Kinetics of Bacillus cereus spore inactivation in cooked rice by combined pressure-heat treatment.

The efficacy of pressure-heat treatment was evaluated for the inactivation of Bacillus cereus spores in cooked rice. The spores of B. cereus ATCC 9818 were inoculated (1.1 × 10(8) CFU/g) in a parboiled rice product (pH 6.0, water activity of 0.95) and inactivated to an undetectable level (<10 CFU/g) by treatment of 600 MPa and process temperatures of 60 to 85 °C or 0.1 MPa and 85 °C. Kinetic inactivation parameters were estimated with linear and nonlinear models. The potential recovery of injured bacteria was also evaluated during storage of the treated product for 4 weeks at 4 and 25 °C. Depending on the process temperature, a 600-MPa treatment inactivated spores by 2.2 to 3.4 log during the 30-s pressure come-up time, and to below the detection limit after 4- to 8-min pressure-holding times. In contrast, a 180-min treatment time was required to inactivate the spores to an undetectable level at 0.1 MPa and 85 °C. The decimal reduction time of spores inactivated by combined pressure-heat treatment ranged from 1.08 to 2.36 min, while it was 34.6 min at 85 °C under atmospheric conditions. The nonlinear Weibull model scale factor increased, and was inversely related to the decimal reduction time, and the shape factor decreased with increasing pressure or temperature. The recovery of injured spores was influenced by the extent of pressure-holding time and process temperature. This study suggests that combined pressure-heat treatment could be used as a viable alternative to inactivate B. cereus spores in cooked rice and extend the shelf life of the product.

Modeling time to inactivation of Listeria monocytogenes in response to high pressure, sodium chloride, and sodium lactate.

A mathematical model was developed to predict time to inactivation (TTI) by high pressure processing of Listeria monocytogenes in a broth system (pH 6.3) as a function of pressure (450 to 700 MPa), inoculum level (2 to 6 log CFU/ml), sodium chloride (1 or 2%), and sodium lactate (0 or 2.5%) from a 4°C initial temperature. Ten L. monocytogenes isolates from various sources, including processed meats, were evaluated for pressure resistance. The five most resistant strains were used as a cocktail to determine TTI and for model validation. Complete inactivation of L. monocytogenes in all treatments was demonstrated with an enrichment method. The TTI increased with increasing inoculum level and decreasing pressure magnitude, from 1.5 min at 700 MPa and 2 log CFU/ml, to 15 min at 450 MPa and 6 log CFU/ml. Neither NaCl nor sodium lactate significantly influenced TTI. The model was validated with ready-to-eat, uncured, Australian retail poultry products, and with product specially made at a U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS)-inspected pilot plant in the United States. Data from the 210 individual product samples used for validation indicate that the model gives "fail-safe" predictions (58% with response as expected, 39% with no survivors where survivors expected, and only 3% with survivors where none were expected). This model can help manufacturers of refrigerated ready-to-eat meats establish effective processing criteria for the use of high pressure processing as a postlethality treatment for L. monocytogenes in accordance with FSIS regulations.

Solute-specific effects of osmotic stress on Staphylococcus aureus.

AIM: To determine if cell death from osmotic stress is because of lack of sufficient energy to maintain cell metabolism. Additionally, the solute-specific effect of five humectants on bacterial osmoregulation and cell survival was examined. METHODS AND RESULTS: Staphylococcus aureus was placed into 84% relative humidity (RH) broth (five humectants used individually). ATP, ADP and cell viability measurements were determined over time. The results indicate that ATP is not the limiting factor for cell survival under excessive osmotic stress. Although the same RH was achieved with various humectants, the rates of cell death varied greatly as did the sensitivities of the cell populations to osmotic stress. CONCLUSIONS: The results from this study provide strong evidence that mechanisms of osmotic inactivation depend on the solute. The molecular mobility of the system may be an important means to explain these differences. SIGNIFICANCE AND IMPACT OF THE STUDY: By bringing together an understanding of solute-specific effects, microbial physiology and genetics, the mechanisms of inactivation of micro-organisms by solute-specific osmotic stress may be elucidated, and this knowledge may then be exploited to ensure the production of high quality, safe foods.

Modeling the growth boundary of Listeria monocytogenes in ready-to-eat cooked meat products as a function of the product salt, moisture, potassium lactate, and sodium diacetate concentrations.

A central composite response surface design was used to determine the time to growth of Listeria monocytogenes as a function of four continuous variables: added sodium chloride (0.8 to 3.6%), sodium diacetate (0 to 0.2%), potassium lactate syrup (60% [wt/wt]; 0.25 to 9.25%), and finished-product moisture (45.5 to 83.5%) in ready-to-eat cured meat products. The design was repeated for ready-to-eat uncured meat products giving a fifth categorical variable for cure status. Products were stored at 4 degrees C. The results were modeled using a generalized regression approach. All five main effects, six two-factor interactions, and two quadratic terms were statistically significant. The model was used to show the boundary between growth and no-growth conditions at 4 degrees C using contour plots of time to growth. It was validated using independent challenge studies of cured and uncured products. Generally, the model predicted well, particularly for cured products, where it will be useful for establishing conditions that prevent the growth of L. monocytogenes. For uncured products, there was good agreement overall between predicted and observed times to growth, but the model is less thoroughly validated than for cured products. The model should initially only be used for screening of formulations likely to prevent growth of Listeria monocytogenes in uncured products, with recommendations subject to confirmation by challenge studies.

Staphylococcus aureus growth boundaries: moving towards mechanistic predictive models based on solute-specific effects.

The formulation of shelf-stable intermediate-moisture products is a critical food safety issue. Therefore, knowing the precise boundary for the growth-no-growth interface of Staphylococcus aureus is necessary for food safety risk assessment. This study was designed to examine the effects of various humectants and to produce growth boundary models as tools for risk assessment. The molecular mobility and the effects of various physical properties of humectants, such as their glass transition temperatures, their membrane permeability, and their ionic and nonionic properties, on S. aureus growth were investigated. The effects of relative humidity (RH; 84 to 95%, adjusted by sucrose plus fructose, glycerol, or NaCl), initial pH (4.5 to 7.0, adjusted by HCl), and potassium sorbate concentration (0 or 1,000 ppm) on the growth of S. aureus were determined. Growth was monitored by turbidity over a 24-week period. Toxin production was determined by enterotoxin assay. The 1,792 data points generated were analyzed by LIFEREG procedures (SAS Institute, Inc., Cary, N.C.), which showed that all parameters studied significantly affected the growth responses of S. aureus. Differences were observed in the growth-no-growth boundary when different humectants were used to achieve the desired RH values in both the absence and the presence of potassium sorbate. Sucrose plus fructose was most inhibitory at neutral pH values, while NaCl was most inhibitory at low pH values. The addition of potassium sorbate greatly increased the no-growth regions, particularly when pH was <6.0. Published kinetic growth and survival models were compared with boundary models developed in this work. The effects of solutes and differences in modeling approaches are discussed.

An immunohistochemical study of osteoprotegerin in the human dental pulp.

This study investigated the expression of osteoprotegerin (OPG) in healthy and inflamed dental pulps. Histological sections 7 microm thick of 47 teeth, either caries-free or affected by gross caries, were used. Sections were stained with hematoxylin-eosin, and other sections of the same specimen were subjected to the avidin-biotin peroxidase complex immunohistochemical procedure for detection of OPG. The study focused on the coronal pulp that was divided into peripheral and central regions. In the peripheral pulp healthy and inflamed specimens showed high OPG immunoreactivity of the odontoblastic layer. When no inflammation was present in the central pulp OPG immunoreactivity was light. Fibroblasts and endothelial cells showed immunoreactivity ranging from none to intense. When inflammation was present in the central pulp the chronic inflammatory cells showed intense immunoreactivity.

Improving recovery of Salmonella enterica serovar typhimurium DT104 cells injured by heating at different water activity values.

This study describes the evaluation of potentially more sensitive methods for the recovery of Salmonella cells injured by heating (54 to 60 degrees C) at different water activity values (0.65 to 0.90, reduced using equal portions of glucose and fructose). These methods included gradual rehydration, the use of diluting media with added solutes or blood, the addition of blood to plating agar, and the use of different incubation temperatures and times. Gradual rehydration of cells that had been challenged at low water activity (0.65 and 0.70) and high temperature markedly improved recovery, measured as a >50% increase in the time to obtain a 3-log10 reduction in cell numbers, compared to dilution into media with a high water activity. Adding sucrose, glycerol, or blood to the diluting media (maximal recovery diluent) did not improve recovery, but a plating agar containing blood recovered approximately 38% more cells than nutrient agar. Prolonged incubation of agar plates allowed recovery of injured Salmonella cells that presumably had extended lag periods, with significantly higher recovery rates after 48 h incubation at 37 degrees C than after 24 h (P = 0.05). This work highlights that by recovering Salmonella using a method specific to the nature of the injury, a better prediction of food safety and the success of food processing can be made.

Effect of challenge temperature and solute type on heat tolerance of Salmonella serovars at low water activity.

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 degrees 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 > or =70 degrees C, Salmonella cells at low a(w) were more heat tolerant than those at a higher a(w) but below 65 degrees 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.

Calculating Salmonella inactivation in nonisothermal heat treatments from isothermal nonlinear survival curves.

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.

Modeling the growth boundary of Staphylococcus aureus for risk assessment purposes.

Knowing the precise boundary for growth of Staphylococcus aureus is critical for food safety risk assessment, especially in the formulation of safe, shelf-stable foods with intermediate relative humidity (RH) values. To date, most studies and resulting models have led to the presumption that S. aureus is osmotolerant. However, most studies and resulting models have focused on growth kinetics using NaCl as the humectant. In this study, glycerol was used to investigate the effects of a glass-forming nonionic humectant to avoid specific metabolic aspects of membrane ion transport. The experiments were designed to produce a growth boundary model as a tool for risk assessment. The statistical effects and interactions of RH (84 to 95% adjusted by glycerol), initial pH (4.5 to 7.0 adjusted by HC1), and potassium sorbate (0, 500, or 1,000 ppm) or calcium propionate (0, 500, or 1,000 ppm) on the aerobic growth of a five-strain S. aureus cocktail in brain heart infusion broth were explored. Inoculated broths were distributed into microtiter plates and incubated at 37 degrees C over appropriate saturated salt slurries to maintain RH. Growth was monitored by turbidity during a 24-week period. Toxin production was explored by enterotoxin assay. The 1,280 generated data points were analyzed by SAS LIFEREG procedures, which showed all studied parameters significantly affected the growth responses of S. aureus with interactions between RH and pH. The resulting growth/no growth boundary is presented.

Habituation of Salmonella spp. at reduced water activity and its effect on heat tolerance.

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 degrees C for up to a week prior to heat challenge at 54 degrees 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 degrees 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 degrees 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.

Tissue reactions after subcutaneous and intraosseous implantation of mineral trioxide aggregate and ethoxybenzoic acid cement.

Biocompatibility of mineral trioxide aggregate and ethoxybenzoic acid cement was investigated by subcutaneous and intraosseous implantation of the materials in rats. Tissue reactions were studied at 15, 30, and 60 days after implantation. Subcutaneous implantation of mineral trioxide aggregate initially elicited severe reactions with coagulation necrosis and dystrophic calcification; the reactions, however, subsided to mostly moderate with time. Subcutaneous implantation of ethoxybenzoic acid cement initially elicited mostly moderate reactions that subsided to mild in time. Osteogenesis was not observed with either material upon subcutaneous implantation indicating that neither material is osteoinductive. Reactions to intraosseous implants of both materials were less intense than with subcutaneous implantation. Osteogenesis occurred in association with intraosseous implants indicating that both materials are osteoconductive.

The application of the Landstuhl frame for air evacuation of patients with femur fractures.

The use of traction to transport patients with femur fractures is well accepted. This paper describes step-by-step the construction of a traction device suitable for use on military aircraft. This "Landstuhl frame" is easily constructed using materials readily available. It is quick and effective for the transportation of patients with lower extremity fractures.

Survival and filamentation of Salmonella enterica serovar enteritidis PT4 and Salmonella enterica serovar typhimurium DT104 at low water activity.

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 degrees C resulted in more rapid loss of viability than incubation at 21 degrees 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 microm 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.

The boundary for growth of Zygosaccharomyces bailii in acidified products described by models for time to growth and probability of growth.

Models to predict days to growth and probability of growth of Zygosaccharomyces bailii in high-acid foods were developed, and the equations are presented here. The models were constructed from measurements of growth of Z. bailii using automated turbidimetry over a 29-day period at various pH, NaCl, fructose, and acetic acid levels. Statistical analyses were carried out using Statistical Analysis Systems LIFEREG procedures, and the data were fitted to log-logistic models. Model 1 predicts days to growth based on two factors, combined molar concentration of salt plus sugar and undissociated acetic acid. This model allows a growth/no-growth boundary to be visualized. The boundary is comparable with that established by G. Tuynenburg Muys (Process Biochem. 6:25-28, 1971), which still forms the basis of industry assumptions about the stability of acidic foods. Model 2 predicts days to growth based on the four independent factors of salt, sugar, acetic acid, and pH levels and is, therefore, much more useful for product development. Validation data derived from challenge studies in retail products from the U.S. market are presented for Model 2, showing that the model gives reliable, fail-safe predictions and is suitable for use in predicting growth responses of Z. bailii in high-acid foods. Model 3 predicts probability of growth of Z. bailii in 29 days. This model is most useful for spoilage risk assessment. All three models showed good agreement between predictions and observed values for the underlying data.

Biocompatibility of two apatite cements.

Biocompatibility and osteogenic potential of two calcium phosphate cements (G-5 and G-6) and Super-EBA were investigated by subcutaneous and intraosseous implantation in 90 rats. Reactions were studied microscopically at 15, 30, and 60 days after implantation. Super-EBA was well tolerated by both soft and hard tissues. G-5 was highly biocompatible with resorption and bone replacement at intraosseous implantation sites. G-6 promoted moderate inflammation and a foreign body giant cell response over the 60-day study period. None of the materials elicited osteogenesis or dystrophic calcification at the subcutaneous implantation sites.

Instrumentation enhances today's endodontic care.

The field of endodontics has virtually exploded in the last several years with technological advances and improvements in many areas. The largest change has occurred with the introduction of nickel-titanium rotary instrumentation, warm gutta-percha obturation, microscopes, and digital imaging. Prominent new devices and instruments are presented with a brief overview of items listed with a source.

In vitro evaluation of effects of ultrasonic root-end preparation on resected root surfaces.

This in vitro study investigated structural alterations in resected roots that had root-end preparations made with a conventional microhead handpiece and ultrasonics at two intensity levels. Root ends were examined with fluorescence confocal microscopy. Serial histologic sections to the 2 mm levels and then at the level of 3 mm and 4 mm from the resected surface were examined. Statistical analysis of the confocal data indicated no significant difference between the groups in the number and length of the fractures. Results of the histologic data indicated that root ends prepared by ultrasonics had a statistically greater number of fractures than both the control and the conventionally prepared groups. The latter did not differ significantly from each other.

Interpretation of chemically created lesions using direct digital imaging.

This study compared conventional radiography to digital imaging in detecting chemically created lesions. Six human cadaver jaw specimens were used. A 70% perchloric acid solution was used to create lesions on the buccal cortical plate of each specimen. Digital imaging and conventional images were created after progressing time increments; each increment represented a more advanced lesion. The images were randomly evaluated by five evaluators. This study concluded: (i) when no lesion existed, there was no significant difference in digital imaging or conventional radiography in early detection; (ii) at 12 and 24 h, digital imaging demonstrated lesions significantly earlier than conventional radiography (p = 0.0001); (iii) no difference could be found between imaging techniques at 36 h and thereafter; and (iv) there were no significant differences in the various RadioVisioGraphy enhancement settings used at any of the time points examined.

An unusual attempt at surgical repair.

Gold foil was used in an attempt to repair a periapical surgical site in the lower anterior region. This method of repair was used in the past for surgical closure of persistent oroantral fistulas with some success. In this case, it met with failure.