Detection of sentinel bacteria in mangrove sediments contaminated with heavy metals.

Mangroves in the Northwest Coast of South America are contaminated with heavy metals due to wastewater discharges from industries, affecting the biota from this environment. However, bacteria proliferate in these harsh environmental conditions becoming possible sentinel of these contaminations. In this study, bacterial community composition was analyzed by throughput sequencing of the 16S rRNA gene from polluted and pristine mangrove sediments affected by marked differences in heavy metal concentrations. Core bacteria were dominated by Proteobacteria, Firmicutes, and Bacteroidetes phyla, with strong differences between sites at class and genus levels, correlated with metal levels. Increment of abundance on specific OTUs were associated with either elevated or decreased concentrations of metals and with the sulfur cycle. The abundance of Sulfurovum lithotrophicum, Leptolinea tardivitalis, Desulfococcus multivorans and Aminobacterium colombiense increases when metals rise. On contrary, Bacillus stamsii, Nioella nitrareducens and Clostridiisalibacter paucivorans abundance increases when metal levels are reduced. We propose these OTUs as bacterial sentinels, whose abundance can help monitor the restoration programs of contaminated mangrove sediments in the future.

Probing the surface fine structure through electrochemical oscillations.

In the course of (electro)catalytic reactions, reversible and irreversible changes, namely the formation of adsorbed poisons, catalyst degradation, surface roughening, etc., take place at distinct time-scales. Reading the transformations on the catalyst surface from the measurement of the reaction rates is greatly desirable but generally not feasible. Herein, we study the effect of random surface defects on Pt(100) electrodes toward the electro-oxidation of methanol in acidic media. The surface defects are gently generated in situ and their relative magnitudes are reproducibly controlled. The system was characterized under conventional conditions and investigated under an oscillatory regime. Oscillatory patterns were selected according to the presence of surface defects, and a continuous transition from large amplitude/low frequency oscillations (type L) on smooth surfaces to small amplitude/high frequency oscillations (type S) on disordered surfaces was observed. Importantly, self-organized potential oscillations were found to be much more sensitive to the surface structure than conventional electrochemical signatures or even other in situ characterization methods. As a consequence, we proved the possibility of following the surface fine structure in situ and in a non-invasive manner by monitoring the temporal evolution of oscillatory patterns. From a mechanistic point of view, we describe the role played by surface defects and of the adsorbed and partially oxidized, dissolved species on the oscillations of type S and L.

Effect of storage conditions in the response of Listeria monocytogenes in a fresh purple vegetable smoothie compared with an acidified TSB medium.

In this study, growth and/or inactivation of Listeria monocytogenes 4032 at different inoculum levels in a vegetable smoothie with purple colour, (previously heat stabilised at 95 °C for 3 min) was evaluated. Growth/inactivation was compared with acidified TSB medium at the same pH level with HCl. Samples were stored at different temperatures (5, 10, 15 and 25 °C). All the smoothies stored at 15 and 25 °C showed growth up to 7.5-8.0 log CFU/mL and at 10 °C growth was only observed at the highest inoculum level. Growth was only observed at 25 °C in acidified TSB. In the case of the smoothies inoculated and stored at 5 °C, L. monocytogenes was not able to grow but survived for a long period of time, whereas at the lower inocula at 10 °C they presented a slow inactivation for an extended time. Acidified TSB inoculated and stored showed inactivation at 5, 10 and 15 °C. Best inactivation modelling alternatives are proposed. The differences between the smoothie and TSB medium about growth or survival in this study, even at relatively low pH values, were due to the favorable nutritional composition of the smoothie compared to a laboratory medium. Results in this study can allow to design safe conditions for smoothie production.

One-step global parameter estimation of kinetic inactivation parameters for Bacillus sporothermodurans spores under static and dynamic thermal processes.

Bacillus sporothermodurans produces highly heat-resistant endospores, that can survive under ultra-high temperature. High heat-resistant sporeforming bacteria are one of the main causes for spoilage and safety of low-acid foods. They can be used as indicators or surrogates to establish the minimum requirements for heat processes, but it is necessary to understand their thermal inactivation kinetics. The aim of the present work was to study the inactivation kinetics under both static and dynamic conditions in a vegetable soup. Ordinary least squares one-step regression and sequential procedures were applied for estimating these parameters. Results showed that multiple dynamic heating profiles, when analyzed simultaneously, can be used to accurately estimate the kinetic parameters while significantly reducing estimation errors and data collection.

Modelling the effects of temperature and osmotic shifts on the growth kinetics of Bacillus weihenstephanensis in broth and food products.

Bacillus weihenstephanensis is a psychrotolerant bacterium belonging to the Bacillus cereus group. Some strains may be cytotoxic although they have not been described as food-poisoning agents so far. The objective of this work is to model the effects of temperature and a(w) downshifts on the lag time of B. weihenstephanensis and the dependence of µ(max) on the growth conditions (temperature and a(w)). Effects of temperature downshifts were studied on 30 experimental conditions (shifts magnitude ranging from 2 to 20 °C, temperature after shift from 10 to 20 °C and a(w) ranging from 0.977 to 0.997). Osmotic shifts were studied for 13 conditions (shift magnitude ranging from 0.008 to 0.020 units of a(w), temperature from 10 to 30 °C, a(w) after shift from 0.977 to 0.997). Experimental results show that temperature downshifts were able to induce considerable lag times (up to 20 days) when occurring near the growth limits. At lower temperatures, osmotic shifts had also a significant effect. Validation experiments in food subjected to changing conditions of temperature showed that the model provided valid predictions in diluted creamed pasta but overestimated bacterial growth in carrot soup (fail safe predictions).

Nonisothermal heat resistance determinations with the thermoresistometer Mastia.

AIMS: To design and build a thermoresistometer, named Mastia, which could perform isothermal and nonisothermal experiments. METHODS AND RESULTS: In order to evaluate the thermoresistometer, the heat resistance of Escherichia coli vegetative cells and Alicyclobacillus acidoterrestris spores was explored. Isothermal heat resistance of E. coli was characterized by D(60 degrees C) = 0.38 min and z = 4.7 degrees C in pH 7 buffer. When the vegetative cells were exposed to nonisothermal conditions, their heat resistance was largely increased at slow heating and fast cooling rates. Isothermal heat resistance of A. acidoterrestris was characterized by D(95 degrees C) = 7.4 min and z = 9.5 degrees C in orange juice. Under nonisothermal conditions, inactivation was reasonably well predicted from isothermal data. CONCLUSIONS: The thermoresistometer Mastia is a very suitable instrument to get heat resistance data of micro-organisms under isothermal and nonisothermal treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: The thermoresistometer Mastia can be a helpful tool for food processors in order to estimate the level of safety of the treatments they apply.

Estimation of the non-isothermal inactivation patterns of Bacillus sporothermodurans IC4 spores in soups from their isothermal survival data.

The isothermal survival curves of the heat resistant spores of Bacillus sporothermodurans IC4, in the range of 117-125 degrees C, were determined in chicken, mushroom and pea soups by the capillary method. They were all non-linear with a noticeable upper concavity and could be described by the equation log(10) [N(t)/N(0)]=-b(T)t(n) with a fixed power, n, of the order of 0.7-0.8. The temperature dependence of b(T) could be described by the equation b(T)=log(e)[1+exp[k(T-T(c))]], where T(c) is the temperature where intensive inactivation starts and k is the slope of b(T) at temperatures well above T(c). They were 121-123 degrees C and 0.2-0.4 degrees C(-1), respectively, depending on the soup. These parameters were used to estimate the survival curves of the spores in two non-isothermal heat treatments using the procedure originally proposed by Peleg and Penchina [Crit. Rev. Food Sci. Nutr. 40 (2000) 159]. The results were compared with experimental survival curves, determined by the direct injection method, in another laboratory. There was a general agreement, although not perfect, between the predicted and experimentally observed survival ratios. Also, the isothermal survival parameters, estimated directly from the non-isothermal inactivation data using the model, were in general agreement with those calculated from the isothermal data. This suggests that the heat inactivation patterns of B. sporothermodurans IC4 spores in soups can be at least roughly estimated using the same general survival model, which has until now only been experimentally validated for vegetative bacterial cells.

Influence of pH and temperature on growth of Bacillus cereus in vegetable substrates.

Bacillus cereus is a food-borne pathogen which most often contaminates foods of plant origin. Spores of psychrotrophic strains have the ability to germinate and grow at refrigeration temperatures in different vegetable substrates, such as carrot broth, zucchini broth, and cooked carrot purée. In some circumstances, factors such as pH, heat treatment, and storage temperature play a fundamental role in controlling the growth of these psychrotrophic strains and in extending the shelf life of refrigerated, minimally processed vegetable-based products in relation to pathogenic spore-forming bacteria. The combination of mild acidification (pH 5.0) and refrigeration (

Effect of heat activation and inactivation conditions on germination and thermal resistance parameters of Bacillus cereus spores.

The effect of isothermal and non-isothermal heat activation on germination and thermoresistance of two strains of Bacillus cereus spores was studied. Results indicated that the germination after isothermal activation was lower than after non-isothermal heating. The activation rate affected the z value, which increased with faster heating rates. For each temperature and inactivation rate, the non-isothermal activation at rate of 2 degrees C/min resulted in larger D values (D90 = 4.70 min) than isothermal activation (D90 = 4.04 min). The two mathematical equations used to analyse non-isothermal data produced similar predicted D and z values, nevertheless the Hayakawa equation modified in this work for non-linear regression analysis, requires less computational effort.

Thermal inactivation kinetics of Bacillus stearothermophilus spores using a linear temperature program.

A systematic study of the inactivation kinetics of Bacillus stearothermophilus spores was carried out in nonisothermic heating conditions using a linear temperature increase program and analyzing the experimental data by means of a one-step nonlinear regression. The D and z values estimated are close to those obtained in isothermic conditions and estimated by using a two-step model, first D values are calculated, and then in the second step a z value is deduced (D(121 degrees C) = 3.08 and 4.38 min, respectively, and z = 7 and 7.9 degrees C, respectively). No convergence problems were observed when using the one-step nonlinear regression proposed. The results indicated that the methodology applied in this study can be used to obtain kinetic data for bacterial spores, which could mean a significant reduction in the amount of experimental work employed to generate these data.

A predictive model that describes the effect of prolonged heating at 70 to 90 degrees C and subsequent incubation at refrigeration temperatures on growth from spores and toxigenesis by nonproteolytic Clostridium botulinum in the presence of lysozyme.

Refrigerated processed foods of extended durability such as cook-chill and sous-vide foods rely on a minimal heat treatment at 70 to 95 degrees C and then storage at a refrigeration temperature for safety and preservation. These foods are not sterile and are intended to have an extended shelf life, often up to 42 days. The principal microbiological hazard in foods of this type is growth of and toxin production by nonproteolytic Clostridium botulinum. Lysozyme has been shown to increase the measured heat resistance of nonproteolytic C. botulinum spores. However, the heat treatment guidelines for prevention of risk of botulism in these products have not taken into consideration the effect of lysozyme, which can be present in many foods. In order to assess the botulism hazard, the effect of heat treatments at 70, 75, 80, 85, and 90 degrees C combined with refrigerated storage for up to 90 days on growth from 10(6) spores of nonproteolytic C. botulinum (types B, E, and F) in an anaerobic meat medium containing 2,400 U of lysozyme per ml (50 microg per ml) was studied. Provided that the storage temperature was no higher than 8 degrees C, the following heat treatments each prevented growth and toxin production during 90 days; 70 degrees C for >/=2,545 min, 75 degrees C for >/=463 min, 80 degrees C for >/=230 min, 85 degrees C for >/=84 min, and 90 degrees C for >/=33.5 min. A factorial experimental design allowed development of a predictive model that described the incubation time required before the first sample showed growth, as a function of heating temperature (70 to 90 degrees C), period of heat treatment (up to 2,545 min), and incubation temperature (5 to 25 degrees C). Predictions from the model provided a valid description of the data used to generate the model and agreed with observations made previously.

Risk analysis of the thermal sterilization process. Analysis of factors affecting the thermal resistance of microorganisms.

A risk analysis was applied to experimental heat resistance data. This analysis is an approach for processing experimental thermobacteriological data in order to study the variability of D and z values of target microorganisms depending on the deviations range of environmental factors, to determine the critical factors and to specify their critical tolerance. This analysis is based on sets of sensitivity functions applied to a specific case of experimental data related to the thermoresistance of Clostridium sporogenes and Bacillus stearothermophilus spores. The effect of the following factors was analyzed: the type of target microorganism; nature of the heating substrate; pH, temperature; type of acid employed and NaCl concentration. The type of target microorganism to be inactivated, the nature of the substrate (reference or real food) and the heating temperature were identified as critical factors, determining about 90% of the alteration of the microbiological risk. The effect of the type of acid used for the acidification of products and the concentration of NaCl can be assumed to be negligible factors for the purposes of engineering calculations. The critical non-uniformity in temperature during thermobacteriological studies was set as 0.5% and the critical tolerances of pH value and NaCl concentration were 5%. These results are related to a specific case study, for that reason their direct generalization is not correct.

Effect of acidification and oil on the thermal resistance of Bacillus stearothermophilus spores heated in food substrate.

The effect of the addition of vinegar and/or oil to a food homogenate (tomato sauce, tuna and vegetables) on the thermal resistance of Bacillus stearothermophilus spores was studied. The results indicated that the food substrate without the addition of vinegar and oil and a pH value of 5.28 reduced the thermal resistance of B. stearothermophilus spores compared with that obtained in double-distilled water, (D121 = 1.41 and 3.08 min respectively). The addition of vinegar reduced the pH of the substrate (4.81) and consequently the D values were reduced (D121 = 1.28 min). The addition of soya oil and vinegar to substrate until a pH of 4.81, further reduced the thermal resistance of the spores, giving a D121 value of 0.93 min.

Predictive model to describe the combined effect of pH and NaCl on apparent heat resistance of Bacillus stearothermophilus.

The combined effect of pH and NaCl on the apparent thermal resistance of Bacillus stearothermophilus ATCC 12980 spores was studied. Spores were heated at different temperatures (115-125 degrees C) in mushroom substrate, acidified using glucono-delta-lactone to different pH levels (from 5.75 to 6.7), which contained concentrations of NaCl that ranged from 0.5 to 3% (w/v). The recovery medium was acidified to the same pH level and contained the same NaCl concentration as the heating menstruum. A factorial experimental design allowed a predictive model to be developed, which described the combined effect of heating temperature, pH and NaCl on the thermal resistance of B. stearothermophilus spores. Predictions from the model provided a valid description of the data used to generate the model, and agreed with observations from the literature and from an independent experiment performed using asparagus and bean substrates.

Predictive model of the effect of CO2, pH, temperature and NaCl on the growth of Listeria monocytogenes.

The growth responses of L. monocytogenes as affected by CO2 concentration (0-100% v/v, balance nitrogen), NaCl concentration (0.5-8.0% w/v), pH (4.5-7.0) and temperature (4-20 degrees C) were studied in laboratory medium. Growth curves were fitted using the model of Baranyi and Roberts, and specific growth rates derived from the curve fit were modelled. Predictions for specific growth rate, doubling time and time to a 1000-fold increase could be made for any combination of conditions within the matrix. Predictions of growth from the model were compared with published data and this showed the model to be suitable for predicting growth of L. monocytogenes in a range of foods packaged under a modified atmosphere.

Thermal Resistance of Bacillus stearothermophilus Heated at High Temperatures in Different Substrates.

The effect of mushroom extract, with or without acidification with glucono-δ-lactone, and the overnight incubation of the spores in CaCl2, on the heat resistance of B. stearothermophilus ATCC 12980 spores was studied. The temperature range considered was 121 to 140°C for mushroom extract and CaCl2 and 121 to 145°C for double-distilled water as a reference substrate. The results indicated that mushroom extract without added acid significantly reduces the thermal resistance of the spores in comparison to the double-distilled water. Acidification of the mushroom extract reduces the heat resistance of spores of B. stearothermophilus at 121 °C. However, above 130°C lowering of the pH did not significantly reduce the thermal resistance of the spores, and so no generalizations should be made with regard to the effect of the pH when high temperature-short time (HTST) processes are being considered. Overnight incubation in CaCl2 and subsequent heat treatment lead to increased heat resistance at 121 °C compared to that observed in double-distilled water. However, at 130°C and above CaCl2 did not increase the apparent heat resistance of the spores.

Mathematical model for the combined effect of temperature and pH on the thermal resistance of Bacillus stearothermophilus and Clostridium sporogenes spores.

Two mathematical models have been studied to establish the relationship between the pH, treatment temperature and thermal destruction constant (k) of Bacillus stearothermophilus and Clostridium sporogenes spores. The study was carried out by heating the spores in mushroom extract acidified with two different acidulants (citric acid and glucono-delta-lactone). Among the models studied, the one that best described the inactivation was a second order polynomial equation, the precision of which depended on the microorganism studied.

The effect of temperature and growth rate on the susceptibility of Listeria monocytogenes to environmental stress conditions.

The effect of growth temperature and growth rate on the susceptibility to heat and pH stress were investigated in Listeria monocytogenes grown in continuous culture where these two growth variables could be varied independently of each other, and in batch culture. After growth at 30 degrees C or 10 degrees C at constant growth rate, or at 30 degrees C at different growth rates, cells did not differ in their resistance to heat at 55 degrees C. Cells grown at 30 degrees C were more resistant to acid stress at pH 2.5 than cells grown at the same growth rates at 10 degrees C. Cells grown at low growth rate at 30 degrees C gave greater resistance to acid stress than those grown at high growth rate. Growth temperature and growth rate had independent effects on the susceptibility of L. monocytogenes to acid stress conditions. This may have implications for the survival of L. monocytogenes in acidic foods.

Effect of lysozyme concentration, heating at 90 degrees C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum.

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10(6) spores per tube. Hen egg white lysozyme (0-50 micrograms ml-1 was added, and the tubes were given a heat treatment equivalent to 19.8 min at 90 degrees C, cooled, and incubated at 8 degrees, 12 degrees, 16 degrees and 25 degrees C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6-D inactivation was therefore achieved. In tubes to which lysozyme (5-50 micrograms ml-1 had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 micrograms ml-1, growth was first observed after 68 d at 8 degrees C, 31 d at 12 degrees C, 24 d at 16 degrees C, and 9 d at 25 degrees C. Thus, in these circumstances, a heat treatment equivalent to 19.8 min at 90 degrees C was not sufficient, on its own, to give a 6-D inactivation. A combination of the heat treatment, maintenance at less than 12 degrees C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10(6).

D Values of Bacillus stearothermophilus Spores as a Function of pH and Recovery Medium Acidulant.

The effect of the pH and the type of acidulant (citric acid or glucono-δ-lactone) of the recovery medium on the thermal resistance of Bacillus stearothermophilus ATCC 12980 was studied. The spores were heated in bidistilled water as a reference substrate and in acidified mushroom extract using citric acid or glucono-δ-lactone as acidulants (pH 6.2) and subcultured in reference (pH 7) and acidified (pH 6.2) media. A period of treatment-dependent pH sensitization was observed in heat-treated spores. In all cases, D values were lower in the acidified recovery medium than those obtained in the reference medium, but the type of acidulant used in the recovery medium had no influence on the D values. No influence on z values was observed as a consequence of the different recovery media, but they changed within a range of 7 to 10°C as a function of the different heating substrates. Glucono-δ-lactone proved to be as effective as citric acid in controlling the microbiological spoilage of foods. The pH has a great influence on decreasing the D values, mainly when acidification of the substrate and acidification of the recovery medium are combined. Consequently, it would be appropriate to take this effect into account in process calculations or validation.