Effect of oil droplet size on activation energy for coalescence of oil droplets in an O/W emulsion.

The activation energy of a reasonable order of magnitude was estimated for the coalescence of oil droplets in an O/W emulsion by formulating the balance of forces acting on a droplet that crosses over the potential barrier to coalesce with another droplet by the DLVO theory and Stokes' law. An emulsion with smaller oil droplets was shown to be more stable.

A novel acylase from Streptomyces mobaraensis that efficiently catalyzes hydrolysis/synthesis of capsaicins as well as N-acyl-L-amino acids and N-acyl-peptides.

A novel enzyme that catalyzes efficient hydrolysis of capsaicin (8-methyl-N-vanillyl-6-nonenamide) was isolated from the culture broth of Streptomyces mobaraensis. The enzyme consisted of two dissimilar subunits with molecular masses of 61 and 19 kDa. The enzyme was activated and stabilized in the presence of Co2+. It showed a pH optimum of about 8 and was stable at temperatures of up to 55 degrees C for 1 h at pH 7.8. The specific activity of the enzyme for the hydrolysis of capsaicin was 10(2)-10(4) times higher than those for the enzymes reported to date. In an aqueous/n-hexane biphasic system, capsaicin analogues such as octanoyl, decanoyl, and lauroyl vanillylamides were synthesized from the corresponding fatty acids and vanillylamine at yields of 50% or greater. In addition, the enzyme catalyzed the deacylation of N-lauroyl-L-amino acids and N-lauroyl-L-dipeptides and the efficient synthesis of Nalpha-lauroyl-L-lysine, Nepsilon-lauroyl-L-lysine, and various N-lauroyl-peptides in aqueous solution in both the absence and the presence of glycerol.

Preparation of finely dispersed O/W emulsion from fatty acid solubilized in subcritical water.

A novel method for preparing a finely dispersed oil-in-water emulsion is proposed. Octanoic acid dissolved in water at a high temperature of 220 or 230 degrees C at 15 MPa was combined with an aqueous solution of a surfactant and then the mixture was cooled. When a nonionic surfactant, decaglycerol monolaurate (ML-750) or polyoxyethylene sorbitan monolaurate (Tween 20), was used, fine emulsions with a median oil droplet diameter of 100 nm or less were successfully prepared at ML-750 and Tween 20 concentrations of 0.083% (w/v) and 0.042%, respectively, or higher. The diameters were much smaller than those of oil droplets prepared by the conventional homogenization method using a rotor/stator homogenizer. However, an anionic surfactant, sodium dodecyl sulfate, was not adequate for the preparation of such fine emulsions by the proposed method. Although the interfacial tensions between octanoic acid and the surfactant solutions were measured at different temperatures, they were not an indication for selecting a surfactant for the successful preparation of the fine emulsion by the proposed method.

Decomposition kinetics of maltose in subcritical water.

The decomposition process of maltose in subcritical water was studied using a tubular reactor in the temperature range of 180 to 260 degrees C and at 10 MPa. The formation of glucose and 5-hydroxymethyl-2-furaldehyde during the maltose decomposition was also observed. The decomposition rate of maltose was faster at higher temperatures. The rate was approximated by first-order kinetics during the early stage of the decomposition, but was accelerated and deviated from these kinetics at the later stage. The effluent pH decreased as the residence time in the reactor increased and the decrease of pH affected the maltose decomposition rate and glucose formation. Low pH of a feed solution accelerated maltose decomposition. A good correlation was obtained between the pH of the effluent and the rate constant of the first-order kinetics.

Relationship between structure and permeability of dipeptide derivatives containing tryptophan and related compounds across human intestinal epithelial (Caco-2) cells.

The permeability of dipeptide derivatives containing tryptophans and indole derivatives through Caco-2 cells was used as an in vitro intestinal absorption model in order to clarify structural factors which influence their intestinal epithelial permeation and metabolism. Most peptide derivatives were hydrolysed not only by the cytosolic enzymes in Caco-2 cells during permeation but also by enzymes released to the apical solution before cell permeation. The N-terminal blocked dipeptides were more resistant to hydrolases expressed in the Caco-2 cells and indole derivatives were not entirely degraded. Based on compound concentration dependency and comparison of permeability coefficients in apical-to-basolateral and basolateral-to-apical directions, the main absorption mechanism of compounds were determined. Compounds were then classified into three groups; (1) passively transported compounds, (2) actively transported compounds and (3) compounds excreted by P-glycoprotein.

Relationships between structure and high-throughput screening permeability of peptide derivatives and related compounds with artificial membranes: application to prediction of Caco-2 cell permeability.

To evaluate absorption of compounds across the membrane via a transcellular route, the permeability of peptide derivatives and related compounds was measured by the parallel artificial membrane permeation assay (PAMPA). The permeability coefficients by PAMPA were analyzed quantitatively using classical QSAR and Volsurf approaches with the physicochemical parameters. The results from both approaches showed that hydrogen bonding ability of molecules in addition to hydrophobicity at a particular pH were significant in determining variations in PAMPA permeability coefficients. The relationship between Caco-2 cell permeability and artificial lipid membrane permeability was then determined. The compounds were sorted according to their absorption pathway in the plot of the Caco-2 cell and PAMPA permeability coefficients.

Hydrolysis of the oil phase of a W/O/W emulsion by pancreatic lipase.

W/O/W emulsions are expected to protect bioactive substances from degradation by pancreatic enzymes. We investigated the enzymatic hydrolysis of the oil phase and release of a marker substance from the inner-aqueous phase to the outer-aqueous phase using an artificial digestive fluid. Octanoic acid triacylglycerol (C8TG) was used as the oil phase. W/O/W emulsions were prepared by two-step homogenization and succeeding membrane filtration. When the artificial digestive fluid containing lipase and gall was added to the emulsion, release of the marker substance from the inner-phase solution, oil-phase hydrolysis, and emulsion coalescence occurred in that order. When a coarse emulsion and 0.2- and 0.8-microm membrane-filtered fine emulsions were treated with the fluid for 1 h, the degrees of C8TG hydrolysis were 3.8%, 55% and 57%, the fractions of the marker substance released from the inner-water phase were 2.7%, 89% and 72%, and the median diameters of the oil droplets were changed from 32 to 23 microm, 0.71 to 27 microm, and from 2.2 to 26 microm, respectively. These results suggested that the diameter of the oil droplets in the W/O/W emulsion significantly affected the release profile of the marker loaded in the inner-water phase of the emulsion.

Anti-oxidant activity of acyl ascorbates in intestinal epithelial cells.

The anti-oxidant activity of acyl ascorbates, with acyl chain lengths of 10 to 18, was measured in an intestinal epithelial cell line. All the acyl ascorbates exhibited the activity for the oxidization induced by three different types of oxidants, at levels higher than ascorbic acid. Myristoyl ascorbate was the most effective and showed activity at 3 x 10(-12) to 3 x 10(-6) M. incubation with myristoyl ascorbate for more than 3 h was needed to sufficiently suppress the oxidization of the intestinal cell monolayers.

Microencapsulation of linoleic acid with low- and high-molecular-weight components of soluble soybean polysaccharide and its oxidation process.

Soluble soybean polysaccharide (SSPS) was fractionated into its low- (LMW) and high-molecular-weight (HMW) components to test their antioxidative and emulsifying properties. Linoleic acid was emulsified with an aqueous solution of SSPS, HMW, a mixture of LMW or HMW with maltodextrin, or maltodextrin alone. The emulsions prepared with SSPS, HWM and the mixture of HMW with maltodextrin were stable. These emulsions were spay-dried to produce microcapsules. The encapsulated linoleic acid was oxidized at 37 degrees C and at various levels of relative humidity. Linoleic acid encapsulated with the mixture of LMW with maltodextrin or HMW was stable to oxidation, and this stability increased as the weight fraction of LMW in the mixture was increased. The LMW components also had high DPPH-radical scavenging activity. These results indicate that LMW played an important role in suppressing or retarding the oxidation of linoleic acid encapsulated with SSPS. The oxidative stability of linoleic acid encapsulated with a mixture of the LMW and HMW components was high at low and high relative humidity, but not at intermediate levels of relative humidity.

Lipase-catalyzed condensation of p-methoxyphenethyl alcohol and carboxylic acids with different steric and electrical properties in acetonitrile.

The substrate selectivity of Candida antarctica lipase for various carboxylic acids was systematically investigated for their condensation with p-methoxyphenethyl alcohol in acetonitrile. The electron density of a carboxylic carbon and the projection area of an acid molecule were numerical indicators for evaluating the substrate selectivity. Both the electron density and projection area had an influence on the V/Km value, while only the projection area affected the Km value. The presence of conjugation with a carboxyl group in an acid and a branched structure in the non-carboxylic region independently lowered the V/Km value by an order of 1.

Preparation of a water-in-oil-in-water (W/O/W) type microcapsules by a single-droplet-drying method and change in encapsulation efficiency of a hydrophilic substance during storage.

Microcapsules of a water-in-oil-in-water (W/O/W) emulsion, which contained a hydrophilic substance, 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (PTSA), in its inner aqueous phase, was prepared by hot-air-drying or freeze-drying the emulsion using a single-droplet-drying method. Pullulan, maltodextrin, or gum arabic was used as a wall material, and the oily phase was tricaprylin, oleic acid, olive oil, or a mixture of tricaprylin and olive oil. An encapsulation efficiency higher than 0.95 was reached except for the microcapsules prepared using gum arabic and oleic acid. The hot-air-dried microcapsules were generally more stable than the freeze-dried microcapsules at 37 degrees C and various relative humidities. The stability was higher for the microcapsules with tricaprylin as the oily phase than for the microcapsules with oleic acid. The higher stability of the microcapsules with tricaprylin would be ascribed to the lower partition coefficient of PTSA to the oily phase. There was a tendency for the stability to be higher at lower relative humidity for both the hot-air- and freeze-dried microcapsules. The volumetric fraction of olive oil in its mixture with tricaprylin did not significantly affect either the encapsulation efficiency or the stability of the hot-air-dried microcapsules.

Prediction of the equilibrium conversion for the synthesis of acyl hexose through lipase-catalyzed condensation in water-miscible solvent in the presence of molecular sieve.

A method is proposed for predicting the equilibrium conversion for the synthesis of monoacyl hexose through the lipase-catalyzed condensation of a fatty acid and a hexose in a water-miscible solvent in the presence of a molecular sieve, based on the apparent reaction equilibrium constant, the adsorption isotherm of water on the molecular sieve, the solubility of hexose in the solvent, and the mass balance with respect of water. Validity of the model was examined for the syntheses of lauroyl mannose, lauroyl glucose, and myristoyl mannose in acetonitrile, 2-methyl-2-propanol, or 2-methyl-2-butanol with molecular sieves 3A 1/16 and 4A 1/16. The predicted conversions agreed well with the experimental values except for the case where a significant amount of diester was formed as the result of the addition of an excess amount of the molecular sieve to the solvent or the high molar ratio of the fatty acid to the hexose.

Relationship between structure and permeability of tryptophan derivatives across human intestinal epithelial (Caco-2) cells.

L-Trp and its derivatives were used as model compounds to clarify structural factors which influence the intestinal epithelial permeation and metabolism of amino-acid derivatives. Permeability of model compounds through Caco-2 cells was used as an in vitro absorption model for human intestinal epithelial cells. The influence of compound concentration, the effects of various transporter substrates on permeability coefficients, and pH dependency of permeability coefficients were investigated. The transcellular permeability of Trp and Trp-NH2 in the direction from the apical side to the basolateral side, in which nutrients and drugs were ordinarily absorbed, declined with increasing concentration and saturated at more than 1 and 0.4 mM, respectively. The permeability coefficients for N-terminal protected Trp derivatives and Ac-Trp-NH2 showed similar and constant values in both from the apical-to-basolateral and basolateral-to-apical directions. In addition, significant inhibition of the apical-to-basolateral permeation of Trp by Leu and Phe was observed. The permeability coefficient ratio at pH 6.3 to that at pH 7.3 was explained by the ratio of the ionic form to the neutral form of the compounds. Based upon these results and the partition coefficients in the 1-octanol/water system, possible absorption mechanism of Trp and its derivatives across Caco-2 cells was proposed.

Transformation of cellobiose to 3-ketocellobiose by the EDTA-treated Agrobacterium tumefaciens cells.

Agrobacterium tumefaciens cells treated with EDTA were used as a biocatalyst for the transformation of cellobiose to 3-ketocellobiose. The effects of pH of the reaction medium and temperature on the reaction rate and the selectivity during the transformation by the cells were examined, and it was found that the optimum reaction conditions were pH 6 and 15 degrees C. The selectivity was about 0.8 under these conditions. The substrate concentration had no significant effect on the selectivity up to 300 mmol/I.

Production of saturated acyl L-ascorbate by immobilized lipase using a continuous stirred tank reactor.

6-O-decanoyl, 6-O-dodecanoyl, or 6-O-tetradecanoyl L-ascorbate was continuously produced at 50 degrees C using a continuous stirred tank reactor (CSTR) with an immobilized lipase, Chirazyme L-2 C2, from Candida antarctica. Acetone was used as the reaction medium. For each saturated acyl L-ascorbate, the productivity of ca. 60 g/L reactor/day was achieved for at least 11 days. The solubility of the saturated acyl L-ascorbate in the soybean oil or water was measured at various temperatures. The solubilities in both the soybean oil and the water were higher for L-ascorbate with a shorter acyl chain. The acyl chain dependence of the solubility in water was stronger than that of the solubility in soybean oil. The temperature dependences of the solubility in both soybean oil and water could be expressed by the van't Hoff equation, and the dissolution enthalpy (DeltaH) values for the soybean oil and water were about 20 and 90 kJ/mol, respectively, irrespective of the acyl chain length. The radical scavenging activities of L-ascorbic acid and the saturated acyl L-ascorbates against 1,1-diphenyl-2-picrylhydrazyl free radical were ca. 95% for all of the compounds, and the introduction of a saturated acyl group to the L-ascorbic acid did not affect the activity.

Oxidation of linoleic acid encapsulated with soluble soybean polysaccharide by spray-drying.

Linoleic acid was encapsulated with a soluble soybean polysaccharide, gum arabic, or a mixture of both together with maltodextrin, and the oxidation process of the encapsulated acid was measured at 37 degrees C and at a relative humidity of 12%. The soybean polysaccharide was more effective for encapsulating the acid and suppressing the oxidation of the encapsulated acid than gum arabic. A mixture of the soybean polysaccharide and maltodextrin was also effective for this purpose when the weight fraction of the polysaccharide was equal to or greater than 0.75.

Solubility of saturated fatty acids in water at elevated temperatures.

The solubility in water of saturated fatty acids with even carbon numbers from 8 to 18 was measured in the temperature range of 60 to 230 degrees C and at a pressure of 5 or 15 MPa. The pressure had no significant effect on the solubility. The solubility of the fatty acids increased with increasing temperature. At temperatures higher than about 160 degrees C, the logarithm of the solubility in mole fraction was linearly related to the reciprocal of the absolute temperature for each fatty acid, indicating that the water containing solubilized fatty acid molecules formed a regular solution at the higher temperatures. The enthalpy of a solution of the fatty acids in water, which was evaluated from the linear relationship at the given temperatures, increased linearly with the carbon number of the fatty acid.

Suppressive effect of saturated acyl L-ascorbate on the oxidation of linoleic acid encapsulated with maltodextrin or gum arabic by spray-drying.

6-O-Palmitoyl L-ascorbate was added to linoleic acid at various molar ratios of the ascorbate to the acid, the mixtures were emulsified with a maltodextrin or gum arabic solution, and the emulsions were spray-dried to produce microcapsules. At higher molar ratios, the oil droplets in the emulsions were smaller, and the oxidative stabilities of the encapsulated linoleic acid were higher for both the maltodextrin- and gum arabic-based microcapsules. 6-O-Capryloyl, caproyl, and lauroyl L-ascorbates, which were synthesized through lipase-catalyzed condensation in acetone, were also used for the microencapsulation of linoleic acid. Except for capryloyl L-ascorbate, the addition of a saturated acyl ascorbate, especially caproyl ascorbate, to linoleic acid was effective for preparing oil droplets of small particle diameter and for suppressing the oxidation of the encapsulated linoleic acid.

Kinetic expression for the oxidation of linoleic and arachidonic acid esters in their mixed system.

Two polyunsaturated fatty acids (PUFAs) or their esters were mixed, and their oxidation processes were measured at 65 degrees C and ca. 0% relative humidity. Except when a PUFA ester was mixed with a free PUFA, the oxidation of the less-oxidative PUFA was promoted as its content in the mixture decreased, while the oxidation of the more-oxidative PUFA was delayed with a decrease in its content. A kinetic model is proposed whereby a PUFA acts as the diluent for another PUFA, and the oxidation rate of the PUFA is proportional to the product of the unoxidized PUFA concentration and the sum of the concentrations of the oxidized PUFA and the other oxidized PUFA. This model well expressed the oxidation processes of the PUFAs in their mixed system.