Effects of heating and recovery media pH on the heat resistance of Alicyclobacillus acidoterrestris Ad 746 spores.

AIMS: Alicyclobacillus acidoterrestris is a sporulating, acidophilic bacterial species which spoils acidic beverages such as fruit juices. This work aims to quantify the heat resistance of A. acidoterrestris spores and their recovery potential as a function of heating and recovery media pH. METHODS AND RESULTS: The heat treatments were carried out with the strain of A. acidoterrestris Ad 746 in Bacillus acidoterrestris thermophilic medium. The pH of the heating medium from pH 7 to pH 2 nonsignificantly reduced the heat resistance. However, the pH levels of the recovery media strongly affected the apparent heat resistance of this strain. The maximum heat resistance was found when the pH was 4·70 and decreased when the pH decreased to pH 2·8, close to the minimum growth pH and when the recovery medium pH increased to pH 5·3. CONCLUSION: The heating medium pH has a slight effect on the spore heat resistances of this acidophilic species. However, the pH of the recovery media strongly affected the apparent heat resistance of this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained parameters quantifying the heat resistance of A. acidoterrestris spores are tools to optimize the heat treatments and to control its development.

Assessing the individual microbial inhibitory capacity of different sugars against pathogens commonly found in food systems.

Highly concentrated sugar solutions are known to be effective antimicrobial agents. However, it is unknown whether this effect is solely the result of the collective osmotic effect imparted by a mixture of sugars or whether the type of carbohydrate used also has an individual chemical effect on bacterial responses, that is, inhibition/growth. In view of this, in this work, the antimicrobial properties of four sugars, namely, glucose, fructose, sucrose and maltose against three common food pathogens; Staphylococcus aureus, Escherichia coli and Salmonella enterica, were investigated using a turbidimetric approach. The results obtained indicate that the type of sugar used has a significant effect on the extent of bacterial inhibition which is not solely dependent on the water activity of the individual sugar solution. In addition, while it was shown that high sugar concentrations inhibit bacterial growth, very low concentrations show the opposite effect, that is, they stimulate bacterial growth, indicating that there is a threshold concentration upon which sugars cease to act as antimicrobial agents and become media instead. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, an analysis on the antimicrobial properties of glucose, fructose, sucrose and maltose in solution was conducted using a turbidimetric approach. Our findings indicate that while, as expected, all of these sugars exhibit significant antimicrobial effects at high concentrations, at low concentrations they appear to act as substrates for the bacteria which results in enhanced microbial growth instead of inhibition. In addition, the results obtained also suggest that the resultant osmotic stress imparted by the sugar solutions is not the only factor which determines their antimicrobial activity and that other chemical factors may be playing a significant role.

Coupling the dynamics of diffused gases and microbial growth in modified atmosphere packaging.

Coupling microbial dynamics with the complete dynamics of the packaging gases is still a challenge. In this work the microbial growth kinetic parameters for Pseudomonas and Lactic Acid Bacteria (LAB) in MAP are identified based on accurate estimation of diffusivity of gases and parameter scaled sensitivity approaches. The microbial dynamics are also compared with those estimated based on partial pressure measurement. Scaled sensitivity coefficient analysis using dissolved gases as variable inputs, shows that in most cases the only coefficients large enough for estimation were those for CO2max-diss, and for µmax. The current data showed that dissolved gases led significant differences on the microbial parameter of CO2max values when compared with the headspace gases. On the other hand, the (so-called) dissolved specific growth rate follows a clear trend down for both microorganisms in relation to the increase of the initial headspace CO2. Finally, current results indicate a possible correlation between CO2max-diss, CO2max-headspace, and µmax as functions of CO2init.

The relevance of supply chain characteristics in GHG emissions: The carbon footprint of Maltese juices.

AIMS: Foods and drinks are major contributors (17%) to the greenhouse gas (GHG) emissions caused by private consumption in Europe. The carbon footprint (CF) of a certain product expresses the total GHG emissions over its whole life cycle, and its calculation for foodstuff is a necessary first step to reduce their contribution to global warming. The calculation of the CF of Maltese food products is especially relevant for two reasons: the economic characteristics of the island, whose food sector is highly dependent on imports, implying longer transport distances; and the Maltese electricity production mix, based almost exclusively on oil combustion. METHODS AND RESULTS: The CF of ten multi-fruit juices marketed in Malta has been determined, covering all the processes from the agricultural stage to the distribution of the final products. As a functional unit (FU), a 250 ml bottle of packaged product arriving at the retailer has been considered. The Maltese orange juice, the only final product in which only local ingredients are used, presents the lowest CF (0.50 kgCO2/FU), while the remaining ones range from 0.67 kgCO2/FU to 0.80 kgCO2/FU. The major contributor to all the CFs is juice processing at the Maltese plant (0.42 kgCO2/FU), mainly due to the use of electricity (78%). CONCLUSIONS: The influence of both the electricity mix and the Maltese supply chain in the CF of the final products has been demonstrated. Alternatives to reduce the impacts of the final products have been proposed and evaluated that could lower the average CF of the juices by 32%. SIGNIFICANCE AND IMPACT OF THE STUDY: The calculation of the CF of Maltese juices represents an innovative case study due to the characteristics of the island, and it is expected to act as a first step to lower their environmental impacts.

Ultrasound processing of liquid system(s) and its antimicrobial mechanism of action.

Ultrasound creates cavitation phenomena, resulting in the formation of several free radicals, namely OH˙ and H˙, due to the breakdown of the H2 O molecule. These radicals affect the cellular integrity of the bacteria, causing the inactivation of several processes, and thus it is important to unravel the mechanism of action of this technology. This research looks into the application and mechanism of action of ultrasound technology as a means of disinfection by acoustic cavitation. Sterile water and synthetic waste water were inoculated with different mutants of Escherichia coli K12 strains containing deletions in genes affecting specific functional properties of E. coli. These were: dnak soxR, soxS, oxyR, rpoS, gadA/gadB, gadC and yneL. Escherichia coli K-12 ΔoxyR appeared to be more resistant to the treatment together with gadW, gadX, gabT and gabD, whereas the mutant K-12 ΔdnaK was more sensitive with c. 2·5 log (CFU per ml) reduction in comparison to their isogenic wild-type E. coli K-12. This indicates that the dnaK gene participates in general stress response and more specifically to hyperosmotic stress. The other E. coli deleted genes tested (soxS, rpoS, gadB, gadC, yneL) did not appear to be involved in protection of microbial cells against ultrasound. SIGNIFICANCE AND IMPACT OF THE STUDY: This study looks at the mechanism of action of ultrasound technology for the disinfection of wastewater. Different mutants with deleted genes were used to study the respective sensitivity or resistance to this treatment. This is essential to characterize changes at the molecular level, which might be occurring during treatment, resulting in bacterial adaptation.

Impact of food model (micro)structure on the microbial inactivation efficacy of cold atmospheric plasma.

The large potential of cold atmospheric plasma (CAP) for food decontamination has recently been recognized. Room-temperature gas plasmas can decontaminate foods without causing undesired changes. This innovative technology is a promising alternative for treating fresh produce. However, more fundamental studies are needed before its application in the food industry. The impact of the food structure on CAP decontamination efficacy of Salmonella Typhimurium and Listeria monocytogenes was studied. Cells were grown planktonically or as surface colonies in/on model systems. Both microorganisms were grown in lab culture media in petri dishes at 20°C until cells reached the stationary phase. Before CAP treatment, cells were deposited in a liquid carrier, on a solid(like) surface or on a filter. A dielectric barrier discharge reactor generated helium-oxygen plasma, which was used to treat samples up to 10min. Although L. monocytogenes is more resistant to CAP treatment, similar trends in inactivation behavior as for S. Typhimurium are observed, with log reductions in the range [1.0-2.9] for S. Typhimurium and [0.2-2.2] for L. monocytogenes. For both microorganisms, cells grown planktonically are easily inactivated, as compared to surface colonies. More stressing growth conditions, due to cell immobilization, result in more resistant cells during CAP treatment. The main difference between the inactivation support systems is the absence or presence of a shoulder phase. For experiments in the liquid carrier, which exhibit a long shoulder, the plasma components need to diffuse and penetrate through the medium. This explains the higher efficacies of CAP treatment on cells deposited on a solid(like) surface or on a filter. This research demonstrates that the food structure influences the cell inactivation behavior and efficacy of CAP, and indicates that food intrinsic factors need to be accounted when designing plasma treatment.

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.

Effect of different modified atmospheric packaging (MAP) gaseous combinations on Campylobacter and the shelf-life of chilled poultry fillets.

Studies were undertaken to investigate the effect of different modified atmospheric packaging (MAP) gaseous combinations on Campylobacter and the natural microflora on poultry fillets. Skinless chicken fillets were stored in gaseous mixtures of 10%, 30%, 50%, 70% and 90% CO2 balanced with N2, 80:20% O2:N2 and 40:30:30% CO2:O2:N2 and control conditions (air) at 2 °C. Samples were analysed periodically for (previously inoculated) Campylobacter, total viable counts (TVC) (mesophiles), TVC (psychrophiles), Enterobacteriaceae, Pseudomonas and lactic acid bacteria (LAB) over 17 days of storage. The carbon dioxide solubility was determined by monitoring the changes in the headspace volume over time using a buoyancy technique and performing calculations based on volumetric measurements and the Henry's constant. Henry's constant was also used to estimate the oxygen solubility in the chicken fillets. The presence of O2 in the MAP gaseous mixtures increased the rate of Campylobacter decline on poultry fillets but in general the counts obtained in aerobic versus anaerobic packs were not significantly (P > 0.05) different. CO2 inhibited the growth of TVC, TEC, LAB and Pseudomonas but only at MAP gaseous combinations containing 50-90% CO2 where concentrations of up to 2000 ppm CO2 were recorded in the fillets after 5 days. Under these conditions a shelf-life in excess of 17 days at 2 °C was obtained. Although, dissolved O2, at levels of 33 ppm in 80:20% O2:N2 packs after 3 days, reduced Campylobacter, it also favoured the growth of the other microbes on the chicken. The optimum gaseous mixture for achieving the combined objectives of reducing Campylobacter and extending shelf was therefore 40:30:30 CO2:O2:N2, which achieved a shelf-life in excess of 14 days.

Assisted ultrasound applications for the production of safe foods.

Ultrasound requires high power and longer treatment times to inactivate micro-organisms when compared to ultrasound combined with other technologies. Previous reports have shown that the effectiveness of ultrasound as a decontamination technology can be increased by combining it with another treatment such as pressure, heat and antimicrobial solutions. Assisted ultrasound, the combination of ultrasound with another technology, is more energy efficient, and it has less impact on the food properties. In this review paper, the power ultrasound antimicrobial mechanisms of action, the antimicrobial effects of ultrasound in combination with other physical processes and antimicrobial solutions are comprehensively discussed. Furthermore, the present interest on using these technologies as alternative processing and decontamination methods is presented. Research outputs on the application of ultrasound combined with physical processes are showcased including applications of thermosonication, manosonication, manothermosonication and osmosonication. Antimicrobial efficacy, energy requirements and optimal operation conditions of the different assisted ultrasound technologies are critically discussed, and their impact on the food industry for future applications is presented. Overall, this review paper highlights the importance and recent developments of assisted ultrasound for enhancing food safety.

Assessing the microbial oxidative stress mechanism of ozone treatment through the responses of Escherichia coli mutants.

AIMS: To investigate the effect of the oxidative stress of ozone on the microbial inactivation, cell membrane integrity and permeability and morphology changes of Escherichia coli. METHODS AND RESULTS: Escherichia coli BW 25113 and its isogenic mutants in soxR, soxS, oxyR, rpoS and dnaK genes were treated with ozone at a concentration of 6 µg ml⁻¹ for a period up to 240 s. A significant effect of ozone exposure on microbial inactivation was observed. After ozonation, minor effects on the cell membrane integrity and permeability were observed, while scanning electron microscopy analysis showed slightly altered cell surface structure. CONCLUSIONS: The results of this study suggest that cell lysis was not the major mechanism of microbial inactivation. The deletion of oxidative stress-related genes resulted in increased susceptibility of E. coli cells to ozone treatment, implying that they play an important role for protection against the radicals produced by ozone. However, DnaK that has previously been shown to protect against oxidative stress did not protect against ozone treatment in this study. Furthermore, RpoS was important for the survival against ozone. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides important information about the role of oxidative stress in the responses of E. coli during ozonation.

Inactivation of Escherichia coli by ozone treatment of apple juice at different pH levels.

This research investigated the efficacy of gaseous ozone on the inactivation of Escherichia coli ATCC 25922 and NCTC 12900 strains in apple juice of a range of pH levels, using an ozone bubble column. The pH levels investigated were 3.0, 3.5, 4.0, 4.5 and 5.0. Apple juice inoculated with E. coli strains (10(6)CFU/mL) was treated with ozone gas at a flow rate of 0.12L/min and ozone concentration of 0.048 mg/min/mL for up to 18 min. Results show that inactivation kinetics of E. coli by ozone were affected by pH of the juice. The ozone treatment duration required for achieving a 5-log reduction was faster (4 min) at the lowest pH than at the highest pH (18 min) studied. The relationship between time required to achieve 5log reduction (t(5d)) and pH for both strains was described mathematically by two exponential equations. Ozone treatment appears to be an effective process for reducing bacteria in apple juice and the required applied treatment for producing a safe apple juice is dependant on its acidity level.

Resistance of Cronobacter sakazakii in reconstituted powdered infant formula during ultrasound at controlled temperatures: a quantitative approach on microbial responses.

Many of the documented outbreaks of Cronobacter sakazakii have been linked to infant formula. The aims of this work are to monitor the inactivation kinetics of C.sakazakii NCTC 08155 and ATCC 11467 and to determine quantitatively the effectiveness of ultrasonic treatments as an alternative to heat processing of reconstituted infant milk formula before feeding of infants at highest risk. Inactivation studies of C. sakazakii inoculated in reconstituted infant formula were performed at the combined conditions of temperature, i.e., 25 degrees C, 35 degrees C, 50 degrees C and amplitude, i.e., 24.4, 30.5, 42.7, 54.9, 61 microm and the kinetics were described by a range of inactivation models. The dependency of the specific inactivation rate with respect to the product of temperature and amplitude was described by a modified Bigelow type model. Ultrasound combined with temperature was efficient to reduce significantly the microbial levels of C. sakazakii. C. sakazakii strain NCTC 08155 was at the same range of temperature and amplitude resistance as strain ATCC 11467. Application of ultrasound is an alternative process for the production of safe reconstituted infant formula. This study contributes on the quantitative assessment of the resistance of C. sakazakii.

Safety and quality assessment during the ozonation of cloudy apple juice.

Traditionally, ozone processing within the food industry has focused on solid foods by either gaseous treatment or washing with ozonized water. However, with the FDA's approval of ozone as a direct additive to food, the potential for liquid applications has emerged. This study investigates the effect of ozone processing on microbial inactivation (E. coli ATCC 25922 and NCTC 12900) and quality parameters (color, phenolic content) of cloudy apple juice. Apple juice samples were ozonated at room temperature (20 ± 1.5 °C) with a generated ozone concentration of 0.048 mg O(3) at a constant flow rate of 0.12 L/min and treatment time of 0 to 10 min. E. coli inactivation kinetics in apple juice were described quantitatively by using the Shoulder log-linear and the Weibull model. Ozone treatment of E. coli in apple juice demonstrate that a desired 5 log reduction can be achieved within 5 min. Apple juice color (L*, a*, and b*) and total phenols were significantly affected by ozone concentration and treatment time.

Identification of non-linear microbial inactivation kinetics under dynamic conditions.

In this study dynamic microbial inactivation experiments are exploited for performing parameter identification of a non-linear microbial model. For that purpose microbial inactivation data are produced and a differential equation exhibiting a shoulder and a loglinear phase is employed. The derived parameter estimates from this method were used to perform predictions on an independent experimental set at fluctuating temperature. Joint confidence regions and asymptotic confidence intervals of the estimated parameters were compared with previous studies originating from parameter identification under isothermal conditions. The developed approach can provide more reliable estimates for realistic conditions compared to the usual or standard two step approach.

Inactivation model equations and their associated parameter values obtained under static acid stress conditions cannot be used directly for predicting inactivation under dynamic conditions.

Organic acids (e.g., lactic acid, acetic acid and citric acid) are popular preservatives. In this study, the Listeria innocua inactivation is investigated under dynamic conditions of pH and undissociated lactic acid ([LaH]). A combined primary (Weibull-type) and secondary model developed for the L. innocua inactivation under static conditions [Janssen, M., Geeraerd, A.H., Cappuyns, A., Garcia-Gonzalez, L., Schockaert, G., Van Houteghem, N., Vereecken, K.M., Debevere, J., Devlieghere, F., Van Impe, J.F., 2007. Individual and combined effects of pH and lactic acid concentration on L. innocua inactivation: development of a predictive model and assessment of experimental variability. Applied and Environmental Microbiology 73(5), 1601-1611] was applied to predict the microbial inactivation under dynamic conditions. Because of its non-autonomous character, two approaches were proposed for the application of the Weibull-type model to dynamic conditions. The results quantitatively indicated that the L. innocua cell population was able to develop an induced acid stress resistance under dynamic conditions of pH and [LaH]. From a modeling point of view, it needs to be stressed that (i) inactivation model equations and associated parameter values, derived under static conditions, may not be suitable for use as such under dynamic conditions, and (ii) non-autonomous dynamic models reveal additional technical intricacies in comparison with autonomous models.

Stress-adaptive responses by heat under the microscope of predictive microbiology.

AIMS: In previous studies the microbial kinetics of Escherichia coli K12 have been evaluated under static and dynamic conditions (Valdramidis et al. 2005, 2006). An acquired microbial thermotolerance following heating rates lower than 0.82 degrees C min(-1) for the studied micro-organism was observed. Quantification of this induced physiological phenomenon and incorporation, as a model building block, in a general microbial inactivation model is the main outcome of this work. METHODS AND RESULTS: The microbial inactivation rate observed (k(obs)) under time-varying temperature conditions is studied and expressed as a function of the heating rate (dT/ dt). Hereto, a model building block related to the microbial physiology (k(phys)) under stress conditions is developed. Evaluation of the performance of the developed mathematical approach depicts that physiological adaptation is an essential issue to be considered when modelling microbial inactivation. CONCLUSIONS: Consideration, at a mathematical level, of microbial responses resulting in physiological adaptations contribute to the reliable quantification of the safety risks during food processing. SIGNIFICANCE AND IMPACT OF THE STUDY: By taking into account the physiological adaptation, the microbiological evolution during heat processing can be accurately assessed, and overly conservative or fail dangerous food processing designs can be avoided.

GInaFiT, a freeware tool to assess non-log-linear microbial survivor curves.

This contribution focuses on the presentation of GInaFiT (Geeraerd and Van Impe Inactivation Model Fitting Tool), a freeware Add-in for Microsoft Excel aiming at bridging the gap between people developing predictive modelling approaches and end-users in the food industry not familiar with or not disposing over advanced non-linear regression analysis tools. More precisely, the tool is useful for testing nine different types of microbial survival models on user-specific experimental data relating the evolution of the microbial population with time. As such, the authors believe to cover all known survivor curve shapes for vegetative bacterial cells. The nine model types are: (i) classical log-linear curves, (ii) curves displaying a so-called shoulder before a log-linear decrease is apparent, (iii) curves displaying a so-called tail after a log-linear decrease, (iv) survival curves displaying both shoulder and tailing behaviour, (v) concave curves, (vi) convex curves, (vii) convex/concave curves followed by tailing, (viii) biphasic inactivation kinetics, and (ix) biphasic inactivation kinetics preceded by a shoulder. Next to the obtained parameter values, the following statistical measures are automatically reported: standard errors of the parameter values, the Sum of Squared Errors, the Mean Sum of Squared Errors and its Root, the R(2) and the adjusted R(2). The tool can help the end-user to communicate the performance of food preservation processes in terms of the number of log cycles of reduction rather than the classical D-value and is downloadable via the KULeuven/BioTeC-homepage at the topic "Downloads" (Version 1.4, Release date April 2005).

Development of predictive modelling approaches for surface temperature and associated microbiological inactivation during hot dry air decontamination.

This research deals with the development of predictive modelling approaches in the field of heat transfer and microbial inactivation. Upon making some backstage microbiological considerations, surface temperature predictions during hot dry air decontaminations are incorporated in a microbial inactivation model, in order to describe inactivation kinetics under realistic (time-varying) temperature conditions. In the present study, the following parts are presented. (i) First, a one-dimensional heat transfer model is developed taking into account exchanges by convection, radiation and evaporation. The model is subsequently validated on a laboratory setup and on a test rig, assuming no water activity changes. This test rig is developed for studying-at a later stage-surface pasteurisation treatment on food products with the use of hot dry air. (ii) Isothermal inactivation data of Escherichia coli K12 MG1655 have been collected and inactivation parameters are accurately estimated by using a primary and a secondary model in a global modelling approach. (iii) Microbiological considerations such as microbial growth effects during come-up times, initial temperature of inactivation, and heat resistance effects, based on experimental observations and on literature studies, are formulated in order to evaluate possible microbial effects arising under the dynamic temperature conditions modelled in step (i). (iv) Microbial inactivation simulations with the incorporation of surface temperature predictions are presented. (v) Finally, the level of the microbial decontamination in an example based on the design of an industrial installation is presented, outlining the importance of the combination of surface temperature and microbial inactivation modelling approaches.

Development of a novel approach for secondary modelling in predictive microbiology: incorporation of microbiological knowledge in black box polynomial modelling.

This research deals with the development of a novel secondary modelling procedure within the framework of predictive microbiology. The procedure consists of three steps: (i) careful formulation of the available microbiological information, both from literature and from the experimental case study at hand, (ii) translation of these requirements in mathematical terms under the form of partial derivatives throughout the complete interpolation region of the experimental design, and (iii) determination of parameter values with suitable optimisation techniques for a flexible black box modelling approach, e.g., a polynomial model or an artificial neural network model. As a vehicle for this procedure, the description of the maximum specific growth rate of Lactobacillus sakei in modified BHI-broth as influenced by suboptimal temperature, water activity, sodium lactate and dissolved carbon dioxide concentration is under study. The procedure results in a constrained polynomial model with excellent descriptive and interpolating features in comparison with an extended Ratkowsky-type model and classical polynomial model, by combining specific properties of both model types. The developed procedure is illustrated on the description of the lag phase as well. It is stressed how the confrontation with experimental data is very important to appreciate the descriptive and interpolating capacities of new or existing models, which is nowadays not always carefully performed. Alternatively, the first two steps of the novel procedure can be used as a tool to demonstrate clearly (possible) interpolative shortcomings of an existing model with straightforward spreadsheet calculations.