We hypothesized that there would be a difference among aw conditions in glass transition temperature woosa (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.
Microbial challenges and you can culturing
Salmonella enterica Typhimurium (RMID 1985009 throughout the Search Institute for Bacterial Problems out-of Osaka University; separated away from customers during the sporadic case), S. enterica Chester, S. enterica Oranienburg (on the Aomori Prefectural Lookup Laboratory of Societal Health; separated off dehydrated squid potato chips for the a break out for the 1999), S. enterica Stanley (RIMD 1981001 in the Lookup Institute having Bacterial Ailment off Osaka University; remote from patients within the sporadic instance), and you will S. enterica Enteritidis (RIMD 1933001 on Browse Institute getting Bacterial Ailment of Osaka University; remote from patients inside the sporadic instance) were chosen for this study.
This type of serovars was was able in the -80°C into the tryptic soy broth (TSB, Merck, Darmstadt, Germany) with which has ten% glycerol. New stresses have been activated immediately following incubating within 37°C for 24 h on tryptic soya ager (TSA, Merck) plates. A remote colony of any bacterium ended up being transferred to 5 mL from TSB within the a great sterile centrifuge tubing, incubated from the 37°C to have twenty four h, then an one hundred ?L aliquot off cultured bacterium is added to help you 400 mL TSB and you may incubated in the 37°C to have 48 h. The fresh cultured structure was basically amassed by centrifugation (3,100 ? g, ten min) and also the pellets was resuspended when you look at the 5 mL from uncontaminated water. Bacterial-cellphone pellets was gotten from the pipetting off the excessive drinking water and you can amassed for the a synthetic plate. The plates have been suspended within -80°C to own 24 h before drying out to own 24 h having fun with a beneficial freeze more dry (FDU-2200, EYELA, Tokyo, Japan). Dehydrated microbial tissue was crushed, placed in an atmosphere-tight basket in the wished relative dampness (% RH), which had been lead using soaked sodium aqueous possibilities (43% RH: potassium carbonate, 57% RH: sodium bromide, 75% RH: sodium chloride, and you may 87% RH: potassium chloride), and kept at cuatro°C to have 48 h. The water craft and temperatures in the air-rigorous container were continuously featured having fun with temperature recorder (TR-72wf, T and you will D, Nagano, Japan). While the water activity of your micro-organisms was verified of the good liquid craft meter (Aqualab 4TE, Decagon Gizmos, Arizona, USA).
Determination from cup 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 .