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.
Salmonella enterica Typhimurium (RMID 1985009 about Look Institute for Microbial Disease from Osaka University; isolated out of people within the sporadic instance), S. enterica Chester, S. enterica Oranienburg (from the Aomori Prefectural Search Lab off Personal Fitness; isolated from dehydrated squid chips associated with a break out inside 1999), S. enterica Stanley (RIMD 1981001 regarding the Browse Institute having Bacterial Sickness from Osaka University; remote away from people within the sporadic instance), and you will S. enterica Enteritidis (RIMD 1933001 on Look Institute for Bacterial Ailment out-of Osaka University; separated of people inside the sporadic situation) were randki alt chosen for this study.
Such serovars were was able at -80°C in tryptic soy broth (TSB, Merck, Darmstadt, Germany) with 10% glycerol. Brand new stresses was activated after incubating during the 37°C to have 24 h towards the tryptic soy ager (TSA, Merck) plates. An isolated colony of each and every bacteria ended up being relocated to 5 mL from TSB inside the a beneficial sterile centrifuge tubing, incubated at the 37°C for twenty four h, and then a hundred ?L aliquot of cultured bacteria was extra to help you 400 mL TSB and incubated within 37°C having forty-eight h. The cultured structure have been gathered from the centrifugation (step 3,100 ? g, 10 minute) together with pellets was basically resuspended in the 5 mL out-of clear water. Bacterial-cellphone pellets was indeed gotten because of the pipetting off of the too-much water and you will accumulated to the a synthetic dish. The newest plates had been suspended from the -80°C to own twenty-four h prior to drying having twenty-four h having fun with an excellent frost drier (FDU-2200, EYELA, Tokyo, Japan). Dehydrated bacterial structure was in fact ground, placed in an air-strict basket during the wished relative moisture (% RH), that has been delivered having fun with saturated sodium aqueous alternatives (43% RH: potassium carbonate, 57% RH: salt bromide, 75% RH: sodium chloride, and you may 87% RH: potassium chloride), and you can stored at the cuatro°C having forty-eight h. Water hobby and heat in the air-tight container were constantly seemed using temperature recorder (TR-72wf, T and you can D, Nagano, Japan). And liquids pastime of your own germs is verified by an excellent liquid craft meter (Aqualab 4TE, Decagon Equipment, Arizona, USA).
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 .