model were kept, with independent local modifications in order to study each factor separately. First, a gastric step was added where lipolysis took place, and only a marginal effect on the following intestinal step was found. Then, the chemical form of vitamin A, either non-hydrolyzed retinyl ester or retinyl ester instantly hydrolyzed into retinol, was investigated by considering different localizations in the droplet, resulting in a higher bioaccessibility for the retinol. The case of a mixture of tricaprylin and triolein indicated an influence of the oil phase viscosity. The consideration of mixed micelles compared to simple bile salt micelles was also investigated, and resulted in a higher vitamin A bioaccessibility, especially with triolein. Finally, a full model including the most influential parameters was tested to simulate the digestion of triglyceride-limonene mixtures, giving bioaccessibility trends in very good agreement with the literature.

A microfluidic device to study the digestion of trapped lipid droplets.

At the junction of chemistry, physics and biology, digestion involves many processes. Studying the mechanisms in such a complex system is challenging because numerous interactions coexist. Even in an apparently simple system such as an emulsion, many physicochemical characteristics affect lipid digestion. Moreover, these characteristics are difficult to control using conventional in vitro techniques. The goal of this work was to design a microfluidic device allowing the study of well-controlled individual oil droplets under gastrointestinal digestion conditions. Different parameters were investigated in order to validate the relevance of this device compared to conventional in vitro techniques using emulsions. Various triglycerides and digestion conditions were tested with droplets of the same initial diameter generated by a flow focusing device, then placed in individual traps of a microfluidic chamber for digestion with continuous digestive juice renewal. The results are in good agreement with those obtained with conventional in vitro techniques and open the way to screening of lipid digestion, in particular, bioaccessibility of lipophilic molecules, a prerequisite for bioavailability studied in nutrition, pharmacology, and toxicology.

A coarse-grained simulation to study the digestion and bioaccessibility of lipophilic nutrients and micronutrients in emulsion.

The digestion of lipophilic nutrients and micronutrients requires numerous and simultaneous processes of chemical, physical and biological nature. Studying these processes experimentally is challenging, explaining why there is only little information about the mechanisms and interactions involved. Nevertheless, the bioaccessibility of lipophilic micronutrients is poorly understood so new investigation approaches are needed, all the more when digestion of lipophilic nutrients is also involved. In this article, the development of a coarse-grained simulation with no adjustable parameter is reported, enabling the study of the chemical and physical processes controlling bioaccessibility in such systems. The intestinal digestion of a droplet of a pure triglyceride containing a lipophilic vitamin was simulated to obtain their bioaccessibility kinetics (via lipolysis and/or solubilization in bile salt). The parameters examined here were the type of triglyceride, the type of vitamin, the digestive fluid amount, the droplet size, and different digestion conditions reflecting the in vitro or in vivo cases. Among these structure and composition parameters, the type of triglyceride and the digestion conditions had the greatest effects on bioaccessibility. An interplay between triglyceride digestion and micronutrient bioaccessibility kinetics was evidenced, highlighting the roles of the different parameters, in agreement with the experimental literature. This new approach is shown to be relevant to both nutrition and pharmacology.

Bioaccessibility of nutrients and micronutrients from dispersed food systems: impact of the multiscale bulk and interfacial structures.

Many food systems are dispersed systems, that is, they possess at least two immiscible phases. This is generally due to the coexistence of domains with different physicochemical properties separated by many interfaces which control the apparent thermodynamic equilibrium. This feature was and is still largely studied to design pharmaceutical delivery systems. In food science, the recent intensification of in vitro digestion tests to complement the in vivo ones holds promises in the identification of the key parameters controlling the bioaccessibility of nutrients and micronutrients. In this review, we present the developments of in vitro digestion tests for dispersed food systems (mainly emulsions, dispersions and gels). We especially highlight the evidences detailing the roles of the constituting multiscale structures. In a perspective section, we show the potential of structured interfaces to allow controlled bioaccessibility.

In vitro digestion of fish oils rich in n-3 polyunsaturated fatty acids studied in emulsion and at the oil-water interface.

The in vitro digestion of ß-lactoglobulin stabilized emulsions rich in the n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), was studied using several physicochemical techniques. Artificial media for the mouth, stomach and small intestine were used in a sequential static in vitro digestion method. Different sizing techniques were compared to follow the droplet size during the digestion steps, including diffusing wave spectroscopy (DWS) which allowed direct measurements on undiluted emulsions. Titration of fatty acids confirmed that the digestion of such emulsified fish oils is partial. The study of the digestion at the oil-water interface using tensiometry revealed specific affinities between lipids and proteins. These could explain the emulsion and the single droplet lipolysis. Nevertheless, by comparing our results to a previous study on fish oil lipolysis, we identified two other important factors. Those were the aqueous solubility and the rate of hydrolysis of the individual fatty acids, the emulsion with the most soluble and hydrolysable ones being digested more quickly.

Relaxation processes of PGPR at the water/oil interface inferred by oscillatory or transient viscoelasticity measurements.

The rheological properties of PolyGlycerol PolyRicinoleate (PGPR) at the oil/water interface were studied using a drop-shaped tensiometer. Small deformation oscillations of the drop area allow the measurement of the interfacial viscoelasticity spectrum, that is, the elastic and viscous moduli as a function of frequency. Another way to obtain such a spectrum is to perform a transient relaxation measurement from which the relaxation modulus as a function of time is deduced and interpreted. Several models containing one or more relaxation times were considered, and their resulting spectra were compared to the oscillatory ones. Similar results suggest that one could in principle use oscillatory or transient relaxations indifferently. However, the transient relaxation technique proved to be more adapted for the determination of the relaxation times. At low PGPR concentrations in oil, the behavior is controlled by long relaxation times, whereas short ones take over when approaching and exceeding the saturation interfacial concentration. This was understood as a shift from a diffusion-dominated regime to a rearrangements-dominated regime.