We hypothesized that there would be a difference among a_w conditions in glass transition temperature (T_g) for bacterial cells. Differential scanning calorimetry (DSC) is widely used as a method for measuring T_g [29,38,39]. However, it is difficult to measure T_g of a composite using DSC because the thermogram shows intricate thermal responses . Therefore, here, thermal rheological analysis (TRA) was used to measure T_g. TRA, which measures T_g by attaching a temperature control device to a rheometer, is based on the principle of thermal mechanical analysis [28–30]. Previous studies used by TRA investigated the effect of water content on the T_g of cookies [29, 40], hazelnuts , and deep-fried food . To conduct the measurements, a sample is compressed at a temperature below T_g, and heated above T_g with compression. Then, the T_g of the sample can be determined as a force drop induced by the glass transition. This is a useful method to apply to amorphous powders. By determining T_g values, we could confirm the glass transition of bacterial cells. In addition, we sought to elucidate the influence of a_w on bacterial survival and its relationship with T_g. Finally, we aimed to resolve the relationship between the state change of several Salmonella serotypes that is known to be present in low water activity foods due to glass transition and the changes in thermal resistance in a desiccation environment. The results obtained here will help to understand bacterial survival in a dry environment, which has not been clarified.

Microbial challenges and you can culturing

Salmonella enterica Typhimurium (RMID 1985009 from the Lookup Institute getting Microbial Disorder off Osaka University; separated from customers in sporadic circumstances), S. enterica Chester, S. enterica Oranienburg (regarding Aomori Prefectural Research Lab off Social Health; separated off dried squid chips for the a break out inside the 1999), S. enterica Stanley (RIMD 1981001 about Lookup Institute having Bacterial Problems of Osaka University; isolated out-of patients for the sporadic circumstances), and you will S. enterica Enteritidis (RIMD 1933001 about Research Institute to have Microbial Disorder off Osaka University; remote out-of customers in the sporadic situation) were used in this research.

These serovars were maintained in the -80°C within the tryptic soya broth (TSB, Merck, Darmstadt, Germany) which includes 10% glycerol. New strains was triggered after incubating in the 37°C to possess 24 h on tryptic soy ager (TSA, Merck) plates. A remote colony of every bacterium was then relocated to 5 mL off TSB in an excellent sterile centrifuge pipe, incubated from the 37°C getting 24 h, after which a hundred ?L aliquot of cultured bacteria was extra so you’re able to 400 mL TSB and you may incubated on 37°C for 48 h. This new cultured structure have been gathered from the centrifugation (step three,100000 ? grams, ten min) plus the pellets was resuspended when you look at the 5 mL out-of clear water. Bacterial-cellphone pellets was received by pipetting from the excess liquid and you may obtained toward a plastic dish. The brand new plates was in fact suspended in the -80°C getting 24 h ahead of drying out to have twenty four h using a freeze drier (FDU-2200, EYELA, Tokyo, Japan). Dried bacterial cells were crushed, listed thaicupid in an air-rigorous basket within wanted relative humidity (% RH), which had been introduced playing with soaked salt aqueous selection (43% RH: potassium carbonate, 57% RH: sodium bromide, 75% RH: salt chloride, and you may 87% RH: potassium chloride), and you will kept from the 4°C for forty-eight h. Water hobby and temperature in the air-rigid container was constantly appeared using thermo recorder (TR-72wf, T and D, Nagano, Japan). Together with liquid craft of your bacteria is affirmed by a beneficial liquid interest meter (Aqualab 4TE, Decagon Gizmos, Arizona, USA).

Determination of glass transition heat (Tg)

Thermal rheological analysis (TRA) was used to measure T_g by attaching a temperature control device to a rheometer (EZ-SX, SHIMADZU, Kyoto, Japan) (illustrated in Fig 1); the analysis is based on the principle of thermal mechanical analysis [28–30]. A dried bacterial cell sample (ca. 100 mg) was placed in the forming die (? = 3 mm) and compacted with a rheometer at ca. 10 MPa. Subsequently, the sample was compressed at ca. 5 MPa ca. for 1 to 3 min and then heated at a rate of approximately 3°C/min until the temperature reached 120°C. Pressure-time data were collected with software attached to the rheometer. In parallel, a thermocouple was attached to the bottom of the forming die and time-temperature data were collected every second using a data logger. Since pressure reduction begins at the point at which the bottom temperature of the sample reaches the mechanical T_g, the onset temperature of pressure reduction could be regarded as the T_g of the sample .