Thermal inactivation kinetics of Escherichia coil β-galactosidase in phosphate buffer was studied for further understanding of non-linear patterns of the thermal inactivation of microorganisms. Thermal inactivation kinetics of the enzyme at constant temperatures were nonlinear; a semilogarithmic plot of residual activity vs. time was upwardly concave. The rate of inactivation was higher at higher pHs, lower phosphate concentrations, or higher temperatures. These inactivation patterns could be well described with a mathematical model. The model consisted of two sequential inactivation processes, each of which follows first-order kinetics. The effects of the temperatures on the values for the model parameters were then analyzed. The inactivation pattern of the enzyme at linearly increasing temperatures was sigmoid. This pattern was almost the same as that predicted with the model.

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One of the mutant non-producers of validamycin fromStreptomyces hygroscopicus subspl imoneus JCM 4911, designated as strain GT 32, showed the accumulation of panose in the culture broth under the conditions for validamycin production by the original strain. Panose production was not detected in the culture broth of the original strain which was known to produce the antibiotic, validamycin, or in that of the other mutant strains studied. The structure of panose, O- α -D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranose, for the oligosaccharide specifically produced by the strain GT-32, was identified by sugar composition analysis, ion-spray mass spectrometry, methylation analysis, and 500 MHz ¹H-NMR. The maximum production (1.59 mg/ml) of panose by the strain GT32 was found in 5 d cultured broth at 37°C on a rotary shaker at 180 rpm.

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The bactericidal activity of the mixed solutions of sodium 1-octanesulfonate (NaOS) and metal ions (Na⁺, Mg²⁺, Al³⁺, La³⁺) againstEscherichia coilATCC 25922 and the leakage of intracellular materials from the organism treated with the mixtures were studied by a quantitative suspension test and turbidimetry, respectively. When the total concentrations of the systems were kept constant, a maximum in bactericidal activity was found in the viciniy of the molar ratio of 2: 1 for NaOS/divalent cations and 3: 1 for NaOS/trivalent cations. Monovalent cations were ineffective at the concentrations tested. In the case of NaOS/LaCl₃, the absorbance at 260 nm (A₂₆₀) also showed a maximum when plotted against the LaCl₃ mole fraction. This mole fraction at the maximum was almost similar to that of the maximum bactericidal activity. The interaction between NaOS and metal cations for the mixture systems was examined by a measurement of surface tension. The surface tension was found to be the lowest at a molar ratio similar to the results observed in the bactericidal test. This lowering of the surface tension seemed to be caused by a strong interaction between NaOS and polyvalent metal cations. These results suggest that the synergistic bactericidal activity in NaOS/polyva lent metal ion mixtures is closely associated with a hydrophobic complex formed between a I-octanesulfonicanion and a polyvalent cation, and with the disorganization of the cell membrane caused by that formation.

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We developed Mn-supplemented, Fe-supplemented, and Ca-supplemented spores of Bacillus subtilis 168 and ATCC9372 in the respective presence of manganese, ferric and calcium ions, and compared their resistance to vapor-phase hydrogen peroxide with that to aqueous phase hydrogen peroxide and heat. Mn-supplemented spores were the most sensitive to heat and hydrogen peroxide solution but was the most resistant to vapor-phase hydrogen peroxide at low relative humidities as compared with Fe-supplemented and Ca-supplemented spores of the two strains. In the vapor-phase hydrogen peroxide treatment, the spore resistance was affected by the relative humidity during the period for spore preparation and was lowest at a relative humidity of 33%. The removal of the spore coat from the spores of stra in 168 markedly sensitized Fe-supplemented and Ca-supplemented spores, and also slightly sensitized Mnsupplemented spores, to vapor-phase hydrogen peroxide. These results indicate that both divalent cations in spores and the relative humidity affect the spore resistance to hydrogen peroxide vapor and suggest that the spore coat plays a role in this resistance.

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We examined the optimal conditions for germination of the spores of Bacillus coagulans and an acidophile Bacillus spp isolated from PET-bottled drinks on the market containing Japanese tea or orange or apple juice. Among the spores of B. coagulans BUB strains, isolated from Japanese tea, BUB46 germinated effectively at an acidic pH in a mixture of D-glucose plus KNO₃ (G+K), and in a mixture of L-asparagine, D-glucose, D-fructose and KCI (AGFK) at 45 or 53°C, but not by L-alanine. The spores of BUB130 germinated in a mixture containing G+K, AGFK or L-alanine, and BUB44 spores also germinated most effectively in an acidic solution of AGFK. Pretreatment of the BUB44 spores with 1/16N NaOH stimulated germination, but that with 1% sodium dodecyl sulfate plus 5 mM dithiothreitol did not. The spores of an acidophile Bacillus spp. isolated from fruit juice germinated at pH 3.0 in a mixture containing G + K, AGFK, L-alanine or potato dextrose.

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Hen egg white lysozyme was more effective than mutanolysin in killing lactic acid bacteria. The lysozyme-reated cell suspension had relatively high levels of turbidity but a low viability. On the other hand, the mutanolysin-reated cell suspension showed rather low levels of turbidity but a high cell survival number. Observations using a scanning electron microscope also showed differences in the appearances of the cells damaged by the treatment with each enzyme. The modes of action of the lysozyme and mutanolysin against the cell wall of lactic acid bacteria appeared to be quite different from each other though these enzymes are both a kind of peptidoglycan N-acetylmuramoylhydrolase. From these facts, it was found that for the killing of lactic acid bacteria, lysozyme is more effective than mutanolysin and the simultaneous use of both enzymes is much more effective than the use of a single type of enzyme.

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Pseudomonas sp. strain no. A-3 (abbreviated as A-3) was able to degrade chlorhexidine to a chlorhexidine degradation intermediate (abbreviated as CHDI-B). We investigated the formation of CHDI-B, and its antibacterial activities. By the addition of [1-¹⁴C] pyruvate, [1-¹⁴C] lactate or [1-¹⁴C] propionate to the incubation mixture, the incorporation of [1-¹⁴C] pyruvate or [1-¹⁴C] -lactate to chlorhexidine was observed. The formation of CHDI-B by the incorporation of lactate was considered to occur after the latter's oxidation to pyruvate. The antibacterial activities of CHDI-B and other related compounds decreased to 1/5 - 1/10 that of chlorhexidine. It was thought that the modification of chlorhexidine is one of the resistant mechanisms of A-3.

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New quaternary ammonium compounds, N-alkyl-4-allylthiopyridinium bromides (ATP-n) having an allylthio group on the pyridine ring, were synthesized from 4-allylthiopyridine and n-alkyl bromides (alkyl group: decyl, dodecyl, tetradecyl, hexadecyl and octadecyl) under 80 MPa of static pressure. ATP-n exhibited a strong bactericidal activity, compared with N-alkyl-4-butenylpyridinium bromides (BNP-n) and N-alkylpyridinium bromides. The bacteriostatic spectrum of ATP-12 was wide and strong. The effect of the allylthio group on the bacteriostatic activity was as high as that of the butenyl group, while the effect on the bacterioclastic activity was higher.

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In the current International Organization for Standardization (ISO) document 11138 series describing biological indicators (BIs), the use of multiple types of microorganisms in a single carrier was rejected because the resistance of individual microorganisms may change due to interference from other strains. In order to confirm the validity of this speculation, we compared the resistance (D value) of a single strain of microorganism with that of the same strain in existence with another strain in identical carrier material. Both BIs were exposed to ethylene oxide gas (EOG) sterilization and moist heat sterilization. D value of B. subtilis and that of B. stearothermophilus spores as a single and as one of two coexistent strains was determined. No statistically significant difference (F-test, p<0.05) ofD value between single and multiple strains was observed in either sterilization method, indicating there was no experimental resistance interference and the speculation at the ISO meeting was unfounded.

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