No abstract

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (a w ). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low a w for long periods, but minimum humectant concentrations of 8% NaCl (a w , 0.95), 96% sucrose (a w , 0.94), and 32% glycerol (a w , 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal a w , incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C. At a w values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 m long. Filamentation was independent of rpoS expression. When the preparations were returned to high-a w conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low a w highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low a w (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-a w storage. If Salmonella strains form filaments in food products that have low a w values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring.

Salmonella spp. are reported to have an increased heat tolerance at low water activity (a w ; measured by relative vapor pressure [rvp]), achieved either by drying or by incorporating solutes. Much of the published data, however, cover only a narrow treatment range and have been analyzed by assuming first-order death kinetics. In this study, the death of Salmonella enterica serovar Typhimurium DT104 when exposed to 54 combinations of temperature (55 to 80°C) and a w (rvp 0.65 to 0.90, reduced using glucose-fructose) was investigated. The Weibull model (LogS ‫؍‬ ؊bt n ) was used to describe microbial inactivation, and surface response models were developed to predict death rates for serovar Typhimurium at all points within the design surface. The models were evaluated with data generated by using six different Salmonella strains in place of serovar Typhimurium DT104 strain 30, two different solutes in place of glucose-fructose to reduce a w , or six low-a w foods artificially contaminated with Salmonella in place of the sugar broths. The data demonstrate that, at temperatures of >70°C, Salmonella cells at low a w were more heat tolerant than those at a higher a w but below 65°C the reverse was true. The same patterns were generated when sucrose (rvp 0.80 compared with 0.90) or NaCl (0.75 compared with 0.90) was used to reduce a w , but the extent of the protection afforded varied with solute type. The predictions of thermal death rates in the low-a w foods were usually fail-safe, but the few exceptions highlight the importance of validating models with specific foods that may have additional factors affecting survival.

The effect of habituation at reduced water activity (a w ) on heat tolerance of Salmonella spp. was investigated. Stationary-phase cells were exposed to a w 0.95 in broths containing glucose-fructose, sodium chloride, or glycerol at 21°C for up to a week prior to heat challenge at 54°C. In addition, the effects of different a w s and heat challenge temperatures were investigated. Habituation at a w 0.95 resulted in increased heat tolerance at 54°C with all solutes tested. The extent of the increase and the optimal habituation time depended on the solute used. Exposure to broths containing glucose-fructose (a w 0.95) for 12 h resulted in maximal heat tolerance, with more than a fourfold increase in D 54 values. Cells held for more than 72 h in these conditions, however, became as heat sensitive as nonhabituated populations. Habituation in the presence of sodium chloride or glycerol gave rise to less pronounced but still significant increases in heat tolerance at 54°C, and a shorter incubation time was required to maximize tolerance. The increase in heat tolerance following habituation in broths containing glucose-fructose (a w 0.95) was RpoS independent. The presence of chloramphenicol or rifampin during habituation and inactivation did not affect the extent of heat tolerance achieved, suggesting that de novo protein synthesis was probably not necessary. These data highlight the importance of cell prehistory prior to heat inactivation and may have implications for food manufacturers using low-a w ingredients.

Salmonella cells in two sugar-rich media were heat treated at various constant temperatures in the range of 55 to 80 degrees C and their survival ratios determined at various time intervals. The resulting nonlinear semilogarithmic survival curves are described by the model log10S(t) = -b(T)tn(T), where S(t) is the momentary survival ratio N(t)/N0, and b(T) and n(T) are coefficients whose temperature dependence is described by two empirical mathematical models. When the temperature profile, T(t), of a nonisothermal heat treatment can also be expressed algebraically, b(T) and n(T) can be transformed into a function of time, i.e., b[T(t)] and n[T(t)]. If the momentary inactivation rate primarily depends on the momentary temperature and survival ratio, then the survival curve under nonisothermal conditions can be constructed by solving a differential equation, previously suggested by Peleg and Penchina, whose coefficients are expressions that contain the corresponding b[T(t)] and n[T(t)] terms. The applicability of the model and its underlying assumptions was tested with a series of eight experiments in which the Salmonella cells, in the same media, were heated at various rates to selected temperatures in the range of 65 to 80 degres C and then cooled. In all the experiments, there was an agreement between the predicted and observed survival curves. This suggests that, at least in the case of Salmonella in the tested media, survival during nonisothermal inactivation can be estimated without assuming any mortality kinetics.