Effect of mechanical properties on in vitro dynamic digestion of starch contained in hydrogels.

BACKGROUND: This study evaluates the effect of mechanical properties on the in vitro dynamic gastrointestinal digestion of hydrogels containing starch (HCSs) as a model for studying the nutrient digestibility of solid foods. It provides a useful theoretical basis for the processing of specific foods. RESULT: Four types of HCSs with two levels of fracture stress (17.4-20.9 kPa and 55.5-57.6 kPa) and two levels of fracture strain (25.4-28.5% and 53.7-57.4%) were prepared. For these HCSs, the degree of gastric disintegration of hydrogels reduced significantly when fracture strain exceeded 30% (P < 0.05). The gastric emptying of HCS particles was also affected by mechanical properties. For example, even at the same level of fracture stress (ca. 20 kPa), the dry solids retention ratio decreased markedly from 0.90 to 0.43 with a decrease in fracture strain from 53.7% to 25.4% (P < 0.05). For the starch hydrolysis of HCSs after gastric digestion, more than 70% of starch in the particles of all types of HCSs emptied did not undergo digestion. The starch hydrolysis of HCSs during small intestinal digestion was also influenced by their mechanical properties. Fracture strains of HCSs, rather than their fracture stress, affected starch digestibility in hydrogels. CONCLUSION: The gastric disintegration, the gastric emptying, and the starch hydrolysis of HCSs are suppressed when fracture strain exceeded 30%. Even with the amount of nutritional components contained in hydrogels being the same, the in vitro gastrointestinal digestion behavior of HCSs depends on their mechanical properties. This behavior has the potential to be used in the design of processed foods with controlled bioaccessibility. © 2023 Society of Chemical Industry.

Vicenistatin induces early endosome-derived vacuole formation in mammalian cells.

Homotypic fusion of early endosomes is important for efficient protein trafficking and sorting. The key controller of this process is Rab5 which regulates several effectors and PtdInsPs levels, but whose mechanisms are largely unknown. Here, we report that vicenistatin, a natural product, enhanced homotypic fusion of early endosomes and induced the formation of large vacuole-like structures in mammalian cells. Unlike YM201636, another early endosome vacuolating compound, vicenistatin did not inhibit PIKfyve activity in vitro but activated Rab5-PAS pathway in cells. Furthermore, vicenistatin increased the membrane surface fluidity of cholesterol-containing liposomes in vitro, and cholesterol deprivation from the plasma membrane stimulated vicenistatin-induced vacuolation in cells. These results suggest that vicenistatin is a novel compound that induces the formation of vacuole-like structures by activating Rab5-PAS pathway and increasing membrane fluidity.

Facilitated encapsulation of a nonionic contrast agent for X-ray computed tomography into lipid vesicles by the multiple emulsification-solvent evaporation method.

We studied the encapsulation of iohexol (Ihex), a nonionic contrast agent used for X-ray computational tomography, into lipid vesicles using the multiple emulsification-solvent evaporation method to formulate a nanosized contrast agent. This lipid vesicle preparation method consists of three steps: (1) primary emulsification for producing water-in-oil (W/O) emulsions containing fine water droplets that will be converted to the internal water phase of the lipid vesicles, (2) secondary emulsification for formulating multiple water-in-oil-in-water (W/O/W) emulsions encapsulating the fine water droplets containing Ihex, and (3) solvent evaporation to remove the oil phase solvent (n-hexane) and to form lipid bilayers surrounding the fine inner droplets, resulting in the formation of lipid vesicles encapsulating Ihex. As the diameter and Ihex concentration of the primary W/O emulsion droplets decreased, a higher Ihex encapsulation yield was obtained for the final lipid vesicles. The entrapment yield of Ihex in the final lipid vesicles varied significantly with the emulsifier (Pluronic® F-68) concentration in the external water phase of W/O/W emulsion, and the highest yield (65%) was obtained when the emulsifier concentration was 0.1 wt%. We also investigated the powderization of lipid vesicles encapsulating Ihex via lyophilization. The powderized vesicles were dispersed in water after rehydration and maintained their controlled diameters. The entrapment yield of Ihex in powderized lipid vesicles was maintained for over 1 month at 25 ˚C, while significant leakage of Ihex was observed in the lipid vesicles suspended in the aqueous phase.

Improvements in Visual Aspects and Chemical, Techno-Functional and Rheological Characteristics of Cricket Powder (Gryllus bimaculatus) by Solvent Treatment for Food Utilization.

This study aimed to improve the visual aspects and chemical, techno-functional and rheological characteristics of Gryllus bimaculatus cricket powder through the use of different solvents, with the objective of using it as a protein source in food production. Four treatments (pH 5 aqueous solution, ethanol 20%, ethanol 99.5%, and hexane) were applied to the powder, and analyses were conducted to assess changes in the previously mentioned parameters. The results showed that the treatments led to an increase in protein concentration (from 55.4 to 72.5%) and a decrease in fat concentration (from 33.0 to 6.8%) in ethanol 99.5% treated powder, as well as a reduction in anti-nutritional compounds concentration, such as tannins (from 13.3 to 5.9 g/kg), in pH 5 treated powder, which is important for the nutritional value of the final product. The color of the powders was improved, being lighter after hexane and ethanol 99.5% treatments due to the removal of melanin with the defatting process. Flowability, water, and oil holding capacity were also improved in the defatted powders. All the results suggest that the main composition of the powder directly influences the analyzed parameters. These findings suggest that cricket powder treated with solvents can be used as a protein source in different food applications.

Efficient preparation of giant vesicles as biomimetic compartment systems with high entrapment yields for biomacromolecules.

The 'lipid-coated ice-droplet hydration method' was applied for the preparation of milliliter volumes of a suspension of giant phospholipid vesicles containing in the inner aqueous vesicle pool in high yield either calcein, α-chymotrypsin, fluorescently labeled bovine serum albumin or dextran (FITC-BSA and FITC-dextran; FITC=fluorescein isothiocyanate). The vesicles had an average diameter of ca. 7-11 µm and contained 20-50% of the desired molecules to be entrapped, the entrapment yield being dependent on the chemical structure of the entrapped molecules and on the details of the vesicle-formation procedure. The 'lipid-coated ice droplet hydration method' is a multistep process, based on i) the initial formation of a monodisperse water-in-oil emulsion by microchannel emulsification, followed by ii) emulsion droplet freezing, and iii) surfactant and oil removal, and replacement with bilayer-forming lipids and an aqueous solution. If one aims at applying the method for the entrapment of enzymes, retention of catalytic activity is important to consider. With α-chymotrypsin as first model enzyme to be used with the method, it was shown that high retention of enzymatic activity is possible, and that the entrapped enzyme molecules were able to catalyze the hydrolysis of a membrane-permeable substrate which was added to the vesicles after their formation. Furthermore, one of the critical steps of the method that leads to significant release of the molecules from the water droplets was investigated and optimized by using calcein as fluorescent probe.

Analysis of Calcium Sulfate Scaling Phenomena on Reverse Osmosis Membranes by Scaling-Based Flux Model.

In this study, the behavior of permeate flux decline due to scale precipitation of calcium sulfate on reverse osmosis membranes was investigated. The proposed scaling-based flux model is able to explain that permeate fluxes attributed to three mechanisms of scale precipitation-cake formation, surface blockage, and mixed crystallization-converge to the same newly defined scaling-based critical flux. In addition, a scaling index is defined, which determines whether scale precipitates on the membrane. The experimental results were analyzed based on this index. The mass-transfer coefficients of flat membrane cells used in the experiments were measured and, although the coefficients differed, they could be summarized in the same form as the Leveque equation. Considering the results of the scale precipitation experiments, where the operating conditions of pressure, solute concentration, temperature, and Reynolds number were varied, the convergent values of the permeate fluxes are explained by the scaling-based critical fluxes and the scale precipitation zones by the scaling indexes.

Characterization of phospholipids in membrane vesicles derived from Pseudomonas aeruginosa.

Many Gram-negative bacteria release membrane vesicles (MVs), but their phospholipid properties are poorly understood. Phosphatidylglycerol was present at high levels in MVs derived from Pseudomonas aeruginosa, but not in the cellular outer membrane. The ratio of stearic acid in MVs was high compared to that in the cellular outer membrane. These findings suggest that membrane rigidity is associated with MV biogenesis.

Formation of monodisperse calcium alginate microbeads by rupture of water-in-oil-in-water droplets with an ultra-thin oil phase layer.

This paper reports a novel formation method of monodisperse calcium alginate microbeads from water-in-oil-in-water (W/O/W) droplets with an ultra-thin oil phase layer. W/O/W droplets containing sodium alginate in an internal aqueous phase were formed as a template of calcium alginate microbeads using a microfluidic device. The ultra-thin oil phase layer of the W/O/W droplets was ruptured by an osmotic pressure difference between the internal and external aqueous phase. Immediately after the rupture, polyanionic alginate in the internal aqueous phase was cross-linked with calcium ion diffused from the external aqueous phase, and monodisperse and spherical calcium alginate microbeads were formed.

Microfluidic preparation of water-in-oil-in-water emulsions with an ultra-thin oil phase layer.

We developed a novel microfluidic device to prepare monodisperse water-in-oil-in-water (W/O/W) emulsions with an ultra-thin (<1 microm) oil phase layer. This microfluidic device was composed of two microchannel junctions, one of which had a step structure, and a uniformly hydrophobic surface for effective oil removal from W/O/W droplets. At the first junction, an internal aqueous phase was transformed into slug-shaped water-in-oil (W/O) droplets by a flow-focusing mechanism. At the second junction equipped with the step structure, the preformed slug-shaped W/O droplets were introduced into an external aqueous phase and were transformed into spherical W/O droplets. In the downstream area of the second junction, the W/O droplets were released from the hydrophobic surface of the microchannel into the external aqueous phase by inertial lift force and were transformed into W/O/W droplets. During this process, most of the oil phase was effectively removed from the W/O droplets: the bulk of the oil phase flowed along the hydrophobic surface of the microchannel. The thickness of the oil phase layer of the resulting W/O/W droplets was ultra-thin, less than 1 microm. The volume of the internal aqueous phase of the W/O/W droplets reflected that of the W/O droplets and was controlled by the flow rates of the internal aqueous phase and oil phase during W/O droplet formation. We successfully demonstrated encapsulation of water-soluble molecules and polymer particles into the prepared W/O/W emulsion.

Variation of physiochemical properties and cell association activity of membrane vesicles with growth phase in Pseudomonas aeruginosa.

Pseudomonas aeruginosa and other Gram-negative bacteria release membrane vesicles (MVs) from their surfaces, and MVs have an ability to interact with bacterial cells. Although it has been known that many bacteria have mechanisms that control their phenotypes with the transition from exponential phase to stationary phase, changes of properties in released MVs have been poorly understood. Here, we demonstrate that MVs released by P. aeruginosa during the exponential and stationary phases possess different physiochemical properties. MVs purified from the stationary phase had higher buoyant densities than did those purified from the exponential phase. Surface charge, characterized by zeta potential, of MVs tended to be more negative as the growth shifted to the stationary phase, although the charges of PAO1 cells were not altered. Pseudomonas quinolone signal (PQS), one of the regulators related to MV production in P. aeruginosa, was lower in MVs purified from the exponential phase than in those from the stationary phase. MVs from the stationary phase more strongly associated with P. aeruginosa cells than did those from the exponential phase. Our findings suggest that properties of MVs are altered to readily interact with bacterial cells along with the growth transition in P. aeruginosa.

Effect of dehydrocholic acid conjugated with a hydrocarbon on a lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine.

The effects of bile acids, dehydrocholic acid (DHA) and DHA conjugated with a hydrocarbon (6-aminohexanoate; 6A-DHA) were evaluated using a lipid bilayer composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). DOPC formed a homogenous thin membrane in presence or absence of the DHA, while 20 mol% 6A-DHA induced phase separation on the DOPC thin membrane. It was observed formation of a stomatocyte-like liposomes when these membranes were suspended in a basic solvent. Generally, liposome formation can be prevented by some bile acids. It was found that DHA and 6A-DHA did not disrupt liposome formation, while DHA and 6A-DHA perturbed the liposomal membrane, resulting in increased local-fluidity due to the bent structure of DHA and 6A-DHA. DHA and 6A-DHA showed completely different effects on the hydrophobicity of the boundary surface of DOPC liposome membranes. The steroidal backbone of DHA was found to prevent the insertion of water molecules into the liposomal membrane, whereas 6A-DHA did not show the same behavior which was attributed to its conjugated hydrocarbon.

Dynamic interaction between oppositely charged vesicles: aggregation, lipid mixing, and disaggregation.

We investigated dynamic interactions between oppositely charged small unilamellar vesicles using positively charged vesicles containing 1,2-dioleoyl-3-trimethylammonium-propane or 3beta-[N-(N('),N(')-dimethylaminoethane)-carbamoyl] cholesterol and negatively charged vesicles containing L-alpha-phosphatidyl-DL-glycerol. Aggregation, lipid bilayer mixing, contents mixing and contents leakage were systematically examined using optical density measurements, fluorescence resonance energy transfer assays, fluorescence quenching assays, light-scattering analyses, and freeze-fracture transmission electron microscopy. The oppositely charged vesicles aggregated immediately. Lipid mixing was observed, but there was no mixing of the contents. The vesicle aggregates disaggregated spontaneously after several minutes. The surface potential of the disaggregated vesicles was neutralized. From these results, we infer that the lipids in the external monolayers were exchanged between the oppositely charged vesicles while the internal monolayers remained intact. The two types of cationic lipids used exhibited different speeds of disaggregation.

[Novel method for preparing vesicles from a monodisperse emulsion aimed at controlling the size and improving the entrapment yield].

A vesicle is a compartment composed of lipid bilayer of amphiphilic molecules. The vesicle is applied to carriers of drugs, cosmetics and functional food ingredients in industries. Vesicles are also applied as a model for artificial cell membrane and expected as micro- and nano-reactors. They are generally prepared by the hydration of dry lipid film, but there is no method to prepare vesicles of a controlled size and high entrapment yield of hydrophilic materials inside them. In this article, a microchannel (MC) emulsification method was applied to prepare vesicles aimed at controlling the size and improving the entrapment yield. Firstly, monodisperse water-in-oil (W/O) emulsions were prepared by the MC emulsification method. In this process, hydrophilic materials to be entrapped were contained inside the water droplets of the emulsions. Keeping the water droplets frozen, the emulsifier was replaced by a bilayer-forming lipid mixture, and then the oil phase was evaporated. After hydration of lipid layers surrounding the water droplets, vesicles were formed. We call this preparation "lipid-coated ice droplet hydration method". The final sizes of the prepared vesicles were comparable to the original emulsion droplet sizes. This means that the size of vesicles can be controlled by controlling the size of original water droplets of the W/O emulsions. Furthermore, calcein as a hydrophilic fluorescent marker and biopolymers, such as enzyme and polysaccharide, were entrapped into the internal water phases of vesicles. The method proposed in this study enables the formation of vesicles with a controlled size and high entrapment yields, potentially useful for expanding the application fields of vesicles as biocompatible carriers and micro- and nano-reactors for biochemical reactions.

Preparation and characterization of beta-carotene nanodispersions prepared by solvent displacement technique.

This work demonstrated the preparation of protein-stabilized beta-carotene nanodispersions using the solvent displacement technique. The emulsifying performance of sodium caseinate (SC), whey protein concentrate (WPC), whey protein isolate (WPI), and a whey protein hydrolysate (WPH, 18% degree of hydrolysis) was compared in terms of particle size and zeta-potential of the nanodispersions. SC-stabilized nanodispersions exhibited a bimodal particle size distribution: large particles (stabilized by casein micelles) with a mean particle size of 171 nm and small particles (stabilized by casein submicelles) of 13 nm. This was confirmed with transmission electron microscopy analysis. Most of the beta-carotene precipitated (87.6%) was stabilized in the small particles. On the other hand, the nanodispersions stabilized by the whey proteins were polydispersed with larger mean particle sizes. The mean particle size of WPC and WPI was 1730 and 201 nm, respectively. The SC-stabilized nanodispersion was expected to be more stable as indicated by its higher absolute zeta-potential value (-31 mV) compared to that of WPC (-15 mV) and WPI (-16 mV). Partially hydrolyzed whey protein possessed improved emulsifying properties as shown by WPH-stabilized samples. It was interesting to note that increasing the SC concentration from 0.05 to 0.5 wt % increased the particle size of beta-carotene stabilized by casein micelles, while the reverse was true for those stabilized by SC submicelles. Microfluidization at 100 MPa of SC solution dissociated the casein micelles, resulting in a decrease in mean particle size of the casein micelle-stabilized particles when the SC solution was used to prepare nanodispersions. The results from this work showed that protein-stabilized beta-carotene nanodispersions could be prepared using the solvent displacement technique.

Freeze-dryable lipid vesicles with size tunability and high encapsulation efficiency prepared by the multiple emulsification-solvent evaporation method.

We investigated the extent of potential applicability of our recently developed method for preparing lipid vesicles [T. Kuroiwa et al., J. Am. Oil Chem. Soc., 93 (2016) 421], designated as the multiple emulsification-solvent evaporation method, with the intention of controlling the vesicle diameter and achieving high entrapment efficiency for water-soluble compounds. Using this method, the diameter of lipid vesicles could be varied by selecting the methods for preparing the primary water-in-oil emulsion, which contained water droplets as templates for the internal water phases of lipid vesicles. We obtained lipid vesicles with mean diameters of 0.2-4.4µm from water-in-oil-in-water multiple emulsions after solvent evaporation. A high entrapment yield of calcein, a water-soluble fluorescent dye, into the lipid vesicles was obtained for each vesicle sample, depending on their diameter and the type of emulsifier added to the external water phase. The use of polymeric emulsifier was more effective in achieving a high entrapment yield. The obtained lipid vesicles were powderized via freeze-drying. Vesicles could be powderized while maintaining their original diameter, as confirmed by scanning electron microscopy. Furthermore, the powderized vesicles could be rehydrated and resuspended without significant change in their diameter. However, the entrapment yield of calcein decreased after freeze-drying and rehydration. The calcein leakage during the freeze-drying followed by rehydration could be suppressed by adding an appropriate amount of trehalose as a lyoprotectant.

Entrapment of some compounds into biocompatible nano-sized particles and their releasing properties.

Two types of biocompatible nanoparticles with an average diameter of around 200 nm were formed only by mixing hydrolysates of chitosan and carboxymethyl cellulose (CMC). Nanoparticle A was produced from chitosanase hydrolysate of chitosan and cellulase hydrolysate of carboxymethyl cellulose, and nanoparticle B was produced from lysozyme hydrolysate of chitosan and the carboxymethyl cellulose hydrolysate. Negatively charged or amphoteric compounds were first mixed with chitosan hydrolysate and then added to carboxymethyl cellulose hydrolysate to effectively entrap them in the particles. Positively charged compounds could also be effectively entrapped by mixing the hydrolysates and the compound in the reverse order. Negatively charged compounds with high molecular weights were maintained in the particles even at the higher pH levels than the pK(a) of the amino groups of chitosan. Entrapped compounds were gradually released from nanoparticle A by lysozyme treatment. In contrast, there was no release from nanoparticle B. These results indicate that nanoparticle A can be applied to controlled-release drug delivery systems, and that nanoparticle B is stably retained in the body without releasing the entrapped compounds.

Formulation and stabilization of nano-/microdispersion systems using naturally occurring edible polyelectrolytes by electrostatic deposition and complexation.

This review paper presents an overview of the formulation and functionalization of nano-/microdispersion systems composed of edible materials. We first summarized general aspects on the stability of colloidal systems and the roles of natural polyelectrolytes such as proteins and ionic polysaccharides for the formation and stabilization of colloidal systems. Then we introduced our research topics on (1) stabilization of emulsions by the electrostatic deposition using natural polyelectrolytes and (2) formulation of stable nanodispersion systems by complexation of natural polyelectrolytes. In both cases, the preparation procedures were relatively simple, without high energy input or harmful chemical addition. The properties of the nano-/microdispersion systems, such as particle size, surface charge and dispersion stability were significantly affected by the concerned materials and preparation conditions, including the type and concentration of used natural polyelectrolytes. These dispersion systems would be useful for developing novel foods having high functionality and good stability.

Factors affecting the composition of oligosaccharides produced in chitosan hydrolysis using immobilized chitosanases.

The hydrolysis reaction of chitosan using immobilized chitosanases with regard to the composition of its products and the yield of the intermediate target products, pentamer and hexamer of chitosan oligosaccharides, was investigated. Chitosanase was immobilized onto agar or agarose gel particles by the multipoint attachment method. In batch experiments, surface enzyme density, support particle size, temperature, agitator speed, and initial substrate concentration significantly affected the composition of the oligosaccharides produced. It was believed that these factors all related to the reaction rate and mass transfer rate at the surface of the support materials immobilizing the enzymes. These effects were summarized as a correlation with Damköhler number (Da), defined as the ratio of the maximum reaction rate to the maximum mass transfer rate. The result showed that the reaction conditions that give a low value of Da provide a high yield of pentamer and hexamer oligosaccharides.

Immobilization and stabilization of chitosanase by multipoint attachment to agar gel support.

Highly stable chitosanase immobilized on an agar gel support was prepared by the multipoint attachment method. The optimum pH range was broadened to between 4 and 6, whereas for free chitosanase, the pH was only 5.6. The optimum temperature was also increased from 60 degrees C to 80 degrees C after the immobilization. The activity of immobilized chitosanase remained at 95% of its initial activity level after 225 h of incubation at 50 degrees C, whereas for free chitosanase, it decreased to 20% after 1 h of incubation. The immobilization markedly increased the thermostability of chitosanase. These changes in the reaction characteristics are favorable for the practical use of chitosanase in industrial processes. The effect of glycidol concentration in the activation of agar gel was also examined. The surface density of the aldehyde residue increased with increasing glycidol concentration. A maximal activity of 11.9 U/g-support was obtained when the glycidol concentration was 0.7 M. At concentrations higher than this, thermostability was almost the same. It was therefore proven that the optimal glycidol concentration in this system is 0.7 M. The effects of glycidol concentration on the activity and the thermostability of chitosanase are discussed in relation to the number of covalent bonds between the chitosanase and its support. Chitosan oligosaccharides were continuously produced using a column reactor packed with the immobilized chitosanase. The percentage of hydrolyzed chitosan after 28 reaction days was 44%. This was a slight decrease from the 48% observed on the first day. The total concentration of pentamer and hexamer ranged from 1.3 mg/ml to 1.5 mg/ml during the 28 reaction days. This was approximately 30% of the chitosan concentration in the supplied substrate solution.

Microcompartmentalized cell-free protein synthesis in semipermeable microcapsules composed of polyethylenimine-coated alginate.

We describe microcompartmentalized cell-free protein synthesis in semipermeable microcapsules prepared from water-in-oil-in-water droplets by a rupture-induced encapsulation method. An aqueous solution of template DNA coding for green fluorescent protein and enzymes for the cell-free protein synthesis was aliquoted into water-in-oil droplets using a microfluidic device, and the droplets were transformed into semipermeable microcapsules. Substrates for protein synthesis diffused into the microcapsules through their semipermeable polyion complex membranes composed of polyethylenimine-coated alginate. Cell-free protein synthesis was confirmed by detection of the fluorescence of the synthesized green fluorescence protein in the microcapsules. We also used this microcompartmentalized system to synthesize protein from a single molecule of template DNA encapsulated by limiting dilution.