A novel procedure for the quantification of antifungal activity against filamentous fungi, mycelial invasion distance (MID) method.

A novel method for the quantification of antifungal activity of fungicides and painted surfaces, mycelial invasion distance (MID) method, was developed and applied to the quantification of activities of parabens and an antifungal paint. In this method, the MID of aerial mycelia on a test paper or a panel placed on a nutrient agar plate was measured with a stereoscopic microscope and a micro-ruler. The antifungal activities of the parabens and painted surfaces were expressed as the MID. The higher the hydrophobicity of parabens, the longer the MID, that is the lower the antifungal activity, were observed. Conversely, relatively polar parabens, such as methyl and ethyl parabens, exhibited stronger antifungal activity, that is shorter MID. The most hydrophobic paraben, benzyl paraben, showed the weakest antifungal activity. Furthermore, it was confirmed that the MID method was effective for the evaluation of the painted surfaces.

A new plate-hanging method for biofilm quantification and its application to evaluate the role of surface hydrophobicity.

A novel procedure for the quantitative analysis of biofilm formation by bacteria and yeasts, the Plate-hanging method, was developed. In this system, various polymer disks were hung from the lid of a 6-well plate, immersed in a cell suspension, and moderately shaken (70 rpm). In order to verify the validity of the procedure, the effects of the solid surface hydrophobicity of the test disks and the cell surface hydrophobicities of microorganisms on biofilm formation were investigated. Biofilm formation of bacteria and yeasts on the solid surface strongly depended on hydrophobic interactions between the solid surface and the cell surface. A positive correlation between the hydrophobic properties of substratum and cell surfaces was observed. On the other hand, hydrophilic yeasts preferentially adsorbed onto relatively hydrophilic surfaces. Moreover, the plate-hanging method coupled with the periodic exchange of the liquid medium enabled the quantification of long-term biofilm growth.

Efficient production of fungal spores by the combination of reduction of nitrogen source content and embedding of hydrophobic polymer in an agar plate.

Fungal sporulation is affected by many environmental factors, for example, we previously observed that embedding of a hydrophobic polymer net in an agar plate medium significantly accelerates spore formation of some fungi. Here, it was found that the fungal spore formation depended on the surface hydrophobicity of cultivation vessels used for the plate cultivation. In a polypropylene (PP) vessel, six fungal strains produced spores of 1.5 to 514.8 times of those growing in a glass vessel. The contact of vegetative hyphae on the surface of the vessels might trigger the fungal spore formation. Moreover, the spore formation was synergistically accelerated by the reduction of nitrogen source content in an agar plate medium and by the contact to hydrophobic polymers. The synergistic effect depended on the surface area of the hydrophobic polymer. Thus, the combination of the reduction of nitrogen source and the embedding of hydrophobic polymer is expected as a novel and effective procedure for production of fungal spores which are useful for the inoculum in fermentation industry and biocontrol agent in agriculture.

Evaluation of Antibacterial and Cytotoxic Properties of a Fluorinated Diamond-Like Carbon Coating for the Development of Antibacterial Medical Implants.

Peri-implant infection is a serious complication in surgical procedures involving implants. We conducted an in vitro study to determine whether the use of a fluorinated diamond-like carbon (F-DLC) coating on a titanium alloy surface can prevent peri-implant infection. After applying the F-DLC, we evaluated its antibacterial and cytotoxic properties. The coating groups, containing controlled fluorine concentrations of 5.44%, 17.43%, 24.09%, and 30%, were examined for the presence of Staphylococcus aureus and Escherichia coli according to ISO 22196 for the measurement of antibacterial activity on plastics and other nonporous surfaces. Biological toxicity was evaluated using Chinese hamster V79 cells according to ISO 10993-5 for the biological evaluation of medical devices. In the control group, populations of S. aureus and E. coli substantially increased from 2.4 × 104 to (1.45 ± 1.11) × 106 colony-forming units (CFUs) and from 2.54 × 104 to (4.04 ± 0.44) × 106 CFUs, respectively. However, no bacteria colonies were detected in any F-DLC group with a fluorine concentration of ≥ 17.43%. In the biological toxicity study, an F-DLC coating with a fluorine concentration of 30% showed a colony formation rate of 105.8 ± 24.1%, which did not differ significantly from the colony formation rate of 107.5 ± 31.1% in the nontoxic control group. An F-DLC coating on titanium alloy discs showed excellent in vitro antibacterial activity with no biological toxicity.

Microbial transformation of water-insoluble substrates by two types of novel interface bioprocesses, tacky liquid-liquid interface bioreactor and non-aqueous sporular bioconversion system.

Although microbial transformation has been expected as a substitution technology for organic synthesis, microbial toxicity and water-insolubility of synthetic substrates prevent the practical application of the technology. For these problems, the authors have developed two types of interfacial bioprocesses, solid-liquid and liquid-liquid interface bioreactors and applied the systems to many microbial transformations. In the bioreactors, addition of substrates and accumulation of products were remarkably enhanced based on the toxicity alleviation effect on the interfaces and solubilization of substrates and/or products in an organic phase of the bioreactors. Recently, a novel tacky liquid-liquid interface bioreactor has been developed and applied to actinomycetes and yeasts. Furthermore, a novel bioconversion system with fungal spores in an organic phase has been constructed based on the combination of two facts as follows: (i) the fungal spores are never resting cells and are active ones like the vegetable cells, (ii) the fungal spores have the excellent solvent-tolerance. In this review, the tacky liquid-liquid interface bioreactor (L-L IBRtac) and the non-aqueous sporular bioconversion system with immobilized fungal spores (NASB) are mainly given outlines.

Solid-liquid Interface Screening SystemーApplication to the Screening of Antibiotic and Cytotoxic Substance-producing Fungi.

　A useful tool for the screening of fungi producing biologically active secondary metabolites such as antibiotics and cytotoxic substances has been developed. An agar plate-organic solvent interface cultivation (A/S-IFC) system, which comprised a hydrophobic organic solvent (upper phase) , a fungal mat (middle phase) and an agar plate (lower phase) , was constructed. The metabolite profiles were compared among the A/S-IFC, a traditional submerged cultivation (SmC) and an extractive liquid surface immobilization (Ext-LSI) system consisted of a hydrophobic solvent (upper phase) , a fungal cells-ballooned microspheres (middle phase) and a liquid medium (lower phase) , with high-performance liquid chromatography-photodiode array detector (HPLC-PDA) . In the A/S-IFC, many hydrophobic metabolites vastly different from those in the SmC were accumulated in the organic phase as with the Ext-LSI. For example, a valuable azaphilone, sclerotiorin, was remarkably produced into the organic phase in the A/S-IFC. The A/S-IFC was applied to the screening of antibiotic-producing fungi. As a result of paper disk method, it was found that 321 isolated among 811 strains produced antifungal metabolites (hit rate, 39.6%) . Furthermore, 8, 23, and 30 strains also produced cytotoxic metabolites against SKOV-3 (human ovary adenocarcinoma) , MESO-1 (human malignant pleural mesothelioma) , and Jurkat cells (immortalized human T lymphocyte) .

Acceleration of fungal spore production by embedding a hydrophobic polymer net in a nutrient agar plate.

A simple and novel procedure for the acceleration of fungal spore production was developed. A net of hydrophobic polymer such as polypropylene (PP) and polytetrafluoroethylene (PTFE) was embedded in a nutrient agar plate, and effect of the polymer net on spore production by 6 fungal strains, such as Aspergillus terreus, Penicillium multicolor, and Trichoderma virens were estimated. The effect of hydrophobic polymer net was insufficient in a liquid-surface immobilization (LSI) system with fungal cells immobilized on a ballooned microsphere layer formed on a liquid medium surface. On the other hand, the embedding of a PTFE net in an agar plate remarkably enhanced the spore production in all 6 strains tested to produce 2.0-8.5 × 107 spores/cm2-agar plate surface. Especially, the spore production by A. terreus ATCC 20542 in the presence of a PTFE net was 7.7 times as much than that in no net. Positive correlations between the hydrophobicity of net and the spore production were observed in all 6 strains (R2, 0.653-0.999).

Novel, Non-aqueous Bioconversion Systems Using Fungal Spores.

Two novel types of non-aqueous bioconversion systems using fungal spores, either adsorbed on the surface of a filter pad or entrapped in calcium alginate beads, were constructed and applied for a model reaction: reduction of benzil to benzoin by Aspergillus sojae NBRC 32074. The spores adsorbed on a filter pad catalyzed the reduction in some toxic organic solvents, such as methylcyclohexane (log P: 3.61) and din-butyl ether (3.21). For the relationship between the reduction activity and the log P value of the organic solvent, a highly positive correlation (R2: 0.815) was observed. Surprisingly, the reduction proceeded in the more hydrophilic and toxic tert-butyl acetate (log P: 1.76). Glycerol was selected as the best hydride source. The higher the glycerol content, the more the benzoin was produced. While the production of benzil by spores was lower than that by mycelia in harmless di-n-hexyl ether (log P: 5.12), mycelia could not catalyze the reduction in the toxic tert-butyl acetate. In contrast, the spores entrapped in the calcium alginate beads could catalyze the reduction. Although the reduction by alginate-entrapped spores could be stably repeated 5 times in di-n-hexyl ether without a decline in the reduction activity, it was observed that the reduction activity of the spores gradually decreased after repeated reduction in tert-butyl acetate.

Production of Valuable Lipophilic Compounds by Using Three Types of Interface Bioprocesses: Solid-Liquid Interface Bioreactor, Liquid-Liquid Interface Bioreactor, and Extractive Liquid-Surface Immobilization System.

Bioconversions such as enzymatic and microbial transformations are attractive alternatives to organic synthesis because of practical advantages such as resource conservation, energy efficiency, and environmentally harmonic properties. In addition, the production of secondary metabolites through microbial fermentation is also useful for manufacturing pharmaceuticals, agricultural chemicals, and aroma compounds. For microbial production of useful chemicals, the authors have developed three unique interfacial bioprocesses: a solid-liquid interface bioreactor (S/L-IBR), a liquid-liquid interface bioreactor (L/L-IBR), and an extractive liquid-surface immobilization (Ext-LSI) system. The S/L-IBR comprises a hydrophobic organic solvent (upper phase), a microbial film (middle phase), and a hydrophilic gel such as an agar plate (lower phase); the L/L-IBR and the Ext-LSI consist of a hydrophobic organic solvent (upper phase), a fungal mat with ballooned microspheres (middle phase), and a liquid medium (lower phase). All three systems have unique and practically important characteristics such as utilization of living cells, high concentration of lipophilic substrates/products in an organic phase, no requirement for aeration and agitation, efficient supply of oxygen, easy recovery of product, high regio- and stereoselectivity, and wide versatility. This paper reviews the principle, construction, characteristics, and application of these interfacial systems for producing lipophilic compounds such as useful aroma compounds, citronellol-related compounds, ß-caryophyllene oxide, and 6-penty-α-pyrone.

Discovery of secondary metabolites in an extractive liquid-surface immobilization system and its application to high-throughput interfacial screening of antibiotic-producing fungi.

An extractive liquid-surface immobilization (Ext-LSI) system, which consists of a hydrophobic organic solvent (an upper phase), a fungal cell-ballooned microsphere layer (a middle phase) and a liquid medium (a lower phase), is a unique interfacial cultivation system for fungi. The fungal cells growing at the interface between the organic and aqueous phases efficiently produce hydrophobic metabolites, which are continuously extracted into the organic phase, and/or hydrophilic metabolites that migrate into the aqueous phase without carbon catabolite repression and product and/or feed-back inhibitions. Application of the system to fermentation of Penicillium multicolor IAM 7153 and Trichoderma atroviride AG2755-5NM398 afforded remarkably different profiles of secondary metabolites in the organic phase compared with those in an aqueous phase in traditional submerged cultivation (SmC). Various hydrophobic metabolites exhibiting unique UV-visible spectra were accumulated into the organic phase. The system was applied to a novel interfacial screening system of antibiotic-producing fungi. Compared with the SmC, the interfacial cultivation system exhibited some interesting and important advantages, such as the higher accumulation of hydrophobic secondary metabolites, the lack of requirement for shaking and troublesome solvent extraction, and the small scale of the vessels (medium, 5 ml; dimethylsilicone oil, 1 ml), as well as the significantly different metabolite profiles. The interfacial screening system yielded a high incidence of antimicrobial activity, with 21.9% of the fungi tested exhibiting antifungal activity against Pichia anomala NBRC 10213. This novel interfacial high-throughput screening approach has the potential to discover new biologically active secondary metabolites even from strains previously found to be unproductive.

Solvent-tolerance of fungi located on an interface between an agar plate and an organic solvent.

While 6 by 20 of type culture fungi could grow on an interface between organic solvent (log P, 4.12) and agar plate, 13 by 20 of strains could form a large colony after the removal of more toxic solvent, such as styrene (log P, 2.95) and tert-butyl acetate (log P, 1.76) because of viability of spores on the interface.

Relationship between interfacial hydrophobicity and hydroxylation activity of fungal cells located on an organic-aqueous interface.

In a liquid-liquid interface bioreactor, fungal cells locate in a hydrophilic polyacrylonitrile microsphere layer on an aqueous-organic interface. In this article, effects of hydrophobicity of the interface on n-decane hydroxylation activity of Monilliera sp. NAP 00702 was examined. (-)-4-Decanol production was significantly enhanced to 132% by addition of polytetrafluoroethylene.

Enhancement of 6-pentyl-α-pyrone fermentation activity in an extractive liquid-surface immobilization (Ext-LSI) system by mixing anion-exchange resin microparticles.

The addition of anion-exchange resin microparticles into a polyacrylonitrile (PAN) ballooned microsphere layer drastically enhanced the fermentative activity of Trichoderma atroviride AG2755-5NM398 in an extractive liquid-surface immobilization (Ext-LSI) system. The production of 6-pentyl-α-pyrone (6PP), a fungicidal secondary metabolite, was 1.92-fold higher than the control (PAN alone).

Derepression of carbon catabolite repression in an extractive liquid-surface immobilization (Ext-LSI) system.

An extractive liquid-surface immobilization (Ext-LSI) system with a fungal mat formed on the surface of a liquid medium effectively enabled derepression of carbon catabolite repression. In this system, a fungicidal secondary metabolite 6-pentyl-α-pyrone was efficiently produced by Trichoderma atroviride AG2755-5NM398 despite the addition of 25% glucose or fructose.

Synthesis of (-)-ß-caryophyllene oxide via regio- and stereoselective endocyclic epoxidation of ß-caryophyllene with Nemania aenea SF 10099-1 in a liquid-liquid interface bioreactor (L-L IBR).

Nemania aenea SF 10099-1, a basidiomycete isolated from a forest soil sample, regio- and stereoselectively epoxidized ß-caryophyllene (Car) to (-)-ß-caryophyllene oxide (Car-Ox) in a liquid-liquid interface bioreactor (L-L IBR) consisted of a liquid medium (a bottom phase), a fungus-ballooned microsphere (MS) mat (a middle phase), and an organic phase containing Car (a top phase). The cultivation conditions, such as carbon and nitrogen sources, kind of MS, initial medium pH and Car concentration, were optimized in the L-L IBR system. The best carbon and nitrogen sources were xylose and tryptone, respectively. The most suitable polyacrylonitrile MS was MMF-DE-1 (former MFL-80SDE; non-coated type). Although the strain could not grow below pH 5.5, the endocyclic epoxidation of Car efficiently proceeded at a wide range of initial medium pH (6.0 to 9.0). The bioconversion system exhibited an excellent alleviation effect toward substrate and product inhibitions. While Car could be added into an organic phase (KF-96L-1CS, dimethyl silicone oil) at 50% (w/v), the accumulation of Car-Ox reached over 30g/l in spite of these strong microbial toxicities. Moreover, the epoxidation reaction smoothly proceeded in a novel L-L IBR system, a multistory L-L IBR systems, consisted of 5 stacked reactor units. The optical rotation of Car-Ox produced was (-) and the enantiomeric excesses of (-)-ß-Car-Ox purified by 1st and 2nd recrystallization from methanol reached 97.51 and 99.33%, respectively.

Efficient hydrolytic reaction of an acetate ester with fungal lipase in a liquid-liquid interface bioreactor (L-L IBR) using CaCO3-coated ballooned microsphere.

In a liquid-liquid interface bioreactor using a CaCO3-coated ballooned microsphere, 2-ethylhexyl acetate was efficiently hydrolyzed to 2-ethyl-1-hexanol with Absidia coerulea NBRC 4423 compared with using talc-coated or non-coated ballooned microsphere. It was assumed that CaCO3 brought about stabilization of lipase by Ca²âº and maintenance of medium pH.

Production of Xylanase with a transformant of Aspergillus oryzae RIB40 in a Liquid-Surface Immobilization (LSI) System.

Xylanase production by a XynF1 (33 kDa)-transformant of Aspergillus oryzae RIB40 was compared between submerged cultivation (SmC) and liquid-surface immobilization (LSI) systems. While the accumulation of xylanase in the SmC decreased by prolonged incubation, LSI system enabled the continuation of xylanase production to afford 4.5-fold xylanase production compared with the SmC system.

Asymmetric reduction of benzil to (S)-benzoin with Penicillium claviforme IAM 7294 in a liquid-liquid interface bioreactor (L-L IBR).

The asymmetric reduction of benzyl to (S)-benzoin with Penicillium claviforme IAM 7294 was applied to a liquid-liquid interface bioreactor (L-L IBR) using a unique polymeric material, ballooned microsphere (MS). The L-L IBR showed superior performance, as compared with suspension, organic-aqueous two-liquid-phase, and solid-liquid interface bioreactor (S-L IBR) systems, affording 14.4 g/l-organic phase of (S)-benzoin (99.0% ee).

Antihypertensive potential and mechanism of action of astaxanthin: III. Antioxidant and histopathological effects in spontaneously hypertensive rats.

We investigated the effects of a dietary astaxanthin (ASX-O) on oxidative parameters in spontaneously hypertensive rats (SHR), by determination of the level of nitric oxide (NO) end products nitrite/nitrate (NO2-/NO3-) and lipid peroxidation in ASX-O-treated SHR. Oral administration of the ASX-O significantly reduced the plasma level of NO2-/NO3- compared to the control vehicle (p<0.05). The lipid peroxidation level, however, was reduced in both ASX-O- and olive oil-treated groups. We also analyzed the post-treatment effects of ASX-O on the vascular tissues by examining the changes in the aorta and coronary arteries and arterioles. The dietary ASX-O showed significant reduction in the elastin bands in the rat aorta (p<0.05). It also significantly decreased the [wall : lumen] aerial ratio of the coronary arteries. These results suggest that ASX-O can modulate the oxidative condition and may improve vascular elastin and arterial wall thickness in hypertension.