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Consumers’ demands for ready-to-eat, fresh-like products are on the rise during the last years. This type of products have minimal processing conditions that can enable the survival and replication of pathogenic microorganisms. Among them, Listeria monocytogenes is of special concern, due to its relatively high mortality rate and its ability to replicate under refrigeration conditions. Previous research works have shown that nanoemulsified essential oils in combination with thermal treatments are effective for inactivating L. monocytogenes. However, previous research works were limited to isothermal conditions, whereas actual processing conditions in industry are dynamic. Under dynamic conditions, microorganism can respond unexpectedly to the thermal stress (e.g. adaptation, acclimation or increased sensitivity). In this work, we assess the combination of nanoemulsified D-limonene with thermal treatments under isothermal and dynamic conditions. The nanoemulsion was prepared following an innovative methodology using soya lecithin, a natural compound as well as the essential oil. Under isothermal heating conditions, the addition of the antimicrobial enables a reduction of the treatment time by a factor of 25. For time-varying treatments, dynamic effects were relevant. Treatments with a high heating rate (20 °C/min) are more effective than those with a slow heating rate (1 °C/min). This investigation demonstrates that the addition of nanoemulsified D-limonene can greatly reduce the intensity of the thermal treatments currently applied in the food industry. Hence, it can improve the product quality without impacting its safety.

Microbial cells respond to sub-lethal stresses with several physiological changes to increase their chance of survival. These changes are of high relevance when combined treatments (hurdle technology) are applied during food production, as the cells surviving the first hurdle may have greater resistance to subsequent treatments than untreated cells. In this study, we analyzed if Salmonella develops increased resistance to thermal treatments after the application of an acid shock. We compared the heat resistance of acid-shocked (pH 4.5 achieved with citric acid) Salmonella cells with that of cells maintained at pH 7 (control cells). Thermal treatments were performed between 57.5 and 65°C. We observed a differential response between the two strains studied. Acid-shocked cells of Salmonella Senftenberg exhibited reduced heat resistance, e.g., for a treatment at 60.0°C and pH 7.0 the time required to reduce the population by 3 log cycles was lowered from 10.75 to 1.98min with respect to control cells. Salmonella Enteritidis showed a different response, with acid-shocked cells having similar resistance than untreated cells (the time required to reduce 3 log cycles at 60.0°C and pH 7.0 was 0.30min for control and 0.31min for acid-shock cells). Based on results by differential plating (with or without adding the maximum non-inhibitory concentration of NaCl to the recovery medium), we hypothesize that the differential response between strains can be associated to sub-lethal damage to the cell membrane of S. Senftenberg caused by the acid shock. These results provide evidence that different strains of the same species can respond differently to an acid shock and highlight the relevance of cross-resistances for microbial risk assessment.

The present work evaluated the antibacterial effect of nanoemulsions from natural compounds d-limonene and nisin against Listeria monocytogenes in tryptic soy broth growth medium, chicken broth, and vegetable cream. Experiments were performed by means of optical density growth curves and studies of viability in culture media and foods. Stability of nanoemulsions was evaluated by a Mastersizer 2000 equipment. Results showed greater effectiveness when applying d-limonene in form of nanoemulsion than when applying it directly, and when using both compounds together, both directly and in the form of nanoemulsion. Concentration of L. monocytogenes decreased at least in three log cycles in all the culture media and foods used within the first 90 min after the addition of the natural antimicrobials combined in form of nanoemulsion. Moreover, the growth of L. monocytogenes was inhibited with the combination of antimicrobials in the four weeks that the experiment lasted. Nanoemulsion technology would solve present problems of solubility and stability of oily antimicrobials in the food industry.

Yeasts are tolerant to acid pH values, are able to grow in anaerobic media and have minimum nutrition requirements. These capabilities enable them to survive and even grow in foods prepared from acid fruits or vegetables. Among yeasts, Candida is one of the genus most frequently isolated from fruit juices. Bacteriocins and essential oils from spices and aromatic herbs are an alternative to preservatives and other technological treatments and have the advantage that their natural origins do not lead to consumer rejection. However, before the food industry uses them on a large scale, it is necessary to know their effects on microorganisms. The objective of this research was to study the effect of different concentrations of nisin, thymol, carvacrol and cymene on the growth of Candida lusitaniae in pH 5 broth at 25 ℃, and their potential uses as food preservatives. The addition of nisin at the concentrations tested (up to 3 µmol L−1) did not affect the yeast growth. Thymol, carvacrol and cymene completely inhibited the yeast growth at concentrations over 1 mM for at least 21 days at 25 ℃. Below this concentration, inhibitions on yeast growth were observed at increasing concentrations. The effect of thymol was also proved in tomato juice. This study indicates the potential use of essential oils for preservation of minimally processed foods.

Strains belonging to Bacillus cereus Group include six different species, among which are Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus cereus sensu stricto, a causative agent of food poisoning. Sequence of the panC-housekeeping gene is used for B. cereus Group affiliation to seven major phylogenetic groups (I-VII) with different ecological niches and variations in thermal growth range and spore heat resistance of B. cereus Group microorganisms varies among phylogenetic groups. We assigned a selection of B. cereus sensu stricto strains related to food poisoning from the Spanish cultivar Collection (Valencia) to Group IV strains based on panC gene sequence. Thermal inactivation assays revealed variability of spore heat resistance within these Group IV strains. Adequate food sanitizing treatments therefore require fast and reliable identification of particular strains. In the present study, feasibility of genotyping via high-resolution melting (HRM) analysis was examined. HRM analysis of amplified polymorphic 16S-23 intergenic spacer region (ISR) region proved to be discriminatory for B. cereus sensu stricto strain typing, while two other polymorphic regions within the bacterial rRNA operon allowed differentiation between Bacillus species, demonstrating its applicability for discrimination on the species and strain level within B. cereus Group.

Stochastic models can be useful to predict the risk of foodborne illness. The presence of Bacillus cereus in liquid egg can pose a serious hazard to the food industry, since a mild heat treatment cannot guarantee its complete inactivation. However, most of the information available in the scientific literature is deterministic, including growth of B. cereus. In this paper, a stochastic approach to evaluate growth of B. cereus cells influenced by different stresses (presence of nisin and lysozyme separately or in combination) was performed, using an individual-based approach of growth through OD measurements. Lag phase duration was derived from the growth curves obtained. From results obtained, histograms of the lag phase were generated and distributions were fitted. Normal and Weibull distributions were ranked as the bestfit distributions in experiments performed at 25 °C. At 16 °C, lag values (obtained in presence of combinations of both antimicrobials) were also fitted by a Gamma distribution. Predictions were compared with growth curves obtained in liquid egg exposed to mild heat, nisin and/or lysozyme to assess their validity.

The microbial safety and stability of minimally processed foods are based on the application of combined preservative factors. Since microorganisms are able to develop adaptive networks to survive under conditions of stress, food safety may be affected, and therefore understanding of stress adaptive mechanisms plays a key role in designing safe food processing conditions. In the present study, the viability and the sublethal injury ofListeria monocytogenesexposed to moderate heat (55°C) and/or essential oil compounds (carvacrol and thymol, 0.3 mM) treatments were studied. Synergistic effects were obtained when combining mild heat (55°C) with one or both essential oil compounds, leading to inactivation kinetics values three to four times lower than when using heat alone. All the treatments applied caused some injury in the population. The injury levels ranged from around 20% of the surviving population under the mildest conditions to more than 99.99% under the most stringent conditions. Protein extracts of cells exposed to these treatments were analysed by two-dimensional gel electrophoresis. The results obtained revealed that stressed cells exhibited differential protein expression to control cells. The proteins upregulated under these stressing conditions were implicated, among other functions, in stress response, metabolism, and protein refolding.

A model for prediction of bacterial spore inactivation was developed. The influence of temperature, pH and NaCl on the heat resistance of Bacillus stearothermophilus spores was described using low-complexity, black box models based on artificial neural networks. Literature data were used to build and train the neural network, and new experimental data were used to evaluate it. The neural network models gave better predictions than the classical quadratic response surface model in all the experiments tried. When the neural networks were evaluated using new experimental data, also good predictions were obtained, providing fail-safe predictions of D values in all cases. The weights and biases values of neurons of the neural network that gave the best results are presented, so the reader can use the model for their own purposes. The use of this non-linear modelling technique makes it possible to describe more accurately interacting effects of environmental factors when compared with classical predictive microbial models.

The antibacterial action of carvacrol and cymene on two Listeria monocytogenes strains (STCC4031 and NCTN4032) was studied. Carvacrol or cymene showed inhibitory effect on the growth of L. monocytogenes during lag and exponential growth phases and was more evident with increasing concentrations in brain heart infusion broth at 30 degrees C. Carvacrol or cymene also decreased the survival of mid-exponential-growth-phase L. monocytogenes STCC4031 cells in potassium-N-2-hydroxy-ethylpiperazine-N-ethanesulfo nic acid, at 30 degrees C. The combination of carvacrol and cymene resulted in an increased antibacterial effect on the growth and a synergistic effect on the viability of L. monocytogenes compared with the natural compounds applied separately. The analysis of survival curves by the Weibull frequency distribution function allowed an accurate prediction of the level of inactivation achieved. Interestingly, an important bactericidal effect (4.7-log reduction) of low concentrations of both antimicrobials combined (0.75 mM) was observed on L. monocytogenes in carrot juice. This study indicates the potential use of carvacrol and cymene applied simultaneously for preservation of minimally processed foods.

The heat-adaptive response of the psychrotrophic spoilage bacterium Bacillus weihenstephanensis DSM11827 is described. It is demonstrated that vegetative cells of B. weihenstephanensis adapts to heat exposure at 47 degrees C by prior exposure to heat at the nonlethal temperature of 38 degrees C. For this adaptive response, protein synthesis is required and maximum adaptation was noted after 15 min to 2 h prior exposure at 38 degrees C. By using two-dimensional gel electrophoresis (2D-E), an overview of the heat-shock proteins (HSPs) of B. weihenstephanensis was obtained and it was shown that the production of 15 proteins increased upon exposure to 38 degrees C. In more detail, the use of specific antibodies revealed induction of the HSPs DnaK, DnaJ, GroEL, ClpC, ClpP and ClpX of B. weihenstephanensis. In addition, also pre-exposure to other stresses than heat, such as exposure to a high salt concentration, low pH, a high ethanol concentration or low temperature, resulted in development of increased heat tolerance of B. weihenstephanensis, and during these conditions, an increased production of some HSPs was noted. This phenomenon of cross-protection might be of substantial importance in relation to the design of safe minimal processing regimes.

To monitor the ability of the food-borne opportunistic pathogen Bacillus cereus to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42°C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50°C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42°C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of B. subtilis . These observations indicate that heat adaptation of B. cereus involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic B. cereus , which may result in increased survival in foods.

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.

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).

The isothermal survival curves of the heat resistant spores of Bacillus sporothermodurans IC4, in the range of 117–125 °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 °C and 0.2–0.4 °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.

The influence of pH and temperature on the bactericidal action of nisin and carvacrol on vegetative cells of different Bacillus cereus strains was studied. The five strains tested showed significant differences in sensitivity towards nisin, at pH 7.0 and 30 degrees C. Carvacrol concentrations of 0.3 mmol l(-1) had no effect on viability of B. cereus cells. When the same carvacrol concentration was combined with nisin, however, it resulted in a greater loss of viability of cells than when nisin was applied alone. The concentration of carvacrol played an important role on the bactericidal effect of nisin and, therefore, on the synergistic action of both compounds combined. At lower pH values (6.30 and 5.75), nisin was more active against B. cereus cells than at pH 7.0 at 30 degrees C, with a different sensitivity of the strains tested. The combined effect of nisin and carvacrol was found to be significantly different at pH 7.0 and 5.75. When the temperature was 8 degrees C, nisin was significantly less active against B. cereus IFR-NL 94-25 than at 30 degrees C, both at pH 7.0 and 6.30. At 8 degrees C, there was a significant increased effect of nisin at lower pH values. Also at this low temperature, a synergistic effect between nisin and carvacrol on B. cereus cells was observed at the pHs tested. This study indicates the potential of nisin and carvacrol at lower pHs to be used for preservation of minimally processed foods.