The study aimed to investigate new wide-spectrum biological control agents against soil-borne fungal plant pathogens. Screening of 336 fungal strains and 256 bacterial strains was carried out in vitro to select the most promising isolates for controlling destructive pathogens of greenhouse-vegetables. In dual cultures, Bacillus subtilis JF419701 and Trichoderma harzianum JF419706 (teleomorph: Hypocrea lixii) inhibited the growth of Alternaria alternata, Fusarium oxysporum, Exserohilum rostratum, Macrophomina phaseolina, Pythium ultimum and Rhizoctonia solani. Microscopic examination showed the ability of T. harzianum JF419706 to parasitize the hyphae of all pathogens and kill them. T. harzianum produced the cell wall degrading enzymes; α-1-3-glucanase (0.83 U/ml) , β-1-3-glucanase (0.89 U/ml) and chitinase (0.86 U/ml) in high concentrations. However, B. subtilis produced proteases in very high concentrations (9341.64 U/ml) . The culture filtrate of T. harzianum did not show any antifungal effect. The cell free extract of B. subtilis, containing cyano-compounds, suppressed the growth of all phytopathogens, especially M. phaseolina. Results proved the efficacy of the two biological control agents to control the common soil pathogens either singly or in combination. We recommend further field experiments to study either the synergistic or antagonistic interactions between them under natural conditions.

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To determine the effects of sporulation temperature and period on Bacillus licheniformis spore heat resistance, B. licheniformis strain No.25 spores were sporulated at 30, 37, 42, or 50°C for 11 d and at 50°C for 1.7, 4, 7, or 11 d. The heat resistance of B. licheniformis strain No.25 spores at 110°C increased with an increase in the sporulation temperature. Spores sporulated at 50°C were 1.4-fold more heat resistant than those sporulated at 30°C. Furthermore, the heat resistance of B. licheniformis strain No.25 spores at 110°C increased with an increase in the sporulation period. Spores sporulated for 11 d were 5.3-fold more heat resistant than those sporulated for 1.7 d. The heat resistance of B. licheniformis strain No.25 spores at 110°C increased with increases in the sporulation temperature and sporulation period. The results presented in this study can be applied to the pasteurization process to control B. licheniformis spores. Pasteurization at 110°C for about 60sec. is effective in controlling B. licheniformis spores isolated from dairy materials in yogurt production.

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DDAC is an effective disinfectant used in the medical and food industries and the environmental field. However, skin irritation in humans occurs at high DDAC concentrations. In this study, we analyzed the combined effect of a low concentration (0.3 ppm) of DDAC and low concentrations (6, 8, and 10 ppm) of 37 products of nonionic surfactants on the bactericidal activity against S. aureus. No bactericidal activity was found at 0.3 ppm DDAC alone. Results showed that a combination of a low concentration of DDAC (0.3 ppm) and some nonionic surfactants tested (synergistic effect of five products ≥ 2.0) improved the bactericidal activity of DDAC. Synergistic effects of DDAC and some nonionic surfactants are desirable and were suggested to occur as follows. Test surfactants acted against the cell walls of S. aureus, which allowed DDAC to act easily on the lipid double membrane in the cell wall, thereby increasing the bactericidal activity of DDAC. In the present study, synergistic effects of a low concentration of DDAC and some nonionic surfactants were observed, a phenomenon that may be considerable value in future developments.

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Growth characteristics of a four-strain cocktail of Salmonella Enteritidis in commercial products of pasteurized and unpasteurized liquid whole egg were studied with a growth model developed by us. The unpasteurized product contained total bacteria at 10^7.3 CFU/g, but no Salmonella. When the products were spiked with Salmonella at various doses ranging from 10¹ to 10⁴ CFU/g, growth curves of the pathogen at 24°C were well described with our model. Salmonella growth curves at constant temperatures from 8°C to 36°C in the two products were also well described with the model. The Baranyi model also described well most of the growth curves. The rate constants of growth for Salmonella at various constant temperatures were well described with the square root model. The maximum cell level, N_max of Salmonella was constant at all temperatures in the pasteurized products, while a linear relationship between log N_max and the temperature was observed in the unpasteurized ones. Salmonella growth in the unpasteurized product was highly suppressed in comparison with that in the pasteurized. This study also suggested the suitability of our model for application in the Salmonella growth analysis in pasteurized and unpasteurized liquid egg products.

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A quantitative and chemical assay of cereulide produced in the cultures by some strains of Bacillus cereus was performed on a HPLC and a ESI electrospray ion trap mass analyzer, using the synthetic cereulide as a standard. All 20 strains of emetic B. cereus were found to produce 27 - 740 ng/ml of cereulide by the LC-MS analysis. In contrast, none of the 10 diarrheal strains produced it. 10² cfu/ml of the cereulide producible strain with a 210 ng/ml yield was inoculated into the 10% suspensions of 14 food products, and was incubated at 32°C for 24h. The B. cereus counts in the cultures grew in the order of 10⁸ to 10⁹ cfu/ml, although the bacteria could not grow in fruits, and the yields of cereulide ranged from 5.18μg in curry to 0.03μg/g of raw material and/or powder material, except for fruits. These culture supernatants were also tested for the biological activity in the HEp-2 cell culture assay. Consequently, a certain correlation was shown between the yields of cereulide and the HEp-2 vacuolation activities. In addition, the supernatants were administered i.p. to 5 Suncus marinus test animals. The emetic dose was calculated to be approximately 16μg/kg.

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