Factors impacting lipid digestion and nutraceutical bioaccessibility assessed by standardized gastrointestinal model (INFOGEST): Emulsifier type.

This paper is part of a series examining the impact of the main factors influencing lipid digestion and nutraceutical bioaccessibility in ß-carotene-loaded oil-in-water emulsions using the harmonized INFOGEST simulated gastrointestinal model. Here, the impact of emulsifier type was examined since food emulsions and nutraceutical delivery systems are often stabilized by various types of emulsifier. The INFOGEST method was adopted to investigate the in vitro gastrointestinal fate of emulsions stabilized by five kinds of food-grade emulsifier representing different classes: synthetic surfactants (Tween 20); natural surfactants (quillaja saponin); proteins (caseinate); polysaccharides (gum arabic); and phospholipids (soy lysolecithin). Microfluidization produced emulsions with small droplet sizes for all emulsifiers, except soy lysolecithin. Within the gastrointestinal model, the caseinate-coated oil droplets had the worst gastric stability, with severe droplet flocculation and coalescence occurring in the stomach. The fraction of the lipid phase that had been digested by the end of the gastrointestinal model was considerably lower for the emulsions stabilized by soy lysolecithin (93%) or caseinate (93%), than those stabilized by gum arabic (99%), quillaja saponin (111%) or Tween 20 (117%). This effect was attributed to lower surface area of lipids available for lipase to attach to for the lysolecithin and caseinate emulsions. The overall bioaccessibility of the ß-carotene increased in this order: lysolecithin (25%) < gum arabic (51%) < caseinate (55%) < quillaja saponin (56%) < Tween 20 (62%). The impact of emulsifier type on carotenoid bioaccessibility was ascribed to various factors: (i) some emulsifiers inhibited lipid digestion and so a fraction of the ß-carotene remained inside the undigested droplets and the mixed micelle phase had less solubilization capacity, i.e., lysolecithin, and caseinate; (ii) some emulsifiers protected ß-carotene from chemical degradation, i.e., lysolecithin and caseinate; and (iii) some emulsifiers promoted sedimentation of the ß-carotene-loaded micelles, i.e., lysolecithin. These results suggest that food emulsion behavior in the human gut may be influenced by the nature of the emulsifier employed, which is important knowledge when creating functional food and beverage products.

Stabilization of soybean oil-in-water emulsions using polypeptide multilayers: Cationic polylysine and anionic polyglutamic acid.

Oil-in-water emulsions are used as delivery systems for non-polar functional ingredients in various industries, including foods, cosmetics, personal care products, agrochemicals, and pharmaceuticals. Emulsions, however, tend to breakdown under the conditions found in many commercial products. In this study, the functional performance of the lipid droplets in emulsions was tailored by sequential layer-by-layer electrostatic deposition of oppositely charged polypeptides onto their surfaces. Cationic poly-L-lysine (PLL) and anionic poly-glutamic acid (PGA) were used as a pair of oppositely charged polypeptides (pH 4.0). First, a primary emulsion (10% w/w soybean oil-in-water emulsion) was formed consisting of small lipid droplets (d32 = 500 µm) coated by a natural surfactant (0.05% w/w quillaja saponin). Second, cationic PLL was deposited onto the surfaces of the anionic saponin-coated droplets. Third, anionic PGA was deposited onto the surfaces of the cationic PLL-saponin-coated droplets. We then assessed the ability of the coatings to protect the lipid droplets from aggregation when the pH (2.0-9.0), ionic strength (0-350 mM), or temperature (30-90 °C) were altered. The properties of the primary, secondary, and tertiary emulsions were monitored by measuring the mean particle diameter (d32), electrical characteristics (ζ-potential), and microstructure of the lipid droplets. The electrical characteristics of the droplets could be modulated by controlling the number and type of layers used. The primary emulsion had the best resistance to varying environmental conditions, while the secondary emulsion had the worst, suggesting electrostatic deposition should only be used to obtain specific functionalities. Interestingly, PLL detached from the surfaces of the secondary emulsions at high salt concentrations due to electrostatic screening, which improved their salt stability. This phenomenon may be useful for some food applications, e.g., having cationic droplets during food storage, but anionic ones inside the human body.

Impact of fat crystallization on the resistance of W/O/W emulsions to osmotic stress: Potential for temperature-triggered release.

Water-in-oil-in-water (W/O/W) emulsions can be designed to encapsulate, protect, and release both hydrophilic and hydrophobic functional compounds. In this study, we examined the impact of crystallizing the fat phase on the resistance of W/O/W emulsions to osmotic stress, with the aim of developing osmotic-responsive systems. Polyglycerol polyricinoleate (PGPR) was used as a hydrophobic surfactant to stabilize the inner water droplets, while Quillaja saponin and whey protein isolate (WPI) were used as hydrophilic surfactants to coat the oil droplets. The impact of fat crystallization was examined by using either a liquid (soybean oil, SO) or semi-solid (hydrogenated soybean oil, HSO) fat as the oil phase. An osmotic stress was generated by establishing a sucrose concentration gradient between the internal and external water phases. Alterations in the droplet size, morphology, and stability of the W/O/W emulsions was measured when the sucrose concentration gradient was changed. The W/O droplets in the SO-emulsions swelled/shrank when the external sucrose concentration was below/above the internal sucrose concentration, which is indicative of water diffusing into/out of the droplets. Conversely, there was no change in the size of the W/O droplets in the HSO-emulsions under the same conditions, which was attributed to the mechanical strength of the fat crystal network resisting swelling or shrinking. HSO-emulsions did exhibit swelling when they were heated above a critical temperature, due to melting of the fat crystals and disruption of the crystal network. Our results demonstrate that crystallization of the oil phase of W/O/W emulsions can prevent water transport due to osmotic stress, which may be useful for developing temperature-triggered delivery systems for application in foods, cosmetics, pharmaceuticals, or personal care products.

Characterization of electrostatic interactions and complex formation of ɣ-poly-glutamic acid (PGA) and ɛ-poly-l-lysine (PLL) in aqueous solutions.

Complex coacervation is a useful approach for creating biopolymer-based colloidal particles for the oral delivery of bioactives, such as nutraceuticals, vitamins, and pharmaceuticals. In this study, we examined the possibility of using anionic É£-poly-glutamic acid (PGA) and cationic ɛ-poly-l-lysine (PLL) to form polyelectrolyte complexes. Initially, the formation and properties of the complexes were characterized using visual observations, UV-visible spectrophotometry, microelectrophoresis (ζ-potential), and isothermal titration calorimetry (ITC). The impact of pH, ionic strength, temperature, and polymer ratio on complex formation was examined. The electrostatic complexes formed had a 1:4 mass ratio of polyanion-to-polycation at saturation (pH 7.4). The surface potential and aggregation stability of the complexes was highly dependent on solution pH (2-12), which was attributed to alterations in the electrical characteristics of the two polyelectrolytes. In particular, insoluble complexes were formed under pH conditions where there was a strong electrostatic attraction between the two polyelectrolytes, whereas soluble complexes were formed when there was only a weak attraction. The addition of salt (≥20 mM NaCl) promoted aggregation of the complexes, presumably due to screening of the electrostatic interactions between them. Conversely, temperature (25-90 °C) did not have a major impact on the stability of the complexes. These results may be useful for the design of effective oral delivery systems for bioactive agents in foods and other products.

Impact of calcium levels on lipid digestion and nutraceutical bioaccessibility in nanoemulsion delivery systems studied using standardized INFOGEST digestion protocol.

Recently, the standardized in vitro digestion model ("INFOGEST method") used to evaluate the gastrointestinal fate of foods has been revised and updated (Brodkorb et al., 2019, Nat. Protoc., 2019, 14, 991-1014). Under fed state conditions, the calcium level used in this model is fixed and relatively low: 0.525 mM. In practice, the calcium concentration in the human gut depends on the nature of the food consumed and may vary from person-to-person. For this reason, we examined the impact of calcium concentration on the gastrointestinal fate of a model nutraceutical delivery system. The effect of calcium level (0.525-10 mM) on lipid digestion and ß-carotene bioaccessibility in corn oil-in-water nanoemulsion was investigated using the INFOGEST method. At all calcium levels, the lipids were fully digested, but this could only be established by carrying out a back titration (to pH 9) at the end of the small intestine phase. Conversely, the bioaccessibility of ß-carotene decreased with increasing calcium levels: from 65.5% at 0.525 mM Ca2+ to 23.7% at 10 mM Ca2+. This effect was attributed to the ability of the calcium ions to precipitate the ß-carotene-loaded mixed micelles by forming insoluble calcium soaps. The ability of calcium ions to reduce carotenoid bioaccessibility may have important nutritional implications. Our results show that the bioaccessibility of hydrophobic carotenoids measured using the INFOGEST method is highly dependent on the calcium levels employed, which may have important consequences for certain calcium-rich foods. Moreover, we have shown the importance of carrying out a back titration to accurately measure free fatty acid levels in the presence of low calcium levels.

Chitosan reduces vitamin D bioaccessibility in food emulsions by binding to mixed micelles.

Consumption of sufficiently high quantities of dietary fibers has been linked to a range of health benefits. Recent research, however, has shown that some dietary fibers interfere with lipid digestion, which may reduce the bioavailability of oil-soluble vitamins and nutraceuticals. For this reason, we examined the impact of a cationic polysaccharide (chitosan) on the bioaccessibility of vitamin D using the standardized INFOGEST in vitro digestion model. The vitamin D was encapsulated within an emulsion-based delivery system that contained whey protein-coated corn oil droplets. Our results showed that chitosan promoted severe droplet flocculation in the small intestine and reduced the amount of free fatty acids detected using a pH-stat method. However, a back-titration of the digested sample showed that the lipids were fully digested at all chitosan levels used (0.1-0.5%), suggesting that chitosan may have bound some of the free fatty acids released during lipid digestion. The presence of the chitosan decreased the bioaccessibility of vitamin D by about 37%, but this effect did not depend strongly on chitosan concentration (0.1-0.5%). It was hypothesized that chitosan bound to the vitamin-loaded mixed micelles and promoted their precipitation. The knowledge gained in this study might provide useful insights in designing emulsion-based delivery systems with high vitamin bioaccessibility.

Modulation of Physicochemical Characteristics of Pickering Emulsions: Utilization of Nanocellulose- and Nanochitin-Coated Lipid Droplet Blends.

Mixed Pickering emulsions were prepared by blending anionic nanocellulose-stabilized lipid droplets with cationic nanochitin-stabilized lipid droplets. Changes in the surface potential, particle size, shear viscosity, and morphology of the mixed emulsions were characterized when the droplet mixing ratio was varied. Emulsion properties could be tailored by altering the pH and mixing ratio. Surface potential measurements suggested that the nanochitin-coated lipid droplets adsorbed to the surfaces of the nanocellulose-coated lipid droplets, thereby dominating the overall electrical characteristics of the mixed emulsions. As a result, the mixed emulsions had better stability to coalescence than the single emulsions containing only nanocellulose-coated lipid droplets. Our results suggest that the physicochemical properties, shelf life, and functional performance of Pickering emulsions may be modulated by blending different kinds of particle-stabilized lipid droplets together.

In situ monitoring of lipid droplet release from biopolymer microgels under simulated gastric conditions using magnetic resonance imaging and spectroscopy.

Functional foods are being designed to breakdown in the human gut in a controlled fashion so as to regulate blood nutrient profiles, hormone release, energy intake, and bioavailability. Biopolymer microgels, small spherical beads made from proteins and/or polysaccharides, are a promising tool that can be used to modulate the gastrointestinal behavior of food components. In this study, lipid droplets were loaded into microgels fabricated from either alginate or carrageenan using an injection-gelation method. The lipid-loaded microgels were then incubated in simulated gastric juices and the impact of biopolymer type on the release of the lipid droplets was observed. Optical microscopy and turbidity measurements showed that lipid droplet release occurred more rapidly for the carrageenan microgels, which was attributed to their partial disruption in the gastric fluids. In contrast, lipid droplet release was relatively slow from the alginate microgels because they remained intact. We also showed that magnetic resonance imaging (MRI) could be used for in situ monitoring of carrageenan microgel disruption and lipid droplet release under simulated stomach conditions. This method was based on quantifying the local lipid levels using T1 images and the magnetic resonance spectroscopy. A water-selected VIBE sequence was optimized for obtaining the T1 images and a stimulated echo acquisition mode (STEAM) sequence was selected for obtaining the MR spectra. The MRI method may be particularly useful for in vivo studies of the behavior of filled microgels.

Impact of an indigestible oil phase (mineral oil) on the bioaccessibility of vitamin D3 encapsulated in whey protein-stabilized nanoemulsions.

These is some interest in replacing digestible fats with indigestible ones to decrease the energy-density of foods. The utilization of indigestible oils, however, may have unforeseen nutritional consequences, such as the reduction of vitamin bioavailability. In this study, the impact of an indigestible oil on the bioaccessibility of emulsified vitamin D3 (VD) was examined. We prepared four kinds of nanoemulsions using different combinations of a digestible oil (DO) and an indigestible oil (IO): DO only; IO only; an oil mixture (OM) consisting of 1:1 DO:IO mixed before homogenization; and, an emulsion mixture (EM) consisting of 1:1 DO:IO nanoemulsions mixed after homogenization. A gastrointestinal tract (GIT) model was employed to elucidate the kinetics of VD bioaccessibility from the nanoemulsions. Both the lipid digestion rate and vitamin bioaccessibility decreased in the same order: DO > OM ≈ EM > IO. The change in vitamin bioaccessibility over time under simulated small intestine conditions was also measured. With the exception of the IO nanoemulsions, the vitamin bioaccessibility increased to a maximum value after around 30 min, but then decreased during the following 24 h. This effect was attributed to an initial solubilization of the vitamin within the mixed micelles, followed by their precipitation during prolonged incubation. Our results show that lipid digestion, micelle solubilization, and micelle aggregation impact the in vitro bioaccessibility of vitamin D. This knowledge may be helpful for designing more efficacious nanoemulsion-based delivery systems for fat-soluble vitamins.

Role of Mucin in Behavior of Food-Grade TiO2 Nanoparticles under Simulated Oral Conditions.

Fine titanium dioxide (TiO2) particles have been used as additives (E171) to modify the optical properties of foods and beverages for many years. Commercial TiO2 additives, however, often contain a significant fraction of nanoparticles (diameter <100 nm), which has led to some concern about their potentially adverse health effects. At present, relatively little is known about how the characteristics of TiO2 particles are altered as they travel through the human gastrointestinal tract. Alterations in their electrical characteristics, surface composition, or aggregation state would be expected to alter their gastrointestinal fate. The main focus of this study was, therefore, to characterize the behavior of TiO2 particles under simulated oral conditions. Changes in the aggregation state and electrical characteristics were monitored using particle size, ζ-potential, turbidity, and electron microscopy measurements, whereas information about mucin-particle interactions were obtained using isothermal titration calorimetry and surface-enhanced Raman spectroscopy. Our results indicate that there was a strong interaction between TiO2 and mucin: mucin absorbed to the surfaces of the TiO2 particles and reduced their tendency to aggregate. The information obtained in this study is useful for better understanding the gastrointestinal fate and potential toxicity of ingested inorganic particles.