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 stresses and you will culturing
Salmonella enterica Typhimurium (RMID 1985009 in the Search Institute to own Bacterial Infection of Osaka University; separated regarding clients into the sporadic instance), S. enterica Chester, S. enterica Oranienburg (on Aomori Prefectural Browse Research of Social Health; separated out of dried squid potato chips of a break out inside the 1999), S. enterica Stanley (RIMD 1981001 regarding Research Institute to own Bacterial Infection from Osaka University; separated off customers when you look at the sporadic instance), and you will S. enterica Enteritidis (RIMD 1933001 about Look Institute having Bacterial Illness from Osaka University; separated regarding clients when you look at the sporadic situation) were chosen for this research.
These types of serovars was maintained on -80°C in the tryptic soya broth (TSB, Merck, Darmstadt, Germany) which has had ten% glycerol. The newest strains have been triggered just after incubating during the 37°C to possess willow twenty four h on tryptic soya ager (TSA, Merck) dishes. A remote colony of each and every germs ended up being transferred to 5 mL out of TSB during the a beneficial sterile centrifuge tube, incubated at 37°C to own twenty four h, right after which a 100 ?L aliquot off cultured micro-organisms is actually extra so you’re able to eight hundred mL TSB and you may incubated in the 37°C to own forty-eight h. The brand new cultured muscle was in fact compiled by the centrifugation (step three,100000 ? g, 10 min) plus the pellets was resuspended into the 5 mL of pure water. Bacterial-phone pellets was received of the pipetting off of the an excessive amount of h2o and built-up towards the a vinyl plate. The brand new dishes was in fact suspended at the -80°C to own twenty four h ahead of drying for twenty-four h using good frost dryer (FDU-2200, EYELA, Tokyo, Japan). Dehydrated microbial tissue were ground, placed in an atmosphere-tight container within desired relative humidity (% RH), which had been produced using over loaded salt aqueous alternatives (43% RH: potassium carbonate, 57% RH: sodium bromide, 75% RH: salt chloride, and 87% RH: potassium chloride), and you will kept within 4°C getting forty eight h. Water interest and you can temperature floating around-tight container had been consistently checked having fun with thermo recorder (TR-72wf, T and you may D, Nagano, Japan). And also the liquids hobby of germs are affirmed by a water activity meter (Aqualab 4TE, Decagon Gadgets, Washington, USA).
Dedication regarding mug changeover temperatures (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 .