PIV and CFD studies on analyzing intragastric flow phenomena induced by peristalsis using a human gastric flow simulator.

This study quantitatively analyzed the flow phenomena in model gastric contents induced by peristalsis using a human gastric flow simulator (GFS). Major functions of the GFS include gastric peristalsis simulation by controlled deformation of rubber walls and direct observation of inner flow through parallel transparent windows. For liquid gastric contents (water and starch syrup solutions), retropulsive flow against the direction of peristalsis was observed using both particle image velocimetry (PIV) and computational fluid dynamics (CFD). The maximum flow velocity was obtained in the region occluded by peristalsis. The maximum value was 9 mm s(-1) when the standard value of peristalsis speed in healthy adults (UACW = 2.5 mm s(-1)) was applied. The intragastric flow-field was laminar with the maximum Reynolds number (Re = 125). The viscosity of liquid gastric contents hardly affected the maximum flow velocity in the applied range of this study (1 to 100 mPa s). These PIV results agreed well with the CFD results. The maximum shear rate in the liquid gastric contents was below 20 s(-1) at UACW = 2.5 mm s(-1). We also measured the flow-field in solid-liquid gastric contents containing model solid food particles (plastic beads). The direction of velocity vectors was influenced by the presence of the model solid food particle surface. The maximum flow velocity near the model solid food particles ranged from 8 to 10 mm s(-1) at UACW = 2.5 mm s(-1). The maximum shear rate around the model solid food particles was low, with a value of up to 20 s(-1).

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.

Electrophoretic behavior of microgel-immobilized polyions.

Electrophoretic behavior was studied for N-isopropylacrylamide (NIPA) microgels, into which different amounts of poly(acrylic acid) (PAAc) were physically entrapped. Copolymer microgels of NIPA with acrylic acid (AAc) were also studied as a control. Electrophoretic mobility was measured in 0.1 M NaCl solution at 25 °C as a function of pH, using an electrophoretic light scattering technique. The mobility of the copolymer microgel whose COOH groups are fully ionized agreed with that of PAAc when its ionization degree (α(n)) is close to the mole fraction (f(AAc)) of the AAc unit in the copolymer gel. There was good agreement between the mobility values of the copolymer microgel and the linear NIPA/AAc copolymer when their AAc contents are very close to each other. However, the mobility of the microgel with immobilized PAAc was higher than that of the copolymer microgel, even when there was no difference in the AAc content for both microgels. Moreover, the immobilized PAAc showed a higher mobility than the free PAAc when its α(n) is equal to f(AAc) in the immobilized system. No correlation was observed between the mobility and the hydrodynamic radius. These results were discussed in terms of the free draining model (FDM) for the electrophoresis of polyelectrolytes. It became apparent that the mobility difference depending upon whether (i) the PAAc ions are in the cage of the NIPA network or (ii) the AAc units are copolymerized with the network chain is due to the structural difference of the segments considered in the FDM.

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.

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.

Optimized culture conditions for the efficient production of porcine adenylate kinase in recombinant Escherichia coli.

Temperature shift cultivations with amino acid supplementation were optimized to produce porcine adenylate kinase (ADK) in recombinant Escherichia coli harboring a pUC-based recombinant plasmid under the control of the trp promoter. With regard to temperature control, the culture condition was initially maintained at 35 degrees C for cellular growth, but ADK expression was suppressed until the late logarithmic growth phase; subsequently, a temperature shift was applied (from 35 degrees C to 42 degrees C), which resulted in maximal ADK production. In addition, supplementation of amino acids, especially valine and leucine, during the temperature shift stimulated ADK expression from 3.5% to 9.2% and 8.6% of the total protein, respectively. After optimization, 1 g ADK per liter was produced within 16 h of cultivation with a dry cell weight of 21.8 g/l. In this system, there was no loss of the recombinant plasmid during cultivation without selective pressure.

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.

Highly productive droplet formation by anisotropic elongation of a thread flow in a microchannel.

We developed a microfluidic device to form monodisperse droplets with high productivity by anisotropic elongation of a thread flow, defined as a threadlike flow of a dispersed liquid phase in a flow of an immiscible, continuous liquid phase. The thread flow was anisotropically elongated in the depth direction in a straight microchannel with a step, where the microchannel depth changed. Consequently, the elongated thread flow was given capillary instability (Rayleigh-Plateau instability) and was continuously transformed into monodisperse droplets at the downstream area of the step in the microchannel. We examined the effects of the flow rates of the dispersed phase and the continuous phase on the droplet formation behavior, including the droplet diameter and droplet formation frequency. The droplet diameter increased as the fraction of the dispersed-phase flow rate relative to the total flow rate increased and was independent of the total flow rate. The droplet formation frequency proportionally increased with the total flow rate at a constant dispersed-phase flow rate fraction. These results are explained in terms of a mechanism similar to that of droplet formation from a cylindrical liquid thread flow by Rayleigh-Plateau instability. The microfluidic device described was capable of forming monodisperse droplets with a 160-microm average diameter and 3-microm standard deviation at a droplet formation frequency of 350 droplets per second from a single thread flow. The highest total flow rate achieved was 6 mL/h using the present device composed of a straight microchannel with a step. We also demonstrated parallel droplet formation by anisotropic elongation of multiple thread flows; the process was applied to form W/O and O/W droplets. The highly productive droplet formation process presented in this study is expected to be useful for future industrial applications.

High cell density cultivation of recombinant E. coli for hirudin variant 1 production by temperature shift controlled by pUC18-based replicative origin.

The copy number of a plasmid, pUC-based vector, was previously shown to be affected by culture temperature. In this study, intracellular hirudin variant 1 (f-HV1) fused to porcine adenylate kinase protein was produced using recombinant Escherichia coli by temperature shift cultivation coupled with a high cell density cultivation technique for E. coli JM109. The optimal temperature for cellular growth suppressing f-HV1 production was 33 degrees C, resulting in a final dried cell concentration of 45.7 g/l, with a specific growth rate of 0.54 1/h. Optimizing the temperature-shift conditions (temperature shifted to an OD660 nm of 15 from 33 degrees C to 37 degrees C) resulted in the production of f-HV1 up to 4763 mg/l as an inclusion body with dried cell concentration of 44 g/l in 18 h.

Novel method for obtaining homogeneous giant vesicles from a monodisperse water-in-oil emulsion prepared with a microfluidic device.

A novel technique called the "lipid-coated ice droplet hydration method" is presented for the preparation of giant vesicles with a controlled size between 4 and 20 microm and entrapment yields for water-soluble molecules of up to about 30%. The method consists of three main steps. In the first step, a monodisperse water-in-oil emulsion with a predetermined average droplet diameter between 4 and 20 microm is prepared by microchannel emulsification, using sorbitan monooleate (Span 80) and stearylamine as emulsifiers and hexane as oil. In the second step, the water droplets of the emulsion are frozen and separated from the supernatant hexane solution by precipitation, followed by a removal of the supernatant and followed by the replacement of Span 80 by using a hexane solution containing egg yolk phosphatidylcholine, cholesterol, and stearylamine (5:5:1, molar ratio). This procedure is performed at -10 degrees C to keep the water droplets of the emulsion in a frozen state and thereby to avoid extensive water droplet coalescence. In the third step, hexane is evaporated at -4 to -7 degrees C and an external water phase is added to the remaining mixture of lipids and water droplets to form giant vesicles that have an average size in the range of that of the initial emulsion droplets (4-20 microm). The entrapment yield and the lamellarity of the vesicles obtained depend on the lipid/water droplet ratio and on the composition of the external water phase. At high lipid/water droplet ratio, the giant vesicles have a thicker membrane (indicating multilamellarity) and a higher entrapment yield than in the case of a low lipid/water droplet ratio. The highest entrapment yield ( approximately 35%) is obtained if the added external water phase contains preformed unilamellar egg phosphatidylcholine vesicles with an average diameter of 50 nm. The addition of these small vesicles minimizes the water droplet coalescence during the third step of the vesicle preparation, thereby decreasing the extent of release of water-soluble molecules originally present in the water droplets. The GVs prepared can be extruded through polycarbonate membranes to yield large unilamellar vesicles with about 100 nm diameter. This size reduction, however, leads to a decrease in the entrapment yield to about 12% due to solute leakage from the vesicles during the extrusion process.

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.

High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production.

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.

On an intraparticle complex of cationic nanogel with a stoichiometric amount of bound polyanions.

A polyelectrolyte nanogel (PENG) particle consisting of lightly cross-linked terpolymer chains of N-isopropylacrylamide, acrylic acid, and 1-vinylimidazole has positive charges in an aqueous medium at pH 3 due to protonation of the imidazole groups, and thereby forms a polyelectrolyte complex with the linear polyanion, potassium poly(vinyl alcohol) sulfate (KPVS). It has been demonstrated that the hydrodynamic radius (Rh), by dynamic light scattering (DLS), and the radius of gyration (Rg), by static light scattering (SLS), of the complex particles are smallest at approximately 1:1 mixing ratio (rm) of anions to cations, in the absence of simple salts such as KCl (Langmuir 2005, 21, 4830). Here, we aimed to study the nature of the complex formed at rm=1 and examined the complex formation process by electrophoretic light scattering (ELS). It was found that the mobility of the cationic PENG with a stoichiometric amount of bound KPVS anions (i.e., the complex formed at rm=1) is positive but not zero at 25 degrees C. This was also the case when the complex was examined by ELS at 45 degrees C, where DLS and SLS show a temperature-driven collapse of the complex. We thus assumed that (a) electroneutrality is maintained in the complex particle with the aid of counterions, but (b) the complex is highly polarizable, and hence (c) during ELS the KPVS anions would dissociate in part from the complex. This hypothesis was supported by the following results: (i) Mixing complexed and uncomplexed PENG particles at different ratios brings about an increase in Rh and a decrease in the light scattering intensity of the complex at the same time, suggesting a polyelectrolyte exchange reaction. (ii) The same phenomenon is seen when poly(diallyldimethylammonium chloride) (PDDA as a polysalt) is added to the complex dispersion, meaning that the PDDA takes out the KPVS from the complex to form a stable PDDA-KPVS complex. (iii) Upon addition of KCl, the complex undergoes little change in Rh (62-67 nm) at a salt concentration (Cs)0.2 M. (iv) The Rh (78 nm) of the soluble complex at Cs from 0.3 to 0.5 M is larger than that at Cs<0.02 M, suggesting dissociation of the KPVS ions. (v) Complexation between KPVS and PDDA as mentioned in (ii) is facilitated in the presence of 0.01 M KCl.

A mycelium with polyelectrolyte complex-bunched hyphae: several important factors affecting on the fermentation performance at a very high cell density.

We have reported in the previous paper (Colloids Surf. B (2006) in press) a marked increase in the rate of gluconic acid production at a very high cell concentration (40 g/l) of filamentous fungus (Aspergillus niger IFO 31012) which was immobilized with polyelectrolyte complex consisting of potassium poly(vinyl alcohol) sulfate and trimethylammonium glycol chitosan iodide [6-O-(2-hydroxyethyl)-2-(trimethylammonio)-chitosan iodide]. The present study was carried out to look at what factors play a crucial role in this enhancement. We measured viscosity of broth, mass-transfer coefficient (k(L)a) for oxygen and diffusion coefficient of glucose (substrate). It has become apparent that there is only a difference in the diffusion coefficient of glucose between the free and immobilized cells. Therefore, we believe that the diffusion limitation by substrates as a problem in submerged mycelial processes is improved by immobilization based on polyelectrolyte complexes.

A mycelium with polyelectrolyte complex-bunched hyphae: preparation and fermentation performance.

We studied the immobilization of a mycelium (Aspergillus niger) using the working hypothesis as follows: (a) when polycation was added to the cell suspension, a few parts of it would bind on the surface of a hypha, allowing to gather the hyphae in part but not all; (b) upon further addition of polyanion, such a gathering of the hyphae is tightly bunched by the polyelectrolyte complex (PEC) which is resulted from the remaining polycation; (c) as a result, a mycelium with partially bunched hyphae can be obtained. Potassium poly(vinyl alcohol) sulfate and trimethylammonium glycol chitosan iodide [6-O-(2-hydroxyethyl-2-(trimethylamonio)-chitosan iodide) were used as the polyanion and the polycation, respectively. The optical and electron microscopic analyses showed that our immobilized cell contains many of PEC-bunched hyphae. The sedimentation rate increased with the weight ratio of PEC to dry cells and leveled off at the weight ratio larger than 0.5. The gluconic acid production from glucose was studied by a semi-large scale (1l) cultivation of the imobilized and free cells using a jar fermentor. It was found that an apparent specific activity of the immobilized cells for glucose oxidation becomes 1.44 times that of the free cells even at a high cell density of 40 g/l.

Formation of intra- and interparticle polyelectrolyte complexes between cationic nanogel and strong polyanion.

Polyelectrolyte complex formation of a strong polyanion, potassium poly(vinyl alcohol) sulfate (KPVS), with positively charged nanogels was studied at 25 degrees C in aqueous solutions with different KCl concentrations (C(s)) as a function of the polyion-nanogel mixing ratio based on moles of anions versus cations. Used as the gel sample was a polyampholytic nanogel consisting of lightly cross-linked terpolymer chains of N-isopropylacrylamide, acrylic acid, and 1-vinylimidazole; thus, the complexation was performed at pH 3 at which the imidazole groups are fully protonated to generate positive charges. Turbidimetric titration was employed to vary the mixing ratio. Also employed for studies of the resulting complexes at different stages of the titration were dynamic light scattering (DLS) and static light scattering (SLS) techniques. It was found from the titration as well as DLS and SLS that there is a critical mixing ratio (cmr) at which both the size and molar mass of the complexed gel particles abruptly increase. The value of the cmr at C(s) = 0 or 0.01 M (mol/L) was observed at approximately 1:1 mixing ratio of anions versus cations but at lower mixing ratios than the 1:1 ratio under conditions of C(s) = 0.05 and 0.1 M. At the mixing ratios less than the cmr, the molar mass of the complex agrees with that of one gel particle with the calculated amount of the bound KPVS ions, indicating the formation of an "intraparticle" KPVS-nanogel complex, by the aggregation of which an "interparticle" complex is formed at the cmr. During the process of the intraparticle complex formation, both the hydrodynamic radius by DLS and the radius gyration by SLS decreased with increasing mixing ratio, demonstrating the gel collapse due to the complexation. At C(s) = 0 or 0.01 M and under conditions where the amount of KPVS bindings was less than half of the nanogel cations, however, the decrease of the hydrodynamic radius was very small, while the radius gyration fell monotonically. These results were discussed in connection with a collapse of dangling chains attached to the nanogel surface by the binding of KPVS.

Improvement of the yield of physiologically active oligosaccharides in continuous hydrolysis of chitosan using immobilized chitosanases.

The continuous production of chitosan oligosaccharides using a packed-bed enzyme reactor was investigated as to the effects of the operation conditions on the yield of pentamers and hexamers of chitosan oligosaccharides. A column reactor packed with immobilized chitosanases prepared by the multipoint attachment method was used for continuous hydrolysis of chitosan. In this reactor, the decrease of the yield of the target intermediate oligosaccharides due to axial mixing was negligible. The surface enzyme density of the support and flow rate of the substrate solution significantly affected the maximum yield of pentamers and hexamers. These effects were summarized as a correlation with the Damköhler number (Da), defined as the ratio of the maximum reaction rate to the maximum mass transfer rate. The optimum condition was determined based on Da. Under the optimized condition (Da = 0.12), pentamers and hexamers could be produced continuously for a month with a yield of over 35% (7 kg/m(3) in concentration).

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.