Biocontrol Science Vol. 22(2017) No. 2

　The effect of the semi purified bacteriocin BacFL31 at 200 and 400 AU/g on the shelf life of refrigerated raw ground turkey meat was investigated. The microbiological, physicochemical, and sensory properties of the meat samples were examined during refrigerated storage. The findings indicated that BacFL31 treatments were effective (p<0.05) against the proliferation of various spoilage microorganisms and suppressed the growth of Listeria monocytogenes and Salmonella Typhimurium. The pH, % Met-MB, and TBA-RS values of the treated samples were lower (p<0.05) than those of their control samples. The addition of BacFL31 extended the shelf life and enhanced the sensory attributes of the turkey meat samples during refrigerated storage. These results suggest that BacFL31 could be considered a promising candidate for future application as an additive to preserve the raw turkey meat during storage at 4℃.

　A marine raphidophyte Heterosigma akashiwo is a causative agent of harmful microalgal blooms, which often cause the massive mortality of aquacultured finfish. In the present study, the Pacific oyster Crassostrea gigas was reared with H. akashiwo, and effect of the microalga on filter-feeding behavior and microflora of the gastrointestinal tract was investigated. The intake of the raphidophyte cells inhibited the molluscan filter-feeding activities, suggesting the negative physiological effect of the microalgal cell contents. However, the bivalves ingested the H. akashiwo cells to the same extent as the diatom Chaetoceros calcitrans, a non-harmful indicator to estimate the filtration rate, showing a continuation of their non-selective ingestion of the phytoplankton. Microflora of the oyster soft tissue was dominated by bacteria affiliated with the family Rhodobacteraceae, some of which are associated with microalgae. In addition, the Bacteroidetes species, in which algicidal bacteria are included, were also found in the bivalve individuals exposed to H. akashiwo. These results suggested that the ingested phytoplankton affected the microbial flora in the gastrointestinal tracts, some constituents of which helped the mollusc assimilate the ingested red tide phytoplankton. This study will provide beneficial information to clarify mechanisms by which the oyster evades the ichthyotoxicity of harmful microalgae and the participation of the intestinal microorganisms in these processes.

　We compared the TBT-resistant ability of resting cells prepared from isolates that formed colonies on nutrient agar plates containing 100 µM tributyltin (TBT) chloride, such as Photobacterium sp. TKY1, Halomonas sp. TKY2, and Photobacterium sp. NGY1, with those from taxonomically similar type strains. Photobacterium sp. TKY1 showed the highest ability among those three isolates. The number of surviving Photobacterium sp. TKY1 cells was hardly decreased after 1 h of exposure to 100 µM TBTCl, regardless of the number of resting cells in the range from 10^9.4 to 10^4.2 CFU mL^-1. In such an experimental condition, the maximum number of TBT molecules available to associate with a single cell was estimated to be approximately 6.0 x 10^11.8. Resting cells prepared from type strains Photobacterium ganghwense JCM 12487^T and P. halotolerans LMG 22194^T, which have 16S rDNA sequences highly homologous with those of Photobacterium sp. TKY1, showed sensitivity to TBT, indicating that TBT-resistant marine bacterial species are not closely related in spite of their taxonomic similarity. We also estimated the impact of TBT-resistant bacterial species to indigenous microbial populations of TBT-polluted surface sediments. The number of surviving TBT-sensitive Vibrio natriegens ATCC 14048^T cells, 10^6.2±0.3 CFU mL^-1, was reduced to 10^4.4±0.4 CFU mL^-1 when TBT-resistant Photobacterium sp. TKY1 cells, 10^9.1±0.2 CFU mL^-1, coexisted with 10^9.4±0.2 CFU mL^-1 of V. natriegens ATCC 14048^T cells in the presence of 100 µM TBTCl. These results indicate that the toxicity of TBT to TBT-sensitive marine bacterial populations might be enhanced when a TBT-resistant marine bacterial species inhabits TBT-polluted surface sediments.

　Loop-mediated isothermal amplification (LAMP) is a useful DNA detection method with high specificity and sensitivity. The LAMP reaction is carried out within a short time at a constant temperature without the need for thermal cycling. We developed a LAMP primer set for detecting a wide range of fungi by aligning the sequences of the large subunit ribosomal RNA gene of Candida albicans (Ascomycota), Cryptococcus neoformans (Basidiomycota), and Mucor racemosus (Mucorales). The threshold of C. albicans rDNA as template with our LAMP primer set was in the range of 10-100 copies per a reaction. In this study, we evaluated the correlation between colony forming units (CFU) and LAMP detection rate using the LAMP method for environmental fungi. The LAMP method should be a useful means of detecting fungi in indoor environments, disaster areas, or even in confined manned spacecraft to prevent allergies or infections caused by fungi.

　Water treatment plants (WTP) are vital in the food, pharmaceutical and chemical process industries. This investigation describes the dense microbial fouling by microbes and organic compounds in a WTP of a heavy water producing industrial unit. On-site observations showed severe algal and bacterial growth in the various units of the WTP which are open to the atmosphere and very dense fungal fouling in the closed vacuum degasser unit. Digital and microscopic images showed that the microbial fouling problem was primarily due to a fungus. Microbiological analysis showed a count of ~10⁵ cfu mL^-1 in various sections of the WTP. On the contrary, slime/biofilm scrapings had very high bacterial populations (>10⁹ cfu cm^-2). High organic carbon values in the system (5.0 to 19.5 ppm) had supported the growth of the fouling fungus in various sections of the WTP along with bacteria. Chlorination was found to be inadequate in controlling the biofouling problem. Consequently chlorine dioxide was tested and found to be a better biocide in controlling the bacterial population. A 2.0% Sodium-2-pyridinethiol-1-oxide solution had completely inhibited the fouling fungus. The paper discusses the importance of fungal adaptation in an industrial unit and highlights the biodeterioration of various sections of the WTP unit.

　The effect of the amount of the proline transporter PutP expression on the mechanism of adaptation of E. coli cells to high salinity was analyzed. The PutP gene derived from the E. coli expression plasmid was introduced into the E. coli cell, and a high PutP expression strain was developed. At 1.2 M NaCl culture condition, the growth of normal E. coli cells was inhibited, whereas high ProP expression cells showed growth under 2.5 M NaCl conditions. The uptake of proline by E. coli as a compatible solute and substrate for metabolization was in good accordance with those seen in cell growth. These data suggested that the amount of the proline transporter PutP expression played an important role in the adaptation of E. coli cells to high saline conditions.

　The growth and survival of Cronobacter isolates were examined under incubation at different temperatures, and their thermal death behavior was investigated at high temperature conditions of above 50℃. Seventy three strains isolated from fresh vegetables, dried foods and soil were tested: 28 of Cronobacter sakazakii, 5 of C. dublienensis, 27 of C. malonaticus and 13 of C. turicensis. All Cronobacter strains grew and multiplied predominantly at 35 and 44℃ until 16 hours of incubation, but showed poor growth at 15℃, and no growth at 5℃. At 48℃, the bacteria grew slightly during 6 to 8 h-incubation but decreased or were inactivated after 16 h-incubation. The heat resistance of Cronobacter spp. was measured under the conditions of 50, 55, 60, 65 and 70℃. Cronobacter strains survived almost without decrement for 30 min at 50℃, but decreased suddenly and perished completely within 10 to 20 min at 55℃ and within 2 - 5 min at above 60℃. Some food materials should be stored below 5℃ until the preparation, and dried food including powdered infant milk formula should be utilized immediately after reconstitution and preparation.

　A novel double subculture method, termed DiVSaL (Differential Viabilities between Solid and Liquid media) method, for the enumeration of injured cell population of a microorganism, which occurs after some sublethal to lethal treatment, was proposed. In this method injured cells were enumerated as the differential value between viabilities determined with two different techniques, the conventional plate counting using a solid agar medium and the growth delay analysis using a liquid medium. In the former technique, the viable cell number is obtained as colony forming unit (CFU) formed on an agar medium where sublethally injured cells are as much rescued as possible. In the latter technique, on the other hand,“ the integrated viability” defined by Takano and Tsuchido (1982) is introduced and is calculated from the growth delay of a stressed population, referred to unstressed one. For the growth delay analysis, in this paper, not only the original theoretical model, where the specific growth rate (and therefore the defined G₁₀ value) does not change after the exposure to a stress treatment, but also a novel modified theory, where the parameter changes, is proposed. On the theoretical background, this DiVSaL method as a double subculture method can be used to enumerate the injured cells without selection by addition of some inhibitor or by nutritional shortage.