Masakazu Furuta

Inactivation of Saccharomyces cerevisiae by ultrasonic irradiation

We have investigated the inactivation of Saccharomyces cerevisiae (yeast cells) by ultrasonic irradiation. The amplitude on the vibration face contacting the sample solution was used as an indication of the ultrasonic power intensity. The effects of the amplitude on the vibration face and the initial cell numbers on the sonolytic inactivation of yeast cells have been investigated using a horn-type sonicator (27.5 kHz). The inactivation of the yeast cells by ultrasonic irradiation shows pseudo first-order behavior. The inactivation rate constant varied from 0.0007 to 0.145 s(-1) when the amplitude on the vibration face was in the range of 1-7 microm(p-p). The change in the inactivation rate constant as a function of the amplitude on the vibration face was similar to that of the OH radical formation rate under the same conditions. The threshold of this sonicator was 3 microm(p-p) with the amplitude on the vibration face. The initial cell numbers (from 10(2) to 10(5) mL(-1)) had an influence on the inactivation of the yeast cells by ultrasonic irradiation. The inactivation rate constants varied from 0.023 to 6.4 x 10(-3) s(-1), and the inactivation by ultrasonic irradiation was fastest at the lowest initial cell numbers. In a squeeze-film-type sonicator (26.6 kHz), 90% inactivation of the yeast cells was achieved by ultrasonic irradiation for 60 min.

A novel collagen hydrogel cross-linked by gamma-ray irradiation in acidic pH conditions

We made a new type of collagen gel by γ-ray irradiation of an acidic solution of type-I collagen, and performed comparative studies on a conventional gel and the new type of gel. The neutral gel, a conventional 0.3% (w/v) collagen gel, was formed at neutral pH and then irradiated by γ-rays. The acidic gel, a 0.3% (w/v) collagen gel, was formed directly from the acidic solution of collagen by γ-ray irradiation. Both types of gel were prepared, swollen in water and then dried for the measurement of specific water content. The neutral gel showed a relatively high specific water content and shrunk moderately, depending on the dose, while the acidic gel showed lower specific water content and shrunk clearly by γ-ray irradiation. A three-dimensional tangled network of microfibrils was clearly observed in the neutral gels by scanning electron microscopy, but not in the acidic gels. From these results, we concluded that the acidic gel was quite different from a conventional collagen gel. Sodium dodecylsulfate-polyacrylamide gel electrophoresis showed that the α 1 subunit and α 2 subunit of the collagen molecule were cross-linked. The triple-helical structure of collagen was only partially perturbed, but not denatured completely, because the circular dichroism spectrum of the collagen solution irradiated at 1.3 kGy was similar to that of native collagen solution. Amino-acid analysis revealed that tyrosine, phenylalanine and histidine decreased by irradiation in the neutral gel. In the case of the acidic gel, these three amino acids and methionine decreased. We considered that these amino acids were cross-linking points between the collagen subunits during the γ-ray irradiation.

Comparison between the effects of ultrasound and γ-rays on the inactivation of Saccharomyces cerevisiae: Analyses of cell membrane permeability and DNA or RNA synthesis by flow cytometry

The effects of 200 kHz ultrasonic irradiation on DNA or RNA formation and membrane permeability of yeast cells were investigated by flow cytometry and compared with those of (60)Co gamma-ray radiation. Colony counting analyses were also performed for comparison. It was observed that the colony-forming activity of yeast cells was not affected by small doses of ultrasonic irradiation, but was closely related to the amounts of sonolytically formed hydrogen peroxide at concentrations of more than 80 microM. On the other hand, gamma-rays directly retarded colony-forming ability in addition to the effects of radiolytically formed hydrogen peroxide. The results obtained by flow cytometry also indicated that the amounts of DNA or RNA formed decreased with an increase in ultrasonic irradiation time without any threshold. These results indicated that flow cytometry can show early growth activities, but that colony counting analyses are insufficient to evaluate continuous and quantitative changes in these activities. In addition, by analyzing the amounts of DNA or RNA formed in the presence of the same amount of hydrogen peroxide, it was found that DNA or RNA formation behavior in the presence of hydrogen peroxide with no irradiation was similar to that following ultrasonic irradiation. These results suggested that similar chemical effects due to the formation of hydrogen peroxide were produced during ultrasonic irradiation. In addition, physical effects of ultrasound, such as shock wave, hardly contributed to cell inactivation and cell membrane damage, because relatively high frequency ultrasound was used here. In the case of gamma-ray radiation, direct physical effects on the cells were clearly observed.

Immobilization of bromelain onto porous copoly(γ-methyl-l-glutamate/l-leucine) beads

Abstract Water-insoluble bromelain was prepared by immobilizing bromelain onto the surface of porous copoly(γ-methyl- l -glutamate/ l -leucine) (ML) beads with and without spacer. The mode of the immobilization between bromelain and porous copolypeptide ML beads was covalent fixation. The relative activity and the stability of the immobilized bromelain was investigated. The retained activity of the bromelain covalently immobilized by the azide method was found to be excellent toward a small ester substrate, N -benzyl- l -arginine ethyl ester, but rather low toward casein, a high molecular weight substrate. The values of the Michaelis constant K m and the maximum reaction velocity V m for free and immobilized bromelain on the porous copolypeptide ML beads were estimated. Apparent K m was larger for immobilized bromelain than for the free one, while V m was smaller for the immobilized bromelain. The thermal stability of the covalently immobilized bromelain was higher than that of the free bromelain. The initial enzymatic activity of the immobilized bromelain remained approximately unchanged with storage time, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.

Inactivation of Escherichia coli Using the Atmospheric Pressure Plasma Jet of Ar gas

Germicidal treatments of Escherichia coli on Langmuir–Blodget (LB) agar were performed using the atmospheric pressure plasma jet sources of Ar gas in the ambient air. Shorter distances from the nozzle of the plasma jet device were more effective in achieving higher bactericidal effects on E. coli grown on LB agar. The surface temperature of the agar was monitored and the spectroscopic analysis of the plasma jet was performed in order to evaluate the factors contributing to the bactericidal effect, such as heating, UV emission, and radical formation caused by the plasma jet. Although the plasma jet raised the surface temperature of LB agar up to about 40 °C, the bactericidal effect was not observed. Moreover, the bactericidal effect of UV (200–300 nm) emitted from the plasma jet was negligible compared with the effects of ions and radical species generated by the atmospheric plasma. The results suggest that the ions and radical species generated by the atmospheric pressure plasma jet are critical for high bactericidal effects on E. coli.

Biodegradable Zein-Based Films : Influence of γ-Irradiation on Structural and Functional Properties

Zein, a predominant corn protein, is an alcohol-soluble protein extracted from corn and is characterized by unique film-forming properties. The characteristic brittleness of zein diminishes its usefulness as a structural material. The objective of this work was to study the effect of gamma-irradiation on improving the performance of zein films in packaging applications. This goal has been achieved by irradiating zein film-forming solutions with various doses of gamma-rays, namely, 10, 20, 30, and 40 kGy at dose rate of 10.5 kGy/h, using a Co(60) gamma-radiation source. The impact of radiation process on the structural properties has been explored through far-UV CD spectral analysis and scanning electron microscopy. Additionally, viscosity changes that reflect the effect of radiation treatment on degradation and/or cross-linking of protein chains have been measured. However, improvements in the performance of zein films as packaging materials that can be accomplished by radiation treatment have been investigated via monitoring of the color, surface density, roughness, mechanical properties (tensile strength and elongation percentage), water uptake, and water barrier properties. The results indicated that gamma-irradiation treatment of the film-forming solution can be used to improve the water barrier properties, as well as color and appearance of the zein films. Moreover, a sterilization effect is considered to be an additional advantage for applying gamma- irradiation.

Gelatin hydrogels cross-linked by γ-ray irradiation : materials for absorption and release of dye

Gelatin hydrogels cross-linked by γ-ray irradiation using 60Co as γ-ray source were prepared. As a model of controlled release of low-molecular-weight compounds, absorption and release of methylene blue, a water-soluble cationic dye, was investigated. Irradiated gelatin hydrogels did not redissolve at temperatures over 40°C, while unirradiated gels were thermoplastic and reversibly changed the stage between gel and sol. Measurement of both the wet weight after swelling in distilled water and dry weight after freeze-drying showed that the higher-dose irradiation gave stiffer and more compact gels with the lower specific water content, irrespective of the absorbed dose rate. The time-course of absorption and release of methylene blue in aqueous solution was measured. Since absorption of dye into gelatin gels was much affected by liquid phase pH, amount of absorption was higher in pH above an IEP of gelatins. Moreover, the absorption and release of methylene blue with Type-B gelatin were higher than with Type-A gelatin, respectively. Therefore, absorption and release of the dye depend on the electrostatic interaction between the dye molecule and gelatin.

2-アルキルシクロブタノンを指標にした放射線照射食肉,魚肉およびその調理品の冷凍保存後の検知

2-Alkylcyclobutanones, such as 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone, were analyzed to assess the irradiation history of irradiated meats or fish, and cooked foods with irradiated ingredients, which had been stored frozen for up to one year. The purpose of the study was to show that irradiated meats could be detected even after having been stored in the distribution system. 2-Alkylcyclobutanones showed a small decrease in irradiated raw meats that had been stored frozen for one year. Cooked foods, such as pancake and fried chicken made with irradiated eggs and chicken, respectively, contained detectable levels of 2-alkylcyclobutanones after storage frozen for one year. The 2-alkylcyclobutanones became undetectable in highly dried samples, such as feed for lab animals, during the same period.

Radiation sterilization of enzyme hybrids with biodegradable polymers

Abstract Ionizing radiations, which have already been utilized for the sterilization of medical supplies as well as gas fumigation, should be the final candidate to decontaminate “hybrid” biomaterials containing bio-active materials including enzymes because irradiation induces neither heat nor substances affecting the quality of the materials and our health. In order to check the feasibility of 60 Co-gamma rays on these materials, we selected commercial proteases including papain and bromelain hybridized with commercial activated chitosan beads and demonstrated that these enzyme-hybrids suspended in water showed the significant radiation durability of more than twice as much as free enzyme solution at 25-kGy irradiation. Enhanced thermal and storage stability of the enzyme hybrids were not affected by the same dose level of irradiation, either, indicating that commercial irradiation sterilization method is applicable to enzyme hybrids without modification.

Calorimetric study on the effect of 60Co γ-rays on the growth of microorganisms

Abstract Using a calorimeter equipped with 24 sample units, the heat evolution from growing Saccharomyces cerevisiae, Escherichia coli and spores of Bacillus pumilus and Bacillus stearothermophilus was detected in the form of growth thermograms. Irradiation with 60Co γ-rays affected the growth pattern, which was used for a quantitative analysis of the effect on microorganisms. Irradiation of B. pumilus and B. stearothermophilus spores led to dose-dependent delays in growth, indicating a bactericidal effect. In case of 60Co γ-irradiated S. cerevisiae, a dose-dependent reduction of the growth rate constant was observed together with the retardation in growth, indicating a combination of bactericidal and bacteriostatic effects. An equation to determine the number of survivors on the basis of the retardation in growth tα and the growth rate constant μ was developed, which proved the opportunity to use the calorimetric technique in predictive microbiology.