Establishment of culture and microinjection methods for false clownfish embryos without parental care.

Anemonefish, including the false clownfish Amphiprion ocellaris, are attractive model organisms because of their unique features, such as sex change and brilliant color patterns in mutants. However, anemonefish are not widely used to study gene function using reverse genetic approaches owing to microinjection difficulties and subsequent rearing and hatching of embryos without parental care. A. ocellaris embryos are spawned on a hard substrate and cared for by their parents until hatching. However, the eggs need to be detached from the substrate and raised without their parents to perform successful microinjection. We established a method to culture and hatch A. ocellaris embryos without spawning substrates or parental care. We found that changing water and generating water flow are critical for culturing the embryos, and that water flow (as physical stimulation) and complete darkness in the dark period are necessary for successful hatching. We further investigated the effectiveness of microinjection into the yolk sac of fertilized eggs rather than into the cytoplasm, which makes microinjection easier. A reporter RNA injected into the yolk sac was transferred to the cytoplasm and translated, indicating that yolk sac microinjection is an efficient alternative as has been used for zebrafish. These findings highlight the potential of A. ocellaris as an experimental model organism for reverse genetics, and our methods could be applied to other anemonefish species.

Micro-particles as interfering substances in colorimetric residual chlorine measurement.

Conventional methods using o-tolidine and N,N-diethyl-p-phenylenediamine as colorimetric reagents have been extensively applied worldwide in residual chlorine measurement for water quality and environmental management. Different types of interferences resulting in erroneous measurements while using colorimetry have been previously reported. In this study, we experimentally demonstrated micro-particles as interfering substances in selected inorganic (five metal oxidants) and organic (microalgae) particles. The results indicated erroneous measurements (viz. colour development) for three of the selected particles. These erroneous measurement levels were evaluated with reference to the chlorine concentration (in mg-Cl2/L, hereafter represented as mg/L) in relation to both representative colorimetric reagents in terms of the amount of particles and time variations. A novel viewpoint that filtration could be a possible solution to the erroneous measurement caused by such micro-particles was proposed.

Sunlight caused interference in outdoor N, N-diethyl-p-phenylenediamine colorimetric measurement for residual chlorine and the solution for on-site work.

Chlorination is the most common method to control water qualities, in some case on-site outdoor measurements are required to measure easily-decaying residual chlorine concentration appropriately without delay. In this study sunlight-induced unexpected colour development (UCD) of N, N-diethyl-p-phenylenediamine (DPD) colorimetric measurement was studied under several sun exposure conditions. The colour development level was evaluated with reference to chlorine concentration (mg/L) and relationships between colour development rate (mg/L min) and intensities of solar were investigated. UCD was found to be related to both exposure intensity and time. By means of exposure experiment under specific wavelength of ultraviolet (UV), it was confirmed that both middle and short wavelength of UV radiation being responsible for such an unexpected measurement. Consequently, a simple device was designed using three commercially available anti-UV films, one of which could effectively prevent the UCD from direct sun exposure.

Omaezallene from red alga Laurencia sp.: structure elucidation, total synthesis, and antifouling activity.

Natural antifouling products have been the subject of considerable attention. We screened marine algae for antifouling activity and discovered omaezallenes, the new bromoallene-containing natural products isolated from the red alga Laurencia sp. Described is the isolation, structure elucidation, and total syntheses of omaezallenes. The relative and absolute configurations of natural omaezallenes were unambiguously established through total synthesis. The antifouling activities and ecotoxicity of omaezallenes were also evaluated.

Kinetic study of thermally stimulated dissociation of inclusion complex of 1-methylcyclopropene with alpha-cyclodextrin by thermal analysis.

The thermally stimulated dissociation of the inclusion complex of 1-methylcyclopropene (1-MCP) with alpha-cyclodextrin (alpha-CD) in solid state was studied by means of thermogravimetry (TG) and differential scanning calorimetry (DSC). The mass loss of 1-MCP/alpha-CD inclusion complex occurs in four separated phases with the thermal dissociation of the inclusion complex and release of 1-MCP taking place in the second phase between 90 and 230 degrees C. The kinetic parameters of the dissociation reaction (the apparent activation energy of dissociation, E(D), the reaction order of thermal dissociation, n, and the pre-exponential factor, k0) were evaluated. The dissociation reaction was satisfactorily described by the unimolecular decay law, where the reaction order, n = 1. The effect of the molar ratio of 1-MCP to alpha-CD (inclusion ratio) in the inclusion complex on the temperature dependence of the dissociation reaction was also studied. The E(D) decreased with increasing inclusion ratio indicating higher complex stability at lower inclusion ratios. The extrapolation of the E(D) of the inclusion complexes with different inclusion ratios to 1 mol 1-MCP/mol alpha-CD yielded the "true" E(D) of 20.9 +/- 2.8 and 18.1 +/- 0.2 kJ/mol for TG and DSC, respectively. The "true" ln k(0TG) and the "true" ln k(0DSC) were also determined by extrapolation, yielding values of +4.5 +/- 1.0 and -0.3 +/- 0.3, respectively.

Improvement of antifungal activity of 10-undecyn-1-ol by inclusion complexation with cyclodextrin derivatives.

The inclusion complexation behavior between 10-undecyn-1-ol and cyclodextrin (CD) derivatives, namely, randomly methylated beta-CD (RM-beta-CD) and hydroxypropyl-beta-CD (HP-beta-CD), was studied in terms of solubility improvement, apparent stability constant, and the inclusion ratios of the resultant inclusion complexes. The aqueous solubility of 10-undecyn-1-ol was greatly improved through complexation with the CD derivatives. RM-beta-CD is comparatively more efficient in solubilizing 10-undecyn-1-ol with an apparent stability constant outstripping that of HP-beta-CD by about an order of magnitude. Comparative in vitro evaluations of the growth inhibition effects of inclusion complex solutions toward Rosellinia necatrix, a phytopathogenic fungus, were performed. In comparison with the positive control, appreciable improvements of the antifungal activity of 10-undecyn-1-ol through the addition of CD derivatives were observed visually. The improvement was evaluated in terms of area covered by the mycelia of Rosellinia necatrix and their growth rate. RM-beta-CD was proven to be more effective compared to HP-beta-CD with regard to the reduction of both fungal mycelium-covered area and growth rate constant, presumably owing to greater solubility enhancement by RM-beta-CD and thus the bioavailability of 10-undecyn-1-ol. Inclusion complexation of 10-undecyn-1-ol with CD derivatives suggests a potential means for production of an environmentally friendly 10-undecyn-1-ol-based fungicide to counteract R. necatrix.

Innovative crystal transformation of dihydrate trehalose to anhydrous trehalose using ethanol.

The crystal transformation of dihydrate trehalose to anhydrous trehalose was investigated using ethanol and a new type of crystal particle with porous structure could be obtained. The specific surface area of the anhydrous crystal transformed at 50 degrees C was 3.3 m(2)/g, with a median pore diameter of 0.21 microm, and void volume of 0.22 mL/g. The crystal transformation was monitored by measuring the crystal moisture content. The crystal transformation rates could be correlated with the Avrami equation, using the mechanism parameter n=11.5, suggesting that the change of surface area occurred during crystal transformation from dehydrate to anhydrous trehalose. The apparent activation energy of the crystal transformation was 132 kJ/mol.

Kinetics of molecular encapsulation of 1-methylcyclopropene into alpha-cyclodextrin.

1-methylcyclopropene (1-MCP), an ethylene inhibiting regulator, is commercially available in the form of an inclusion complex with alpha-cyclodextrin (alpha-CD). In this study, molecular encapsulation of gaseous 1-MCP into aqueous alpha-CD was investigated in a closed, agitated vessel with a flat gas-liquid interface. Molecular encapsulation of gaseous 1-MCP by alpha-CD is a simultaneous two-step reaction which involves the aqueous dissolution of gaseous 1-MCP and the encapsulation of the dissolved molecules by alpha-CD. The kinetics and mechanism of molecular encapsulation were analyzed based on the depletion rate of 1-MCP in the headspace of the vessel. The encapsulation rates could be explained quantitatively by the gas absorption theory with a pseudo-first-order reaction between 1-MCP and alpha-CD. The negative value of the calculated apparent activation energy of encapsulation (-24.4 kJ/mol) implied the significant effect of exothermic aqueous dissolution of 1-MCP. An encapsulation temperature of 15 degrees C was optimal; at this temperature, the highest 1-MCP yield and best inclusion ratio of inclusion complex were obtained. Changes in the X-ray diffraction pattern suggested that the crystal lattice structure of alpha-CD was altered upon inclusion of 1-MCP.

Effect of water activity on the release characteristics and oxidative stability of D-limonene encapsulated by spray drying.

The stability of encapsulated D-limonene, which was prepared by spray drying, was studied in view of the release characteristics and oxidation stability. Gum arabic, soybean water-soluble polysaccharide, or modified starch, blended with maltodextrin were used as the wall materials. The powders were stored under the conditions of 23-96% relative humidity at 50 degrees C. The release rate and the oxidation rate were closely related to the relative humidity. The relationship was not simple. Initially, the release rate and the oxidation rate increased with increasing water activity, but around the glass transition temperature, the rates decreased sharply to increase again at a further increase of water activity. The results could be explained by a change in the powder structure, where a glass capsule matrix was changed into rubbery state during storage.

Encapsulation of alcohol dehydrogenase in mannitol by spray drying.

The retention of the enzyme activity of alcohol dehydrogenase (ADH) has been studied in various drying processes such as spray drying. The aim of this study is to encapsulate ADH in mannitol, either with or without additive in order to limit the thermal denaturation of the enzyme during the drying process. The retention of ADH activity was investigated at different drying temperatures. When mannitol was used, the encapsulated ADH was found inactive in all the dried powders. This is presumably due to the quick crystallization of mannitol during spray drying that resulted in the impairment of enzyme protection ability in comparison to its amorphous form. Maltodextin (dextrose equivalent = 11) was used to reduce the crystallization of mannitol. The addition of maltodextrin increased ADH activity and drastically changed the powder X-ray diffractogram of the spray-dried powders.

High-oil-load encapsulation of medium-chain triglycerides and D-limonene mixture in modified starch by spray drying.

UNLABELLED: Oil mixtures of medium-chain triglycerides (MCT) and D-limonene in mixing ratios from 10 to 100 wt% were encapsulated in modified starch (wall material) by spray drying to produce oil-rich powders. The oil load (mass ratio of oil mixture to wall material) of the infeed emulsion markedly influenced the properties of the infeed liquid and the characteristics of the resulting powder. The viscosity of the infeed liquid and the particle size of the powder exponentially decreased with increasing oil load, while the emulsion droplet size in the infeed liquid increased. In addition, retention of D-limonene during spray drying also decreased markedly with increasing oil load. Irrespective of the different oil loads and concentrations of the wall material, D-limonene retention was well correlated with the emulsion droplet diameter of the infeed liquid. The encapsulation efficiency of the oil mixture exhibited a maximum value (almost 100%) at an oil load between 0.5 and 1.0, before decreasing at higher oil loads. At an oil load of 2.0, the encapsulation efficiency of D-limonene was reduced to almost zero, while around 40% of the initial MCT was encapsulated in the powder. The increase in oil load also led to increased amounts of surface oil of MCT and D-limonene in the resulting powder due to the increasing emulsion droplet diameter of the infeed liquids. PRACTICAL APPLICATION: This study proposes the microencapsulation of medium-chain triglycerides under high-oil-load conditions by spray drying. The powders prepared by this process provide significant benefits in terms of rapid energy conversion after consumption without accumulation in the body. Important quality factors of the powder products such as the encapsulation efficiency and the amount of surface oil were examined to understand the optimum process conditions for spray drying.

Formation of a new crystalline form of anhydrous ß-maltose by ethanol-mediated crystal transformation.

ß-Maltose monohydrate was transformed into an anhydrous form by ethanol-mediated method under several temperatures with agitation. A new stable anhydrous form of ß-maltose (Mß(s)) was obtained, as substantiated by the X-ray diffraction patterns. Mß(s) obtained by this method presented a fine porous structure, resulting in greater specific surface area compared to those of ß-maltose monohydrate and anhydrous ß-maltose obtained by vacuum drying (Mß(h)). The crystal transformation presumably consisted of two steps: dehydration reaction from the hydrous to amorphous forms and crystal formation from the amorphous forms to the noble anhydrous form. The kinetics of these reactions were determined by thermal analysis using Jander's equation and Arrhenius plots. The overall activation energies of the dehydration reaction and the formation of anhydrous maltose were evaluated to be 100 and 90 kJ/mol, respectively.

Dissociation characteristic of the inclusion complex of cyclomaltohexaose (alpha-cyclodextrin) with 1-methylcyclopropene in response to stepwise rising relative humidity.

The dissociation of a crystalline complex of cyclomaltohexaose (alpha-cyclodextrin) and 1-methylcyclopropene has been studied in response to stepwise rising relative humidity at 50 degrees C using a dynamic vapor sorption instrument. The dissociation of the inclusion complex was monitored with a proton transfer reaction mass spectrometer. The increase in relative humidity generally triggered the complex dissociation. However, the dissociation was greatly retarded at 80% relative humidity, presumably owing to collapse of the crystalline structure. Abrupt dissociation was observed at 90% relative humidity which corresponded to complex dissolution. The changes in powder X-ray diffraction pattern of the inclusion complex during the storage period were also investigated.

Formation of spray-dried powder of S-adenosyl-L-methionine.

S-Adenosyl-L-methionine (SAM) is an essential metabolite in all living organisms. In clinical research, SAM has also been suggested as a chemotherapeutic agent in various diseases. The main problem of SAM is its instability at high temperatures, at neutral and alkaline pH, and in the presence of humidity. SAM retention in spray-dried powder was determined under various conditions of spray-drying. The highest SAM retention was obtained when maltodextrin (dextrose equivalent, DE, of 25) was used as the carrier solid with the SAM feed liquid at pH 4.0. The water content in the powder had a significant effect on the stability of SAM. SAM powder with lower water content exhibited higher stability.

Interaction of modified cyclodextrins with cytochrome P-450.

The effects of modified cyclodextrins (CDs) hydroxypropyl-beta-CD and methyl-beta-CD were studied in vitro on cDNA-expressed human cytochrome P-450 (CYP) activities (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). The modified CDs inhibited the activities of CYP2C19 and CYP3A4 while enhancing CYP2C9 activity by 140 to 176% relative to the control values at lower concentrations. In addition, methyl-beta-CD inhibited CYP1A2 and CYP2D6 at higher concentrations.

Release characteristics of flavor from spray-dried powder in boiling water and during rice cooking.

The release characteristics of flavor in boiling water and the flavor retention in the rice after cooking were investigated by using spray dried powder in encapsulated in or emulsified with d-limonene or ethyl n-hexanoate in cyclodextrin and maltodextrin, or in gum arabic and maltodextrin. The behavior of flavor release into the boiling water was well simulated by Avrami's equation. The retention of d-limonene and ethyl n-hexanoate in cooked rice was correlated in each case with the flavor amount of spray-dried powder added.

Autoxidation kinetic analysis of docosahexaenoic acid ethyl ester and docosahexaenoic triglyceride with oxygen sensor.

The application of omega-3 polyunsaturated fatty acids (PUFAs) as food additives is restricted by their chemically quite reactive properties. However, quantitative analyses of the oxidative kinetics of PUFAs are very few compared to other studies on food chemistry. In this study, the autoxidation kinetics of ethyl docosahexaenoate (DHAEE), docosahexaenoic triglyceride (DHA oil), and emulsified DHA oil were investigated with an oxygen sensor. The autocatalytic reaction rate constants for DHAEE, DHA oil, and the emulsified DHA oil with 20% (w/v) GA, 20% SSPS, or 20% SSPS containing 5% soy protein were obtained at 35, 50, and 70 degrees C. A plot of the natural logarithm of the frequency factor, In ka0, vs. the activation energy, Ea, demonstrated that In ka0 against Ea fitted well with a single straight line both for the data from this study and for other reported results. This implies that the chemical compensation relationship holds between ka0 and Ea for PUFA and emulsified DHA oil.

Encapsulation of shiitake (Lenthinus edodes) flavors by spray drying.

Powdery encapsulation of shiitake flavors, extracted from dried shiitake, was investigated by spray drying. Flavor retention increased with an increase in drying air temperature and solid content, and decreased with an increase in dextrose equivalents of maltodextrin. A heat-treatment of the extract liquid made the lenthionine concentration increase, but did not influence the concentrations of the other flavors. The formation of lenthionine with heat-treatment could be described by the consecutive unimolecular-type first order reaction. Lenthionine content in a spray-dried powder prepared with the heated extracted liquid significantly increased. alpha-Cyclodextrin was the most suitable encapsulant of alpha-, beta-, and gamma-cyclodextrins to prepare the spray-dried powder, including lenthionine. The flavor retentions were markedly increased by using of alpha-cyclodextrin and maltodextrin in combination as an encapsulant.