Impact of sporulation temperature on germination of Bacillus subtilis spores under optimal and adverse environmental conditions.

Bacillus subtilis spores are important food spoilage agents and are occasionally involved in food poisoning. In foods that are not processed with intense heat, such bacterial spores are controlled by a combination of different hurdles, such as refrigeration, acidification, and low water activity (aw), which inhibit or delay germination and/or growth. Sporulation temperature has long been regarded as a relevant factor for the assessment of germination in chemically defined media, but little is known about its impact on food preservation environments. In this study, we compared germination dynamics of B. subtilis spores produced at optimal temperature (37 °C) with others incubated at suboptimal (20 °C) and supraoptimal (43 °C) temperatures in a variety of nutrients (rich-growth medium, L-alanine, L-valine, and AGFK) under optimal conditions as well as under food-related stresses (low aw, pH, and temperature). Spores produced at 20 °C had a lower germination rate and efficiency than those incubated at 37 °C in all the nutrients, while those sporulated at 43 °C displayed a higher germination rate and/or efficiency in response to rich-growth medium and mostly to L-alanine and AGFK under optimal environmental conditions. However, differences in germination induced by changes in sporulation temperature decreased when spores were activated by heat, mainly due to the greater benefit of heat for spores produced at 20 °C and 37 °C than at 43 °C, especially in AGFK. Non-heat-activated spores produced at 43 °C still displayed superior germination fitness under certain stresses that had considerably impaired the germination of the other two populations, such as reduced temperature and aw. Moreover, they presented lower temperature and pH boundaries for the inhibition of germination in rich-growth medium, while requiring a higher NaCl concentration threshold compared to spores obtained at optimal and suboptimal temperature. Sporulation temperature is therefore a relevant source of variability in spore germination that should be taken into account for the accurate prediction of spore behaviour under variable food preservation conditions with the aim of improving food safety and stability.

Comparative study on the impact of equally stressful environmental sporulation conditions on thermal inactivation kinetics of B. subtilis spores.

Control of bacterial spores continues to be one of the main challenges for the food industry due to their wide dissemination and extremely high resistance to processing methods. Furthermore, the large variability in heat resistance in spores that contaminate foods makes it difficult to establish general processing conditions. Such heterogeneity not only derives from inherent differences among species and strains, but also from differences in sporulation environments that are generally ignored in spores encountered in foods. We evaluated heat inactivation kinetics and the thermodependency of resistance parameters in B. subtilis 168 spores sporulated at adverse temperatures, water activity (aw), and pH, applying an experimental approach that allowed us to quantitatively compare the impact of each condition. Reduction of incubation temperature from the optimal temperature dramatically reduced thermal resistance, and it was the most influential factor, especially at the highest treatment temperatures. These spores were also more sensitive to chemicals presumably acting in the inner membrane. Reducing sporulation aw increased heat resistance, although the magnitude of that effect depended on the solute and the treatment temperature. Thus, changes in sporulation environments varied 3D100°C values up to 10.4-fold and z values up to 1.7-fold, highlighting the relevance of taking such a source of variability into account when setting heat processing conditions. UV-C treatment and sodium hypochlorite efficiently inactivated all spore populations, including heat-resistant ones produced at low aw.

Optimization and Validation of a New Microbial Inhibition Test for the Detection of Antimicrobial Residues in Living Animals Intended for Human Consumption.

Even though antibiotics are necessary in livestock production, they can be harmful not only due to their toxicity, but also in view of their contribution to the emergence of antimicrobial resistance. Screening tests based on microbial growth inhibition appeared to be useful tools to prevent its entry into the food chain. They have nevertheless been traditionally carried out post mortem, leading to great economical loss and harm to the environment in case a positive sample is found. Hence, the objective was to evaluate the use of a screening test as an ante mortem alternative for the detection of antibiotic residues in meat: thus, Explorer®-Blood test was optimized and validated. After adapting the procedure for matrix preparation, the assay parameters were assessed from 344 antibiotic-free blood serum samples. Limits of Detection (LoDs) were defined by spiking blood serum with several of the most common antimicrobials used in veterinary practice. LoDs were similar to those obtained for meat and were at or below the maximum residue limits set by EU legislation for muscle. Analyses of in vivo injected samples, previously characterized by LC-MS/MS, demonstrated the method's accuracy and proved that Explorer®-Blood can be considered a suitable alternative to conventional post mortem screening methods.

Antibacterial Residue Excretion via Urine as an Indicator for Therapeutical Treatment Choice and Farm Waste Treatment.

Many of the infectious diseases that affect livestock have bacteria as etiological agents. Thus, therapy is based on antimicrobials that leave the animal's tissues mainly via urine, reaching the environment through slurry and waste water. Once there, antimicrobial residues may lead to antibacterial resistance as well as toxicity for plants, animals, or humans. Hence, the objective was to describe the rate of antimicrobial excretion in urine in order to select the most appropriate molecule while reducing harmful effects. Thus, 62 pigs were treated with sulfamethoxypyridazine, oxytetracycline, and enrofloxacin. Urine was collected through the withdrawal period and analysed via LC-MS/MS. Oxytetracycline had the slowest rate of degradation (a half-life time of 4.18 days) and the most extended elimination period in urine (over 2 months), followed by enrofloxacin (a half-life time of 1.48 days, total urine elimination in ca. 3 weeks) and sulfamethoxypyridazine (a half-life time of 0.49 days, total urine elimination in ca. 1 week). Bacterial sensitivity and recommendations for responsible use are limiting when selecting the treatment. Nevertheless, with similar effectiveness, sulfamethoxypyridazine would be the choice, as waste treatment would only need to be implemented for 1 week after treatment. Thus, more in-depth knowledge regarding antibacterial elimination would improve resource management, while protecting animals and consumers' health.

Is Blood a Good Indicator for Detecting Antimicrobials in Meat? Evidence for the Development of In Vivo Surveillance Methods.

The introduction of antimicrobial residues in the food chain has a significant impact on human health. An innovative solution to avoid their presence in meat is the adaptation of current control methods for use with in vivo matrixes. Thus, the aim was to obtain paired blood and muscle samples from pigs treated with some of the main antimicrobials currently used in veterinary medicine (oxytetracycline, sulfamethoxypyridazine, enrofloxacin, amoxicillin), and to compare their rate of depletion in both matrixes. Antimicrobial concentrations in paired samples of blood and muscle were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or high performance liquid chromatography with fluorescence detection (HPLC-FLD). A comparison between values obtained in muscle and blood showed a similar distribution in both matrixes for oxytetracycline; for sulfamethoxypyridazine, a similar decrease rate but a concentration three times higher in blood compared to muscle was found; for enrofloxacin, we found significant differences in the rate of depletion, with similar antimicrobial concentrations in both matrixes with values close to the maximum residue limit (MRL) and higher amounts in muscle for values that lay considerably over the MRL. Conversely, amoxicillin depletion was so rapid that its appearance in carcasses does not seem to pose a risk. Therefore, blood would be a feasible matrix for the development of new in vivo tests.

Cellular events involved in E. coli cells inactivation by several agents for food preservation: A comparative study.

Traditional and novel technologies for food preservation are being investigated to obtain safer products and fulfil consumer demands for less processed foods. These technologies inactivate microorganisms present in foods through their action on different cellular targets, but the final cause of cell loss of viability often remains not well characterized. The main objective of this work was to study and compare cellular events that could play a role on E. coli inactivation upon exposure to treatments with technologies of different nature. E. coli cells were exposed to heat, high hydrostatic pressure (HHP), pulsed electric fields (PEF) and acid treatments, and the occurrence of several alterations, including presence of sublethal injury, membrane permeabilization, increased levels of reactive oxygen species (ROS), DNA damage and protein damage were studied. Results reflected differences among the relevance of the several cellular events depending on the agent applied. Sublethally injured cells appeared after all the treatments. Cells consistently recovered in a higher percentage in non-selective medium, particularly in minimal medium, as compared to selective medium; however this effect was less relevant in PEF-treated cells. Increased levels of ROS were detected inside cells after all the treatments, although their order of appearance and relationship with membrane permeabilization varied depending on the technology. A high degree of membrane permeabilization was observed in PEF treated cells, DNA damage appeared as an important target in acid treatment, and protein damage, in HHP treated cells. Results obtained help to understand the mode of action of food preservation technologies on bacterial cells.

Protective effect of glutathione on Escherichia coli cells upon lethal heat stress.

Heat treatments are widely used by the food industry to obtain safe and stable products, therefore a deeper knowledge of its mode of action on microorganisms would allow a better profit of this technology. Heat shows a multitarget mechanism of action on bacteria, affecting various cellular structures, and causing unbalances in several homeostatic parameters. The aim of this work was to study the effect of glutathione on bacterial survival against heat treatments, in order to acquire knowledge about the mode of action of heat on bacterial cells. Cells were treated in presence or absence of added glutathione and the level of inactivation, soluble protein concentration, enzymatic activities, intracellular ROS level and membrane damages were studied. Results showed that glutathione protected microorganisms against heat inactivation. Moreover, glutathione in the treatment medium preserved intracellular enzyme activity, membrane structure and reduced ROS detection. Besides, glutathione decreased sublethal injury in E. coli. Data presented in this work add new knowledge about bacterial inactivation and survival by heat.

Physiology of the Inactivation of Vegetative Bacteria by Thermal Treatments: Mode of Action, Influence of Environmental Factors and Inactivation Kinetics.

Heat has been used extensively in the food industry as a preservation method, especially due to its ability to inactivate microorganisms present in foods. However, many aspects regarding the mechanisms of bacterial inactivation by heat and the factors affecting this process are still not fully understood. The purpose of this review is to offer a general overview of the most important aspects of the physiology of the inactivation or survival of microorganisms, particularly vegetative bacteria, submitted to heat treatments. This could help improve the design of current heat processes methods in order to apply milder and/or more effective treatments that could fulfill consumer requirements for fresh-like foods while maintaining the advantages of traditional heat treatments.

Oxidative stress in E. coli cells upon exposure to heat treatments.

Heat treatments are widely used by the food industry to inactivate microorganisms, however their mode of action on microbial cells is not fully known. In the last years, it has been proposed that the generation of oxidative species could be an important factor contributing to cell death by heat and by other stresses; however, investigations in this field are scarce. The present work studies the generation of reactive oxygen species (ROS) upon heat treatment in E. coli, through the use of cell staining with specific fluorochromes. Results obtained demonstrate that ROS are detected in E. coli cells when they are subjected to heat exposure, and the amount of fluorescence increases with temperature and time, as does the cellular inactivation. The addition of glutathione or tiron, a potent antioxidant and a superoxide quencher, respectively, to the heating medium protected E. coli against heat inactivation and concurrently reduced the detection of ROS, especially in the case of glutathione. Finally, recovery of heated cells under conditions that relief oxidative stress produced an increase in cell survival. Data presented in this work support the view that ROS generation and subsequent control in bacterial cells could be an essential factor determining inactivation and survival upon exposure to heat, and it could be a potential target to increase the efficacy of current treatments.

Modeling optimal process conditions for UV-heat inactivation of foodborne pathogens in liquid foods.

The combination of ultraviolet radiation and heat (UV-H treatment) has been demonstrated as a promising strategy to overcome the limited UV germicidal effect in fruit juices. Nonetheless, there are so far no data regarding the efficacy of the combined process for the inactivation of bacterial foodborne pathogens in other liquid foods with different pH and composition. In this investigation, the optimum UV-H processing conditions for the inactivation of Escherichia coli, Salmonella Typhimurium, Listeria monocytogenes, and S. aureus in chicken and vegetable broth, in addition to juices, were determined. From these data models that accurately predict the most advantageous UV-H treatment temperature and the expected synergistic lethal effect from UV and heat resistance data separately were constructed. Equations demonstrated that the optimum UV-H treatment temperature mostly depended on heat resistance, whereas the maximum synergistic lethal effect also was affected by the UV resistance of the microorganism of concern in a particular food.

Comparative Resistance of Bacterial Foodborne Pathogens to Non-thermal Technologies for Food Preservation.

In this paper the resistance of bacterial foodborne pathogens to manosonication (MS), pulsed electric fields (PEFs), high hydrostatic pressure (HHP), and UV-light (UV) is reviewed and compared. The influence of different factors on the resistance of bacterial foodborne pathogens to these technologies is also compared and discussed. Only results obtained under harmonized experimental conditions have been considered. This has allowed us to establish meaningful comparisons and draw significant conclusions. Among the six microorganisms here considered, Staphyloccocus aureus is the most resistant foodborne pathogen to MS and HHP and Listeria monocytogenes to UV. The target microorganism of PEF would change depending on the treatment medium pH. Thus, L. monocytogenes is the most PEF resistant microorganism at neutral pH but Gram-negatives (Escherichia coli, Salmonella spp., Cronobacter sakazakii, Campylobacter jejuni) would display a similar or even higher resistance at acidic pH. It should be noted that, in acidic products, the baroresistance of some E. coli strains would be comparable to that of S. aureus. The factors affecting the resistance of bacterial foodborne pathogens, as well as the magnitude of the effect, varied depending on the technology considered. Inter- and intra-specific differences in microbial resistance to PEF and HHP are much greater than to MS and UV. Similarly, both the pH and aw of the treatment medium highly condition microbial resistance to PEF and HHP but no to MS or UV. Growth phase also drastically affected bacterial HHP resistance. Regarding UV, the optical properties of the medium are, by far, the most influential factor affecting its lethal efficacy. Finally, increasing treatment temperature leads to a significant increase in lethality of the four technologies, what opens the possibility of the development of combined processes including heat. The appearance of sublethally damaged cells following PEF and HHP treatments could also be exploited in order to design combined processes. Further work would be required in order to fully elucidate the mechanisms of action of these technologies and to exhaustively characterize the influence of all the factors acting before, during, and after treatment. This would be very useful in the areas of process optimization and combined process design.

Influence of dimethyl dicarbonate on the resistance of Escherichia coli to a combined UV-Heat treatment in apple juice.

Commercial apple juice inoculated with Escherichia coli was treated with UV-C, heat (55°C) and dimethyl dicarbonate - DMDC (25, 50, and 75 mg/L)-, applied separately and in combination, in order to investigate the possibility of synergistic lethal effects. The inactivation levels resulting from each treatment applied individually for a maximum treatment time of 3.58 min were limited, reaching 1.2, 2.9, and 0.06 log10 reductions for UV, heat, and DMDC (75 mg/L), respectively. However, all the investigated combinations resulted in a synergistic lethal effect, reducing the total treatment time and UV dose, with the synergistic lethal effect being higher when larger concentrations of DMDC were added to the apple juice. The addition of 75 mg/L of DMDC prior to the combined UV-C light treatment at 55°C resulted in 5 log10 reductions after only 1.8 min, reducing the treatment time and UV dose of the combined UV-Heat treatment by 44%.

Effect of pressure-induced changes in the ionization equilibria of buffers on inactivation of Escherichia coli and Staphylococcus aureus by high hydrostatic pressure.

Survival rates of Escherichia coli and Staphylococcus aureus after high-pressure treatment in buffers that had large or small reaction volumes (ΔV°), and which therefore underwent large or small changes in pH under pressure, were compared. At a low buffer concentration of 0.005 M, survival was, as expected, better in MOPS (morpholinepropanesulfonic acid), HEPES, and Tris, whose ΔV° values are approximately 5.0 to 7.0 cm(3) mol(-1), than in phosphate or dimethyl glutarate (DMG), whose ΔV° values are about -25 cm(3) mol(-1). However, at a concentration of 0.1 M, survival was unexpectedly better in phosphate and DMG than in MOPS, HEPES, or Tris. This was because the baroprotective effect of phosphate and DMG increased much more rapidly with increasing concentration than it did with MOPS, HEPES, or Tris. Further comparisons of survival in solutions of salts expected to cause large electrostriction effects (Na2SO4 and CaCl2) and those causing lower electrostriction (NaCl and KCl) were made. The salts with divalent ions were protective at much lower concentrations than salts with monovalent ions. Buffers and salts both protected against transient membrane disruption in E. coli, but the molar concentrations necessary for membrane protection were much lower for phosphate and Na2SO4 than for HEPES and NaCl. Possible protective mechanisms discussed include effects of electrolytes on water compressibility and kosmotropic and specific ion effects. The results of this systematic study will be of considerable practical significance in studies of pressure inactivation of microbes under defined conditions but also raise important fundamental questions regarding the mechanisms of baroprotection by ionic solutes.

UV-C inactivation of Cronobacter sakazakii.

The purpose of this research was to study the effect of different factors on the resistance of Cronobacter sakazakii NCTC 9238 to UV-C light (which includes germicidal ultraviolet light at 254 nm) and to determine whether a combined treatment with heat would produce a synergistic effect for its inactivation. Growth temperature between 10°C and 37°C did not change the UV-C resistance of C. sakazakii. On the contrary, cells in the logarithmic phase of growth were more sensitive to UV-C light than in the stationary phase. The lethality of UV-C was independent of pH (between 3.0 and 7.0) and a(w) (between 0.94 and > 0.99) of the treatment media, but it exponentially decreased with the absorption coefficient (α). When applying a UV-C treatment of 27.1 J/mL at 25°C to C. sakazakii suspended in vegetable soup and apple juice, 1 and 2 log10 cycles of inactivation were barely achieved, respectively. However, the bactericidal effect of UV-C light increased with temperature. The lethality of the combined process was the result of a synergistic effect that was maximum at 52.5°C for apple juice and 55°C for vegetable soup. In conclusion, these results indicate that UV-C efficacy may be influenced by microbial growth conditions and food characteristics, and that its combination with heat may act synergistically against C. sakazakii.

Inactivation of Escherichia coli O157:H7 in fruit juices by combined treatments of citrus fruit essential oils and heat.

This work approaches the possibility of combining mild heat treatments with citrus fruit essential oils (EOs) to improve the effectiveness of heat treatments and thus to reduce treatment intensity. Concentrations between 10 and 200 µL/L of lemon, mandarin, or orange EO were tested at 54 °C for 10 min in laboratory media, determining that 200 µL/L of each EO was necessary to achieve a 5 log(10) reduction of the initial Escherichia coli O157:H7 concentration. A relationship could be established between sublethally injured cells after the heat treatment and inactivated cells after the combined process. In apple juice, the synergism in the inactivation of E. coli O157:H7 when adding 200 µL/L of lemon EO might suppose a reduction in the treatment temperature (of 4.5 °C) or in the treatment time (by 5.7 times) within the range of temperature assayed (54-60 °C). Addition of 75 µL/L of lemon EO was determined to achieve the same synergistic effect of the combined treatment when the initial inoculum was reduced from 3×10(7) to 3×10(4) CFU/mL. Since the addition of lemon EO did not decrease the hedonic acceptability of apple juice, the proposed combined treatment could be further studied and optimized for the production of new minimally processed juices.

Biological approach to modeling of Staphylococcus aureus high-hydrostatic-pressure inactivation kinetics.

Graphs for survival under high hydrostatic pressure (450 MPa; 25°C; citrate-phosphate buffer, pH 7.0) of stationary-growth-phase cells of eight Staphylococcus aureus strains were found to be nonlinear. The strains could be classified into two groups on the basis of the shoulder length. Some of them showed long shoulders of up to 20 min at 450 MPa, while others had shoulders of <3.5 min. All strains showed tails. No significant differences in the inactivation rate were found during the log-linear death phase among the eight strains. The entry into stationary growth phase resulted both in an increase in shoulder length and in a decrease in the inactivation rate. However, whereas shoulder length proved to depend on sigma B factor activity, the inactivation rate did not. Recovery in anaerobiosis decreased the inactivation rate but did not affect the shoulder length. Addition of the minimum noninhibitory concentration of sodium chloride to the recovery medium resulted in a decrease in shoulder length and in an increase in the inactivation rate for stationary-growth-phase cells. In the tail region, up to 90% of the population remained sensitive to sodium chloride.

Thermostability of oxytetracycline, tetracycline, and doxycycline at ultrahigh temperatures.

The thermostability parameters of three tetracycline antibiotics at high and ultrahigh temperatures (110-140 degrees C) as well as the influence of treatment medium pH and water activity on their thermotolerance have been investigated. The thermal degradation of the three antibiotics followed a first-order reaction kinetic within the 1.5-2 log(10) cycles investigated. A linear relationship was observed between the log of the DT values and the treatment temperature. The temperature dependence of the DT values was similar for the three molecules (z=28+/-2 degrees C). DT values of doxycycline were approximately 1.5 and 3 times higher than those of tetracycline and oxytetracycline, respectively. Changes in the treatment medium pH (7.0-4.0) and water activity (0.99-0.93) scarcely varied the antibiotics' thermal stability. Only when doxycycline was heat-treated at pH 4.0 did its thermal resistance increase by 3 times. The thermostability parameters obtained would allow the effect of different cooking and sterilization procedures to be estimated. Whereas low-temperature-long-time treatments (conventional sterilization) would destroy >98% of the initial concentration of the residues of the three antibiotics, high-temperature-short-time treatments (UHT) would leave unaltered residues in the 50-90% range.

Predicting microbial heat inactivation under nonisothermal treatments.

The aim of this study was to develop an equation that accurately predicts microbial heat inactivation under nonisothermal treatments at constantly rising heating rates (from 0.5 to 5 degrees C/min) in media with different pH values (4.0 or 7.4). The survival curves of all bacteria (Enterococcus faecium, Escherichia coli, Listeria monocytogenes, Salmonella Senftenberg 775W, Salmonella Typhimurium, and Staphylococcus aureus) tested under isothermal treatments were nearly linear. For the most heat-resistant microorganism (E. faecium), the estimated DT-values at pH 7.4 were at least 100 times those of the second most thermotolerant microorganism (Salmonella Senftenberg 775W). The heat resistance of E. faecium was up to 30 times lower at pH 4.0 than at pH 7.4. However, E. faecium was still the most heat-resistant microorganism under nonisothermal treatments at both pH values. Inactivation under nonisothermal conditions was not accurately estimated from heat resistance parameters of isothermal treatments when microbial adaptation or sensibilization occurred during the heating up lag phases. The under-prediction of the number of survivors might be greater than 15 log CFU within the nonisothermal treatment conditions investigated. Therefore, the nonisothermal survival curves of the most heat-resistant microorganisms were fitted with the following equation: log S(t) = -(t/delta)P. This equation accurately described the survival curves of all the bacteria tested. We observed a linear relationship between the log of the scale parameter (delta) and the log of the heating rate. A p value characteristic of each microorganism and pH tested was calculated. Two equations capable of predicting the inactivation rate of all bacteria tested under nonisothermal treatments at pH 7.4, 5.5, or 4.0 were developed. The model was evaluated in skim milk and apple juice. The results of this study could be used to help minimize public health risks and to extend the shelf life of those foods requiring long heating up lag phases during processing.

Predicting heat inactivation of Staphylococcus aureus under nonisothermal treatments at different pH.

The aim was to assess whether heat resistance data obtained from isothermal treatments allow the estimation of survivors of Staphylococcus aureus under nonisothermal conditions and to find a model that accurately predicts its heat inactivation at constantly rising heating rates (0.5-9 degrees C/min) in media of different pH (4.0-7.4). S. aureus showed a higher heat resistance under isothermal treatments at pH 4.0 than at pH 5.5-7.4. However, under nonisothermal treatments S. aureus increased its heat resistance at pH 5.5-7.4 and became more thermotolerant than at pH 4.0. Estimations of survival curves under nonisothermal treatments obtained from heat resistance parameters of isothermal treatments did not adequately fit experimental values. Whereas the number of survivors was much higher than estimated at pH 5.5-7.4, that obtained at the slower heating rates at pH 4.0 was lower. An equation based on the Weibullian-like distribution (log10 S(t) = (t/delta)p) accurately described survival curves obtained under nonisothermal conditions. A nonlinear relationship was observed among the scale parameter (delta) and the heating rate which allowed the development of two equations capable of predicting the inactivation rate of S. aureus under nonisothermal treatments. This study might contribute to prevent public health risks in foods requiring long heating lag phases during their processing.

Predicting thermal inactivation in media of different pH of Salmonella grown at different temperatures.

The influence of the growth temperature and the pH of the heating medium on the heat resistance at different temperatures of Salmonella typhimurium ATCC 13311 was studied and described mathematically. The shift of the growth temperature from 10 to 37 degrees C increased heat resistance of S. typhimurium fourfold. The pH of the heating medium at which heat resistance was maximum was pH 6 for cells grown at 37 degrees C, but changed with growth temperature. The alkalinization of the heating medium from pH 6 to pH 7.7 decreased the heat resistance of cells grown at 37 degrees C by a factor of 3. Neither the growth temperature nor the pH modified the z values significantly (4.9 degrees C). The decimal reduction times at different treatment temperatures, in buffers of different pH of cells of S. typhimurium grown at different temperatures, were accurately described by a mathematical equation (correlation coefficient of 0.97). This equation was also tested for Salmonella senftenberg 775W (ATCC 43845) and Salmonella enteritidis ATCC 13076, strains in which the correlation coefficients between the observed and the theoretically calculated values were 0.91 and 0.98, respectively.