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.
Microbial strains and you can culturing
Salmonella enterica Typhimurium (RMID 1985009 from the Research Institute to own Microbial Disease regarding Osaka University; separated out-of patients inside the sporadic instance), S. enterica Chester, S. enterica Oranienburg (throughout the Aomori Prefectural Search Lab off Social Fitness; remote out of dehydrated squid potato chips associated with a break out inside 1999), S. enterica Stanley (RIMD 1981001 regarding the Browse Institute getting Microbial Problems regarding Osaka University; isolated out-of clients when you look at the sporadic instance), and you will S. enterica Enteritidis (RIMD 1933001 regarding the Lookup Institute getting Bacterial Illness of Osaka University; remote away from patients from inside the sporadic circumstances) were used in this study.
This type of serovars was indeed handled at -80°C in the tryptic soy broth (TSB, Merck, Darmstadt, Germany) which has had 10% glycerol. The newest stresses were triggered shortly after incubating on 37°C for twenty four h towards tryptic soy ager (TSA, Merck) plates. An isolated colony of any bacterium ended up being relocated to 5 mL regarding TSB inside the good sterile centrifuge tubing, incubated in the 37°C to own twenty-four h, immediately after which a 100 ?L aliquot out of cultured micro-organisms is actually added in order to 400 mL TSB and you may incubated during the 37°C to have forty eight h. This new cultured cells were compiled by centrifugation (step 3,000 ? grams, 10 min) together with pellets were resuspended during the 5 mL off uncontaminated water. Bacterial-phone pellets had been obtained from the pipetting off the way too much h2o and you can accumulated towards the a plastic dish. The newest plates have been frozen at -80°C to own twenty four h before drying out to own 24 h playing with an excellent freeze more dry (FDU-2200, EYELA, Tokyo, Japan). Dried microbial tissues was in fact soil, placed in an atmosphere-rigid container during the wanted cousin dampness (% RH), that was brought using soaked salt https://datingranking.net/pl/amateurmatch-recenzja/ aqueous choices (43% RH: potassium carbonate, 57% RH: salt bromide, 75% RH: salt chloride, and you can 87% RH: potassium chloride), and kept at cuatro°C for forty eight h. Water activity and you can temperature floating around-tight basket was basically consistently looked having fun with thermo recorder (TR-72wf, T and you may D, Nagano, Japan). Additionally the liquids craft of your own germs try confirmed because of the good liquids interest meter (Aqualab 4TE, Decagon Devices, Arizona, USA).
Commitment out of glass 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 .