We hypothesized that there would be a difference among aw conditions in glass transition temperature (Tg) for bacterial cells. Differential scanning calorimetry (DSC) is widely used as a method for measuring Tg [29,38,39]. However, it is difficult to measure Tg of a composite using DSC because the thermogram shows intricate thermal responses . Therefore, here, thermal rheological analysis (TRA) was used to measure Tg. TRA, which measures Tg 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 Tg of cookies [29, 40], hazelnuts , and deep-fried food . To conduct the measurements, a sample is compressed at a temperature below Tg, and heated above Tg with compression. Then, the Tg 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 Tg values, we could confirm the glass transition of bacterial cells. In addition, we sought to elucidate the influence of aw on bacterial survival and its relationship with Tg. 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.
Bacterial strains and culturing
Salmonella enterica Typhimurium (RMID 1985009 on the Look Institute getting Microbial Diseases out-of Osaka University; separated from clients in sporadic instance), S. enterica Chester, S. enterica Oranienburg (on the Aomori Prefectural Search Research from Public Health; remote off dehydrated squid chips with the an outbreak for the 1999), S. enterica Stanley (RIMD 1981001 regarding the Research Institute to have Microbial Infection out of Osaka University; separated out-of people inside sporadic instance), and S. enterica Enteritidis (RIMD 1933001 from the Search przeglÄ…d bristlr Institute to possess Microbial Ailment off Osaka University; remote from customers within the sporadic situation) were used in this study.
This type of serovars was indeed was able at -80°C from inside the tryptic soya broth (TSB, Merck, Darmstadt, Germany) which includes ten% glycerol. The fresh stresses were triggered once incubating on 37°C to own twenty four h towards tryptic soya ager (TSA, Merck) dishes. An isolated colony of every micro-organisms was then relocated to 5 mL away from TSB during the a beneficial sterile centrifuge pipe, incubated at 37°C for twenty-four h, right after which a hundred ?L aliquot of cultured bacteria are added to help you 400 mL TSB and you can incubated from the 37°C getting 48 h. The newest cultured structure had been compiled because of the centrifugation (step 3,100 ? grams, 10 minute) therefore the pellets was basically resuspended when you look at the 5 mL off uncontaminated water. Bacterial-cellphone pellets were obtained from the pipetting from the too-much h2o and amassed towards a plastic material plate. The newest plates had been suspended at the -80°C for twenty four h prior to drying to possess twenty-four h using good freeze more dry (FDU-2200, EYELA, Tokyo, Japan). Dried bacterial tissue was in fact crushed, listed in an atmosphere-strict basket within need cousin dampness (% RH), which was put playing with saturated sodium aqueous choice (43% RH: potassium carbonate, 57% RH: salt bromide, 75% RH: salt chloride, and 87% RH: potassium chloride), and stored during the 4°C to possess forty-eight h. The water activity and you will heat in the air-strict basket was basically continuously appeared playing with thermo recorder (TR-72wf, T and you may D, Nagano, Japan). And the drinking water passion of one’s micro-organisms is actually confirmed because of the a good liquid craft meter (Aqualab 4TE, Decagon Equipment, Washington, USA).
Commitment out of mug change temperature (Tg)
Thermal rheological analysis (TRA) was used to measure Tg 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 Tg, the onset temperature of pressure reduction could be regarded as the Tg of the sample .