Plant and animal protein mixed systems as wall material for microencapsulation of Manuka essential Oil: Characterization and in vitro release kinetics.

Combination of plant and animal protein diet is becoming a valuable source of nutrition in the modern diet due to the synergistic functional properties inherent in these protein complexes. Moreover, the synergy between animal and plant proteins can contribute to the high stability and improved solubility of the encapsulated bioactive ingredients (e.g., essential oils). Therefore, the study was designed to evaluate the plant (pea protein (PP) and lupine protein (LP)) and animal protein (whey protein, WP) mixed systems as a wall material for microencapsulation of manuka essential oil, as an example of bioactive compound. Moreover, physicochemical properties and in vitro release profile of encapsulated manuka essential oil were studied. Manuka essential oil microcapsules exhibited low moisture content (5.3-7.1 %) and low water activity (0.33-0.37) with a solubility of 53.7-68.1 %. Change in wall material ratio significantly affected the color of microcapsules, while microcapsules prepared with 1:1 protein/oil ratio demonstrated a high encapsulation efficiency (90.4 % and 89.4 %) for protein mixed systems (PP + WP and LP + WP), respectively. Microcapsules further showed low values for lipid oxidation with a high oxidative stability and antioxidant activity (62.1-87.0 %). The zero order and Korsmeyer-Peppas models clearly explained the release mechanism of encapsulated oil, which was dependent on the type and concentration of the protein mixed used. The findings demonstrated that the protein mixed systems successfully encapsulated the manuka essential oil with controlled release and high oxidative stability, indicating the suitability of the protein mixed systems as a carrier in encapsulation and application potential in development of encapsulated functional foods.

Influence of pH and lipid membrane on the liquid-liquid phase separation of wheat γ-gliadin in aqueous conditions.

Protein body (PB) formation in wheat seeds is a critical process influencing seed content and nutritional quality. In this study, we investigate the potential mechanisms governing PB formation through an in vitro approach, focusing on γ-gliadin, a key wheat storage protein. We used a microfluidic technique to encapsulate γ-gliadin within giant unilamellar vesicles (GUVs) and tune the physicochemical conditions in a controlled and rapid way. We examined the influence of pH and protein concentration on LLPS and protein-membrane interactions using various microscopy and spectroscopy techniques. We showed that γ-gliadin encapsulated in GUVs can undergo a pH-triggered liquid-liquid phase separation (LLPS) by two distinct mechanisms depending on the γ-gliadin concentration. At low protein concentrations, γ-gliadins phase separate by a nucleation and growth-like process, while, at higher protein concentration and pH above 6.0, γ-gliadin formed a bi-continuous phase suggesting a spinodal decomposition-like mechanism. Fluorescence and microscopy data suggested that γ-gliadin dense phase exhibited affinity for the GUV membrane, forming a layer at the interface and affecting the reversibility of the phase separation.

In Situ ATR Spectroscopy Study of the Interaction of Acacia senegal Gum with Gold Nanoparticles Films at the Solid-Liquid Interface.

The adsorption process of Acacia gum (A. senegal), a complex heteropolysaccharide, was followed by using a spectroscopic method to unravel the relative contribution of the protein moieties and the carbohydrate blocks on the adsorption process. In situ ATR-FTIR was used to investigate the kinetics and conformational changes associated with the adsorption of A. senegal gum on gold nanoparticle films (Au-NPs) at different pHs. The results of this thorough study highlighted the adsorption of A. senegal gum through its protein moieties, in particular, AGPs of low molecular weight and high protein content, close to the Au-NPs surface. Isotherm experiments, by gradually increasing the concentration, showed that the gum adsorption was heterogeneous and followed the Freundlich model for the amide part, while the polysaccharide part followed the Langmuir model. In addition, the hydration and structural organization of the gum layer depended on the gum concentration. A. senegal gum adsorbed irreversibly on Au-NPs whatever the pHs, but the adsorbed layer presented a different behavior depending on pH. A more aggregated and less hydrated structure was observed at acidic pH, while a very hydrated and continuous layer was detected at higher pH. The secondary structure analysis through amide III band revealed a change in the gum secondary structure at high pH with the increase in ß-turn while random coil decreased.

Micromolding-based encapsulation of mesenchymal stromal cells in alginate for intraarticular injection in osteoarthritis.

Osteoarthritis (OA) is an inflammatory joint disease that affects cartilage, subchondral bone, and joint tissues. Undifferentiated Mesenchymal Stromal Cells are a promising therapeutic option for OA due to their ability to release anti-inflammatory, immuno-modulatory, and pro-regenerative factors. They can be embedded in hydrogels to prevent their tissue engraftment and subsequent differentiation. In this study, human adipose stromal cells are successfully encapsulated in alginate microgels via a micromolding method. Microencapsulated cells retain their in vitro metabolic activity and bioactivity and can sense and respond to inflammatory stimuli, including synovial fluids from OA patients. After intra-articular injection in a rabbit model of post-traumatic OA, a single dose of microencapsulated human cells exhibit properties matching those of non-encapsulated cells. At 6 and 12 weeks post-injection, we evidenced a tendency toward a decreased OA severity, an increased expression of aggrecan, and a reduced expression of aggrecanase-generated catabolic neoepitope. Thus, these findings establish the feasibility, safety, and efficacy of injecting cells encapsulated in microgels, opening the door to a long-term follow-up in canine OA patients.

Recent trends in design of healthier plant-based alternatives: nutritional profile, gastrointestinal digestion, and consumer perception.

In recent years, various types of plant-based meat, dairy, and seafood alternatives merged in the health-conscious consumer market. However, plant-based alternatives present complexity in terms of nutritional profile and absorption of nutrients after food ingestion. Thus, this review summarizes current strategies of plant-based alternatives and their nutritional analysis along with gastrointestinal digestion and bioavailability. Additionally, regulatory frameworks, labeling claims, and consumer perception of plant-based alternatives are discussed thoroughly with a focus on status and future prospects. Plant-based alternatives become a mainstream of many food-processing industries with increasing alternative plant-based food manufacturing industries around the world. Novel food processing technologies could enable the improving of the taste of plant-based foods. However, it is still a technical challenge in production of plant-based alternatives with authentic meaty flavor. In vitro gastrointestinal digestion studies revealed differences in the digestion and absorption of plant-based alternatives and animal-based foods due to their protein type, structure, composition, anti-nutritional factors, fibers, and polysaccharides. Overall, plant-based alternatives may facilitate the replacement of animal-based foods; however, improvements in nutritional profile and in vitro digestion should be addressed by application of novel processing technologies and food fortification. The specific legislation standards should be necessary to avoid consumer misleading of plant-based alternatives.

Adsorption Behavior of Arabinogalactan-Proteins (AGPs) from Acacia senegal Gum at a Solid-Liquid Interface.

Adsorption of five different hyperbranched arabinogalactan-protein (AGP) fractions from Acacia senegal gum was thoroughly studied at the solid-liquid interface using a quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), and atomic force microscopy (AFM). The impact of the protein/sugar ratio, molecular weight, and aggregation state on the adsorption capacity was investigated by studying AGP fractions with different structural and biochemical features. Adsorption on a solid surface would be primarily driven by the protein moiety of the AGPs through hydrophobic forces and electrostatic interactions. Increasing ionic strength allows the decrease in electrostatic repulsions and, therefore, the formation of high-coverage films with aggregates on the surface. However, the maximum adsorption capacity was not reached by fractions with a higher protein content but by a fraction that contains an average protein quantity and presents a high content of high-molecular-weight AGPs. The results of this thorough study highlighted that the AGP surface adsorption process would depend not only on the protein moiety and high-molecular-weight AGP content but also on other parameters such as the structural accessibility of proteins, the molecular weight distribution, and the AGP flexibility, allowing structural rearrangements on the surface and spreading to form a viscoelastic film.

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.

New exploration of the γ-gliadin structure through its partial hydrolysis.

The partial enzymatic hydrolysis of wheat gliadins constitutes an interesting tool to unravel their structural specificity. In this work, the structure and conformation of γ-gliadin were investigated through its limited chymotrypsic digestion. Using a combination of computational, biochemical and biophysical tools, we studied each of its N and C terminal domains. Our results reveal that γ-gliadin is a partially disordered protein with an unfolded N-terminal domain surprisingly resistant to chymotrypsin and a folded C-terminal domain. Using spectroscopic tools, we showed that structural transitions occured over the disordered N-terminal domain for decreasing ethanol/water ratios. Using SAXS measurements, low-resolution 3D structures of γ-gliadin were proposed. To relate the repeated motifs of the N-terminal domain of γ-gliadin to its structure, engineered peptide models PQQPY/F were also studied. Overall results demonstrated similarities between the N-terminal domain and its derived model peptides. Our findings support the use of these peptides as general templates for understanding the wheat protein assembly and dynamics.

Semi-permeable vesicles produced by microfluidics to tune the phase behaviour of encapsulated macromolecules.

Understanding the dynamics of macromolecular assemblies in solution, such as Liquid-Liquid Phase Separation (LLPS), represents technologic and fundamental challenges in many fields. In cell biology, such dynamics are of great interest, because of their involvement in subcellular processes. In our study, we aimed to control the assembly of macromolecules in aqueous semi-permeable vesicles, that we named osmosomes, using microfluidics. We developed a microfluidic chip that allows for producting and trapping Giant Unilamellar Vesicles (GUVs) encapsulating macromolecules. This device also allows for modification of the composition of the inner phase and of the membranes of the trapped GUVs. The vesicles are produced from water-in-oil-in-water (w/o/w) double emulsions in less than 20 min after discarding the oil phase. They are highly monodisperse and their diameter can be modulated between 20 and 110 µm by tuning the flow rates of fluid phases. Their unilamellarity is proofed by two techniques: (1) fluorescence quenching experiments and (2) the insertion of the α-hemolysin membrane protein pore. We demonstrate that the internal pH of osmosomes can be tuned in less than 1 min by controlling solvent exchanges through the α-hemolysin pores. The detailed analysis of the exchange kinetics suggests that the microfluidic chip provides an efficient pore formation due to the physical trapping of vesicles and the constant flow rate. Finally, we show a proof of concept for macromolecular assembly within osmosomes by pH-triggered LLPS of wheat proteins within a few minutes.

Role of protein conformation and weak interactions on γ-gliadin liquid-liquid phase separation.

Wheat storage proteins, gliadins, were found to form in vitro condensates in 55% ethanol/water mixture by decreasing temperature. The possible role of this liquid-liquid phase separation (LLPS) process on the in vivo gliadins storage is elusive and remains to be explored. Here we use γ-gliadin as a model of wheat proteins to probe gliadins behavior in conditions near physiological conditions. Bioinformatic analyses suggest that γ-gliadin is a hybrid protein with N-terminal domain predicted to be disordered and C-terminal domain predicted to be ordered. Spectroscopic data highlight the disordered nature of γ-gliadin. We developed an in vitro approach consisting to first solubilize γ-gliadin in 55% ethanol (v/v) and to progressively decrease ethanol ratio in favor of increased aqueous solution. Our results show the ability of γ-gliadin to self-assemble into dynamic droplets through LLPS, with saturation concentrations ranging from 25.9 µM ± 0.85 µM (35% ethanol (v/v)) to 3.8 µM ± 0.1 µM (0% ethanol (v/v)). We demonstrate the importance of the predicted ordered C-terminal domain of γ-gliadin in the LLPS by highlighting the protein condensates transition from a liquid to a solid state under reducing conditions. We demonstrate by increasing ionic strength the role displayed by electrostatic interactions in the phase separation. We also show the importance of hydrogen bonds in this process. Finally, we discuss the importance of gliadins condensates in their accumulation and storage in the wheat seed.

Soft-Matter Approaches for Controlling Food Protein Interactions and Assembly.

Animal- and plant-based proteins are present in a wide variety of raw and processed foods. They play an important role in determining the final structure of food matrices. Food proteins are diverse in terms of their biological origin, molecular structure, and supramolecular assembly. This diversity has led to segmented experimental studies that typically focus on one or two proteins but hinder a more general understanding of food protein structuring as a whole. In this review, we propose a unified view of how soft-matter physics can be used to control food protein assembly. We discuss physical models from polymer and colloidal science that best describe and predict the phase behavior of proteins. We explore the occurrence of phase transitions along two axes: increasing protein concentration and increasing molecular attraction. This review provides new perspectives on the link between the interactions, phase transitions, and assembly of proteins that can help in designing new food products and innovative food processing operations.

Application of Millifluidics to Encapsulate and Support Viable Human Mesenchymal Stem Cells in a Polysaccharide Hydrogel.

Human adipose-derived stromal cells (hASCs) are widely known for their immunomodulatory and anti-inflammatory properties. This study proposes a method to protect cells during and after their injection by encapsulation in a hydrogel using a droplet millifluidics technique. A biocompatible, self-hardening biomaterial composed of silanized-hydroxypropylmethylcellulose (Si-HPMC) hydrogel was used and dispersed in an oil continuous phase. Spherical particles with a mean diameter of 200 µm could be obtained in a reproducible manner. The viability of the encapsulated hASCs in the Si-HPMC particles was 70% after 14 days in vitro, confirming that the Si-HPMC particles supported the diffusion of nutrients, vitamins, and glucose essential for survival of the encapsulated hASCs. The combination of droplet millifluidics and biomaterials is therefore a very promising method for the development of new cellular microenvironments, with the potential for applications in biomedical engineering.

Oil encapsulation techniques using alginate as encapsulating agent: applications and drawbacks.

Oils are used in agriculture, nutrition, food and cosmetics; however, these substances are oxidisable and may readily lose their properties. To reduce their degradation or to mask certain undesirable aspects, one strategy consists in encapsulating the oil in inert structures (capsules). The capsules are classified according to the morphology, the number of cores and size, can be produced by several techniques: jet-cutting, vibrating jet, spray-drying, dispersion and milli-microfluidic. Among the polymers used as a membrane in the capsules, alginates are used in oil encapsulation because of their high gelling capacity, biocompatibility and low toxicity. In the presence of calcium ions, the alginate macromolecules crosslink to form a three-dimensional network called hydrogel. The oil encapsulation using alginate as encapsulating material can be carried out using technologies based on the external, internal or inverse gelation mechanisms. These capsules can found applications in areas as cosmetics, textile, foods and veterinary, for example.

Oil encapsulation in core-shell alginate capsules by inverse gelation II: comparison between dripping techniques using W/O or O/W emulsions.

In the first part of this article, it was described an innovative method of oil encapsulation from dripping-inverse gelation using water-in-oil (W/O) emulsions. It was noticed that the method of oil encapsulation was quite different depending on the emulsion type (W/O or oil-in-water (O/W)) used and that the emulsion structure (W/O or O/W) had a high impact on the dripping technique and the capsules characteristics. The objective of this article was to elucidate the differences between the dripping techniques using both emulsions and compare the capsule properties (mechanical resistance and release of actives). The oil encapsulation using O/W emulsions was easier to perform and did not require the use of emulsion destabilisers. However, capsules produced from W/O emulsions were more resistant to compression and showed the slower release of actives over time. The findings detailed here widened the knowledge of the inverse gelation and gave opportunities to develop new techniques of oil encapsulation.

Oil encapsulation in core-shell alginate capsules by inverse gelation. I: dripping methodology.

The production of capsules by inverse gelation consists of adding dropwise oil containing calcium dispersion into an alginate bath. A dripping technique to produce capsules from oil-in-water (O/W) emulsions was proposed by Abang. However, little is known about the oil encapsulation using water-in-oil (W/O) emulsions. This work aims to develop a new method of W/O emulsions encapsulation by inverse gelation. The success of the W/O emulsion encapsulation is due to three factors: 1) use of an emulsion with moderate stability (50 min); 2) production of an emulsion with at least 90 g/L of CaCl2 and 3) addition of ethanol (20% v/v) into the alginate bath. Both wet and dry capsules were obtained with a spherical shape with diameters of 7 and 3.6 mm, respectively. All volume of oil was encapsulated and the oil loading in the wet and dry capsules was of 23 and 68% v/v, respectively.

Silica nanofibers as a new drug delivery system: a study of the protein-silica interactions.

Drug delivery systems are proposed for the in situ controlled delivery of therapeutic molecules in the scope of tissue engineering. We propose herein silica nanofibers as carriers for the loading and release of bioactive proteins. The influence of pH, time and concentration on the amount of adsorbed proteins was studied. The interactions allowing loading were then studied by means of electron microscopy, zeta potential measurements, electron energy loss spectroscopy and attenuated total reflectance Fourier transform infrared analysis. Release profiles were determined and biological activities were enzymatically assessed. The first part of the work was carried out with lysozyme as a model protein, and then bioactive growth factors TGF-ß1 and GDF-5 were used because their significance in human adipose stromal cell differentiation towards intervertebral disc nucleopulpocytes was previously assessed. It is demonstrated that protein-silica nanofiber interactions are mainly driven by hydrogen bonds and local electrostatic interactions. The present data thus provide a better understanding of the adsorption phenomenon involved, as well as a method to control protein adsorption and release. It is worth pointing out that the kinetic release of growth factors, up to 28 days, and their biological activity maintenance seem to be compatible with intervertebral disc regenerative medicine.

Oil core microcapsules by inverse gelation technique.

A promising technique for oil encapsulation in Ca-alginate capsules by inverse gelation was proposed by Abang et al. This method consists of emulsifying calcium chloride solution in oil and then adding it dropwise in an alginate solution to produce Ca-alginate capsules. Spherical capsules with diameters around 3 mm were produced by this technique, however the production of smaller capsules was not demonstrated. The objective of this study is to propose a new method of oil encapsulation in a Ca-alginate membrane by inverse gelation. The optimisation of the method leads to microcapsules with diameters around 500 µm. In a search of microcapsules with improved diffusion characteristics, the size reduction is an essential factor to broaden the applications in food, cosmetics and pharmaceuticals areas. This work contributes to a better understanding of the inverse gelation technique and allows the production of microcapsules with a well-defined shell-core structure.

Microfluidics-assisted diffusion self-assembly: toward the control of the shape and size of pectin hydrogel microparticles.

We demonstrated the generation of pectin hydrogel microparticles having complex shapes either by combining the phenomenon of gelation and water diffusion-induced self-assembly in microfluidic channels (on-chip) or by the deformation of the pregelled droplets outside the channels (off-chip) at a fluid-fluid interface. We proved that by tuning the mode of pectin cross-linking (CaCl2 vs CaCO3) and the degree of shrinking (water content in the dimethyl carbonate (DMC) organic continuous phase) we can control the shape of the final particle. Sphere, doughnut, oblate ellipsoid, or mushroom-type morphologies were thus produced, demonstrating the ability to control the formation of anisotropic biopolymer-based hydrogel microparticles using microfluidics. Shape changes were explained by the redistribution of calcium ions in combination with the local Peclet number experienced by the microdroplets during the on-chip process. Moreover, during the off-chip process, the interplay between elastic and viscous forces for microdroplets entering the CaCl2-DMC interface caused deformation of the pregelled droplets to occur and therefore resulted in the formation of microparticles with a mushroom-like morphology.

Microfluidic generation and selective degradation of biopolymer-based Janus microbeads.

We describe a microfluidic approach for generating Janus microbeads from biopolymer hydrogels. A flow-focusing device was used to emulsify the coflow of aqueous solutions of one or two different biopolymers in an organic phase to synthesize homo or hetero Janus microbeads. Biopolymer gelation was initiated, in the chip, by diffusion-controlled ionic cross-linking of the biopolymers. Pectin-pectin (homo Janus) and, for the first time, pectin-alginate (hetero Janus) microbeads were produced. The efficiency of separation of the two hemispheres, which reflected mixing and convection phenomena, was investigated by confocal scanning laser microscopy (CSLM) of previously labeled biopolymers. The interface of the hetero Janus structure was clearly defined, whereas that of the homo Janus microbeads was poorly defined. The Janus structure was confirmed by subjecting each microbead hemisphere to specific enzymatic degradation. These new and original microbeads from renewable resources will open up opportunities for studying relationships between combined enzymatic hydrolysis and active compound release.

Soluble and filamentous proteins in Arabidopsis sieve elements.

Phloem sieve elements are highly differentiated cells involved in the long-distance transport of photoassimilates. These cells contain both aggregated phloem-proteins (P-proteins) and soluble proteins, which are also translocated by mass flow. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to carry out a proteomic survey of the phloem exudate of Arabidopsis thaliana, collected by the ethylenediaminetetraacetic acid (EDTA)-facilitated method. We identified 287 proteins, a large proportion of which were enzymes involved in the metabolic precursor generation and amino acid synthesis, suggesting that sieve tubes display high levels of metabolic activity. RNA-binding proteins, defence proteins and lectins were also found. No putative P-proteins were detected in the EDTA-exudate fraction, indicating a lack of long-distance translocation of such proteins in Arabidopsis. In parallel, we investigated the organization of P-proteins, by high-resolution transmission electron microscopy, and the localization of the phloem lectin PP2, a putative P-protein component, by immunolocalization with antibodies against PP2-A1. Transmission electron microscopy observations of P-proteins revealed bundles of filaments resembling strings of beads. PP2-A1 was found weakly associated with these structures in the sieve elements and bound to plastids. These observations suggest that PP2-A1 is anchored to P-proteins and organelles rather than being a structural component of P-proteins.