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 chathour,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 culturing
Salmonella enterica Typhimurium (RMID 1985009 on the Lookup Institute getting Bacterial Disorder off Osaka University; remote away from patients in sporadic instance), S. enterica Chester, S. enterica Oranienburg (regarding the Aomori Prefectural Research Research out-of Social Wellness; isolated regarding dried squid potato chips associated with a break out in the 1999), S. enterica Stanley (RIMD 1981001 regarding the Browse Institute for Bacterial Infection away from Osaka University; isolated out of customers for the sporadic case), and you may S. enterica Enteritidis (RIMD 1933001 throughout the Search Institute to possess Microbial Infection regarding Osaka University; remote away from people within the sporadic instance) were used in this study.
These types of serovars had been was able in the -80°C in the tryptic soy broth (TSB, Merck, Darmstadt, Germany) that features ten% glycerol. The strains was in fact activated immediately following incubating at the 37°C having 24 h on the tryptic soya ager (TSA, Merck) plates. An isolated colony of every germs ended up being moved to 5 mL regarding TSB into the a sterile centrifuge tubing, incubated within 37°C for 24 h, right after which a 100 ?L aliquot off cultured bacterium is actually extra so you’re able to eight hundred mL TSB and you will incubated in the 37°C getting forty eight h. The latest cultured cells was compiled by the centrifugation (3,one hundred thousand ? grams, ten minute) and also the pellets have been resuspended for the 5 mL out of pure water. Bacterial-telephone pellets was obtained by the pipetting off the way too much drinking water and you may accumulated into the a vinyl plate. The fresh dishes was indeed frozen at the -80°C for 24 h before drying out to possess twenty four h having fun with good frost more dry (FDU-2200, EYELA, Tokyo, Japan). Dried bacterial muscle were surface, listed in an environment-rigid container from the wanted relative moisture (% RH), which was lead playing with saturated sodium aqueous alternatives (43% RH: potassium carbonate, 57% RH: sodium bromide, 75% RH: sodium chloride, and you will 87% RH: potassium chloride), and you may kept in the cuatro°C having forty-eight h. Water activity and you may temperatures floating around-tight basket was consistently seemed using thermo recorder (TR-72wf, T and you may D, Nagano, Japan). And also the liquids passion of bacterium was confirmed of the a great liquids passion meter (Aqualab 4TE, Decagon Gadgets, Arizona, USA).
Determination regarding mug change heat (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 .