To establish the effects of type I antifreeze protein (AFP) on E. coli cells, we have focused on the survival rate of the E. coli cells using type I AFP at various concentrations under rapid cooling conditions using liquid N₂ at atmospheric or low pressure. The survival rate of E. coli was enhanced by the addition of type I AFP at a concentration of 10 μg/ml, and its value shifted from 0.73% to 2.96%. When the concentration of type I AFP was 100 μg/ml, the cell survival rate markedly decreased to 0.090%. This low survival rate was further decreased (0.022%) by the application of the same freeze-thaw treatment for four times. Also, the effect of type I AFP as a bactericidal agent did not vary according to the varying initial cell densities from 10⁴ to 10⁸ cells/ml. Furthermore, the effects of using type I AFP at 1.0 MPa with N₂ gas under conditions of low pressure and low oxygen tension using a simple device were examined. When the actions of type I AFP as a cryoprotectant were stimulated, the survival rate of the E. coli cells increased to 57.8%. In addition, the bactericidal effect of type I AFP at 100 μg/ml of protein concentration could also be enhanced. The survival rate using 100 μg/ml of type I AFP under low pressure was 0.35% of that using 10 μg/ml under the same conditions. This is the first report on the cryoprotectant and cryosterilization effects of type I AFP of E. coli cells under various conditions.

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This research investigated the antimicrobial activities of unnatural nucleosides. We tested the MIC and MBC of 17 synthetic nucleoside analogues against 10 microbial strains. These nucleoside analogues were classified into four groups according to their structural characteristics. Inhibition was observed with compounds 1-1, 3-1, and 4-3. In particular, 5'-deoxythymidine (3-1) was most effective at 50μg/mL against Bacillus subtilis and Staphylococcus aureus. This analogue has had the hydroxyl group at the 5' position replaced with a hydrogen atom. All compounds had weak effects against various species of mold. The MBC of 5'-deoxythymidine was 50μg/mL in 0.5 h against S. aureus. These results showed that 5'-deoxythymidine had the most effective antimicrobial activity of the 17 different unnatural nucleosides. The inhibitory effect of 3-1 suggests that it may be useful as an antibacterial agent in medical situations.

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The responses of Aspergillus oryzae to the stimuli from near-UV irradiation were investigated by using a black light fluorescent lamp in the presence or absence of TiO₂ particles. Light irradiation at an intensity of 6 W/m² strongly inhibited the growth of germinated pellets of A. oryzae. This growth inhibition was weakened by TiO₂ particles (0.05 g/ι ), especially in the initial growth phase, in which the expression level of catalase gene (catB) was approximately three times higher than that in the absence of TiO₂ particles. However, the initial induction of catB expression by H₂O₂ pretreatment did not restore the growth under the black light irradiation. The weakening of growth inhibition is thought to result from alternative physiological responses of A. oryzae against stimulus by photo-excited TiO₂.

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Forty-five strains of Pseudomonas aeruginosa isolated from cockroaches captured in hospitals were investigated with regard to their biofilm-forming ability and resistance to various disinfectants in various cellular states. The hydrophobicity value varied among the test strains from 0.001 to 0.241, and the mean±standard deviation (SD) was 0.101±0.074. The relative viscosity of the extracellular product was measured. All test strains produced a mucous substance, and the value was 1.05-1.30 (mean±SD: 1.11±0.06), showing that all test strains had a biofilm-forming ability.
Bactericidal experiments were performed using 6 disinfectants: ethanol, Hibitane, Isojin, Osvan, Tego 51, and Welpas. When bacteria in suspension were exposed to the disinfectants for 1 min (suspension test), 5 of the 6 disinfectants excluding Tego 51 completely killed all test strains, showing high bactericidal effects. In contrast, when adherent bacteria were exposed to the disinfectants for 1 min (adhesion test), the killing rate by Welpas was 100 %, but those by Isojin and ethanol were lower (88.9 and 60.0 %, respectively), that by Osvan was only 4.4 %, and no bacteria were killed by Hibitane or Tego 51. These findings showed that the bactericidal effects markedly decreased in the case of adherent P. aeruginosa.

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The antimicrobial activities of fullerene C₆₀ and its derivatives against 6 kinds of bacteria and 2 kinds of fungi were evaluated. The tested samples were water-soluble fullerenes (polyvinylpyrrolidone (PVP)/C₆₀, γ-cyclodextrin (γ-CD)/C₆₀, and nano-C₆₀) and 3 types of fullerenols (C₆₀(OH)₁₂, C₆₀(OH)₃₆·8H₂O, and C₆₀(OH)₄₄·8H₂O). Their activities were compared with those of (+)-catechin and hinokitiol from the viewpoint of future application to cosmetics. Although pristine C₆₀ demonstrated no antimicrobial activity, fullerenols exhibited good antimicrobial activity against Propionibacterium acnes, Staphylococcus epidermidis, Candida albicans, and Malassezia furfur. In particular, C₆₀(OH)₄₄ exhibited a strong and wide-ranging antimicrobial activity comparable to that of catechin. This compound exhibits antimicrobial activity via inhibition of microbial cell growth and not via bactericidal activity.

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Loop-mediated isothermal amplification (LAMP) assay was applied to the detection of Salmonella in food and human materials. It was possible for the assay to detect Salmonella within 60min. All of 54 serovars of Salmonella tested were amplified, but all bacteria tested other than Salmonella were not. The LAMP assay could detect 10² cfu/ml levels of Salmonella. The specificity was similar to that of a PCR assay, but the sensitivity of LAMP was considered to be greater. Thus, the LAMP assay was confirmed to be a rapid, specific and sensitive detection method for Salmonella.

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The sporicidal activity of an improved iodide formulation based on a previously reported agent (Kida et al., 2004, tentatively designated as the KMT reagent) which is composed of 50 mM EDTA-2Na, 50 mM ferric chloride hexahydrate (FeCl₃·6H₂O), 50 mM potassium iodide (KI) and 50% ethanol in 0.85% NaCl solution at pH 0.3 with hydrochloric acid, was examined in the liquid and vapor phases. The improved iodide formulation subject to distillation (tentatively designated as the distilled KMT reagent: pH around 3) showed comparable sporicidal activity with the KMT reagent. As for the dilution effect, dilution at 1:2 showed more potent sporicidal activity than the undiluted one. It achieved complete disinfection with a treatment for 5 min at 20°C and for 60 min at 5°C. Even at a ratio of 1:100, the dilutions showed significant sporicidal activities at 37°C. The experiment on the disinfection of the biological safety cabinet (Class II type A) as a practical possibility showed that pretreatment with 400 ml of water vapor treatment, and a mixture of 300 ml of this reagent and 150 ml of water in vapor phase achieved complete disinfection after a 24 h-decontamination process. The distilled KMT reagent may be useful for disinfecting against various contaminated materials and sites in both the liquid phase and vapor phase.

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Recently we developed a mathematical model for microbial growth in food. The model successfully predicted microbial growth at various patterns of temperature. In this study, we developed a program to fit data to the model with a spread sheet program, Microsoft Excel^®. Users can instantly get curves fitted to the model by inputting growth data and choosing the slope portion of a curve. The program also could estimate growth parameters including the rate constant of growth and the lag period. This program would be a useful tool for analyzing growth data and further predicting microbial growth.

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