Fat and fibre interfere with the dramatic effect that nanoemulsified d-limonene has on the heat resistance of Listeria monocytogenes.

The application of d-limonene in form of nanoemulsion has been proved to reduce dramatically the thermal resistance of Listeria monocytogenes in culture media. The present research shows very promising results on the application in food products. The thermal resistance of L. monocytogenes was reduced 90 times when 0.5 mM nanoemulsified d-limonene was added to apple juice. This is the biggest reduction in the heat resistance of a microorganism caused by an antimicrobial described ever. However, no effect was found in carrot juice. A carrot juice system was prepared in an attempt to unravel which juice constituents were responsible for the lack of effect. When fat and fibre were not included in the carrot juice system formulation, the thermal resistance of L. monocytogenes was, again, dramatically reduced in presence of nanoemulsified d-limonene, so these components were shown to interfere with the effect. Once this interaction with food constituents becomes solved, the addition of nanoemulsified antimicrobials would allow to reduce greatly the intensity of the thermal treatments currently applied in the food processing industry.

Effect of thymol in heating and recovery media on the isothermal and non-isothermal heat resistance of Bacillus spores.

Members of the genus Bacillus include important food-borne pathogen and spoilage microorganisms for food industry. Essential oils are natural products extracted from herbs and spices, which can be used as natural preservatives in many foods because of their antibacterial, antifungal, antioxidant and anti-carcinogenic properties. The aim of this research was to explore the effect of the addition of different concentrations of thymol to the heating and recovery media on the thermal resistance of spores of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis at different temperatures. While the heat resistance was hardly reduced when thymol was present in the heating medium, the effect in the recovery medium was greater, reducing the D100 °C values down to one third for B. subtilis and B. cereus when 0.5 mM thymol was added. This effect was dose dependent and was also observed at other heating temperatures.

Inactivation kinetics of foodborne pathogens by UV-C radiation and its subsequent growth in fresh-cut kailan-hybrid broccoli.

The inactivation of Escherichia coli, S. Enteritidis and Listeria monocytogenes after UV-C radiation with 0, 2.5, 5, 7.5, 10 and 15 kJ UV-C m(-2) on fresh-cut kailan-hybrid broccoli was explored. Inactivation did not follow linear kinetics. Hence, it was modelled by using the Weibull distribution function, obtaining adjusted R(2) values higher than 94%, indicative of the accuracy of the model to the experimental data. The UV-C doses needed to reduce 1 log cycle the E. coli, S. Enteritidis and L. monocytogenes counts were 1.07, 0.02 and 9.26 kJ m(-2), respectively, being S. Enteritidis the most sensitive microorganism to UV-C radiation while L. monocytogenes was the most resistant. According to experimental data, UV-C doses higher than 2.5 kJ m(-2) did not achieve great microbial reductions. No differences in the growth behaviour of these microorganisms was observed in the treated samples stored under air conditions at 5, 10 and 15 °C, compared to the control. Conclusively, low UV-C doses are effective to reduce E. coli, S. Enteritidis and L. monocytogenes populations in fresh-cut kailan-hybrid broccoli keeping such counts stable during shelf life at 5-10 °C. The current study provides inactivation models for these foodborne pathogens that can be used in microbial risk assessment.

Biofilm busters: Exploring the antimicrobial and antibiofilm properties of essential oils against Salmonella Enteritidis.

This study delves into an exploration of the antimicrobial and antibiofilm properties of the essential oils (EOs) of cinnamon, garlic, and onion on Salmonella Enteritidis. Firstly, disc diffusion and minimum inhibitory concentration (MIC) techniques were employed to assess the antibacterial activity of the EOs. Additionally, the study explored the effect of these EOs on both initial cell attachment and 24 h-preformed biofilms. The crystal violet assay was implemented to evaluate biofilm biomass. The findings revealed that cinnamon EO exhibited the highest anti-biofilm activity. Furthermore, initial cell attachment inhibition at MIC ranged between 50 and 65% for the three oils, while inhibition rates on preformed structures were lower than 40% for all EOs at this MIC concentration. The study also found that the effects of these oils were dosage- and time-dependent (p < 0.05), thereby urging the adoption of these natural extracts as effective strategies for combating Salmonella biofilms.

Effect of the medium characteristics and the heating and cooling rates on the nonisothermal heat resistance of Bacillus sporothermodurans IC4 spores.

In recent years, highly thermo-resistant mesophilic spore-forming bacteria belonging to the species Bacillus sporothermodurans have caused non-sterility problems in industrial sterilization processes. The aim of this research was to evaluate the effect of the heating medium characteristics (pH and buffer/food) on the thermal inactivation of B. sporothermodurans spores when exposed to isothermal and non-isothermal heating and cooling treatments and the suitability of non-linear Weibull and Geeraaerd models to predict the survivors of these thermal treatments. Thermal treatments were carried out in pH 3, 5 and 7 McIlvaine buffer and in a courgette soup. Isothermal survival curves showed shoulders that were accurately characterized by means of both models. A clear effect of the pH of the heating medium was observed, decreasing the D120 value from pH 7 to pH 3 buffer down to one third. Differences in heat resistance were similar, regardless of the model used and were kept at all temperatures tested. The heat resistance in courgette soup was similar to that shown in pH 7 buffer. When the heat resistance values obtained under isothermal conditions were used to predict the survival in the non-isothermical experiments, the predictions estimated the experimental data quite accurately, both with Weibull and Geeraerd models.

Combined effect of nisin, carvacrol and a previous thermal treatment on the growth of Salmonella enteritidis and Salmonella senftenberg.

The aim of this study was to evaluate the combined effect of a previous mild heat treatment (15 min at 55 ) with the use of antimicrobials, nisin and carvacrol, on the growth of Salmonella enteritidis and Salmonella senftenberg. Natural antimicrobials, alone or combined with a previous mild heat treatment, affected the growth of these two serovars in Tryptone Soy Broth at 37 . Increasing concentrations of carvacrol had a significant effect on both growth rate and lag phase duration of both strains. The time to reach stationary phase was almost doubled in the case of S. enteritidis when a concentration of 0.77 mM in carvacrol was added. For S. senftenberg the effect was smaller. The effect of nisin and of heat, applied individually, was lower for both microorganisms. A combination of 1.2 µM nisin with 0.77 mM carvacrol significantly delayed the growth of heat treated cells, compared to the control without antimicrobials, showing additive effects.

Antibacterial and antibiofilm activity of essential oil of clove against Listeria monocytogenes and Salmonella Enteritidis.

The antibacterial and antibiofilm activity of essential oil of clove against Listeria monocytogenes and Salmonella Enteritidis were investigated. The chemical composition of the oil was characterized by gas chromatography-mass spectrometry. Stock solution of the essential oil of clove was prepared in 95% (v/v) ethanol (EOC). The antibacterial assays were performed by disk diffusion assay and minimal inhibitory concentration (MIC). The biomass of adhered cells and preformed biofilms after incubation with different concentrations of EOC was assessed by crystal violet. Eugenol was the major bioactive compound of clove essential oil, accounting for 78.85% of the total composition. The MIC values for L. monocytogenes and S. Enteritidis were 0.05 mg/ml and 0.1 mg/ml, respectively. The initial cell adhesion at MIC was inhibited by 61.8% for L. monocytogenes and 49.8% for S. Enteritidis. However, the effect of EOC was less marked on biofilm eradication than on cell adhesion. At MIC and within 1 hour of incubation with the EOC, the preformed biofilms were reduced by 30.2% and 20.3% for L. monocytogenes and S. Enteritidis, respectively. These results suggest that sanitizers based on clove essential oil could be a potential strategy to control biofilms in food-related environments.

Applications of Natural Antimicrobials in Food Packaging and Preservation.

In the food science field, the term "antimicrobial" basically refers to active substances of synthetic or natural origin, that are directly or indirectly present in a specific food, packaging material or food contact surface that affect the viability or the growth of microorganisms in that matrix [...].

Impact of Heating Rates on Alicyclobacillus acidoterrestris Heat Resistance under Non-Isothermal Treatments and Use of Mathematical Modelling to Optimize Orange Juice Processing.

Alicyclobacillus acidoterrestris is a spoilage microorganism responsible for relevant product and economic losses in the beverage and juice industry. Spores of this microorganism can survive industrial heat treatments and cause spoilage during posterior storage. Therefore, an effective design of processing treatments requires an accurate understanding of the heat resistance of this microorganism. Considering that industrial treatments are dynamic; this understanding must include how the heat resistance of the microorganism is affected by the heating rate during the heating and cooling phases. The main objective of this study was to establish the effect of heating rates and complex thermal treatments on the inactivation kinetics of A. acidoterrestris. Isothermal experiments between 90 and 105 °C were carried out in a Mastia thermoresistometer, as well as four different dynamic treatments. Although most of the inactivation takes place during the holding phase, our results indicate the relevance of the heating phase for the effectiveness of the treatment. The thermal resistance of A. acidoterrestris is affected by the heating rate during the heating phase. Specifically, higher heating rates resulted in an increased microbial inactivation with respect to the one predicted based on isothermal experiments. These results provide novel information regarding the heat response of this microorganism, which can be valuable for the design of effective heat treatments to improve product safety and stability. Moreover, it highlights the need to incorporate experimental data based on dynamic treatments in process design, as heating rates can have a very significant effect on the thermal resistance of microorganisms.

The Different Response to an Acid Shock of Two Salmonella Strains Marks Their Resistance to Thermal Treatments.

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.

Prediction of time to growth of Listeria monocytogenes using Monte Carlo simulation or regression analysis, influenced by sublethal heat and recovery conditions.

Stochastic models, including the variability in extent and probability of microbial growth, are useful for estimating the risk of foodborne illness (i.e. Nauta, 2000). Risk assessment typically has to embrace all sources of variability. In this paper, a stochastic approach to evaluate growth of heat damaged Listeria monocytogenes cells influenced by different stresses (pH and presence of eugenol) was performed, using an individual-based approach of growth through OD measurements. Both the lag phase duration and the "work to be done" (h(0) parameter) were derived from the growth curves obtained. From results obtained histograms of the lag phase were generated and distributions were fitted. Histograms showed a shift to longer lag phases and an increase in variability with high stress levels. Using the distributions fitted, predictions of time to unacceptable growth (10(2) cfu/g) of L. monocytogenes were established by Monte Carlo simulation and they were compared with results from statistical methods. It was evidenced that both methods (Monte Carlo and regression analysis) gave a good indication of the probability of a certain level of growth other than the average. Tornado plots were obtained to establish a sensitivity analysis of the influence of the conditions tested (heat, pH, eugenol) applied to the microorganism and their combinations.

Variability in the heat resistance of Listeria monocytogenes under dynamic conditions can be more relevant than that evidenced by isothermal treatments.

Heterogeneity in the response of microbial cells to environmental conditions is inherent to every biological system and can be very relevant for food safety, potentially being as important as intrinsic and extrinsic factors. However, previous studies analyzing variability in the microbial response to thermal treatments were limited to data obtained under isothermal conditions, whereas in the reality, environmental conditions are dynamic. In this article we analyse both empirically and through mathematical modelling the variability in the microbial response to thermal treatments under isothermal and dynamic conditions. Heat resistance was studied for four strains of Listeria monocytogenes (Scott A, CECT 4031, CECT 4032 and 12MOB052), in three different matrices (buffered peptone water, pH 7 Mcllvaine buffer and semi-skimmed milk). Under isothermal conditions, between-strain and between-media variability had no impact in the heat resistance, whereas it was very relevant for dynamic conditions. Therefore, the differences observed under dynamic conditions can be attributed to the variability in the ability for developing stress acclimation. The highest acclimation was observed in strain CECT 4031 (10-fold increase of the D-value), while the lowest acclimation was observed in strain CECT 4032 (50% increase of the D-value). Concerning the different media, acclimation was higher in buffered peptone water and semi-skimmed milk than in Mcllvaine buffer of pH 7.0. To the knowledge of the authors, this is the first research work that specifically analyses the variability of microbial adaptation processes that take place under dynamic conditions. It highlights that microbial heat resistance under dynamic conditions are sometimes determined by mechanisms that cannot be observed when cells are treated in isothermal conditions (e.g. acclimation) and can also be affected by variability. Consequently, empirical evidence on variability gathered under isothermal conditions should be extrapolated with care for dynamic conditions.

Garlic, Onion, and Cinnamon Essential Oil Anti-biofilms' Effect against Listeria monocytogenes.

Biofilms represent a serious problem for food industries due to their persistence in processing surfaces, from which they can cause food spoilage or, even worse, lead to foodborne diseases. Microorganisms immersed in biofilms are more resistant to biocides. The search for natural effective alternatives for the prevention and the control of biofilms has increased lately. The aim of this research was to test the antibacterial and the anti-biofilm activities of cinnamon, onion, and garlic essential oils against Listeria monocytogenes. The methodology highlighted first the effect of these essential oils on L. monocytogenes using disc diffusion and minimum inhibitory concentration (MIC) methods and then on initial cell attachment and six hours preformed biofilms. The inhibition of biofilms was assessed by crystal violet assay. Sulfides were the most abundant compounds present in onion and garlic essential oils, while cinnamaldehyde was predominant in cinnamon essential oil. MIC values were of 0.025 mg mL-1 for onion essential oil and 0.100 mg mL-1 for cinnamon and garlic. Onion essential oil inhibited initial cell attachment by 77% at 0.5 of the MIC dose, while at MIC, cinnamon and garlic essential oils inhibited the initial microbial adhesion completely. All three essential oils completely inhibited initial cell attachment when applied at 2 MIC. On the contrary, preformed biofilms were more resistant, and the inhibition rate ranged from 33% to 78%. In summary, this investigation revealed that the essential oils of garlic, onion, and cinnamon show an effective antibiofilm activity against L. monocytogenes and are promising natural antimicrobial alternatives for food processing facilities.

Limonene nanoemulsified with soya lecithin reduces the intensity of non-isothermal treatments for inactivation of Listeria monocytogenes.

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.

Tail or artefact? Illustration of the impact that uncertainty of the serial dilution and cell enumeration methods has on microbial inactivation.

The estimation of the concentration of microorganisms in a sample is crucial for food microbiology. For instance, it is essential for prevalence studies, challenge tests (growth and/or inactivation studies) or microbial risk assessment. The application of serial dilutions followed by viable counts in Petri dishes is probably the most extended experimental methodology for this purpose. However, this enumeration technique is also a source of uncertainty. In this article, the uncertainty of the serial dilution and viable count methodology related to the sampling error is analyzed, as well as the approximation of the microbial concentration by the number of colonies in a Petri dish. We analyze from a theoretical point of view (statistical analysis) the application of the binomial and Poisson models, demonstrating that the Poisson distribution increases the variance when used to model individual serial dilutions. On the other hand, the binomial model produces unbiased results. Therefore, the Poisson distribution is only applicable when it is a good approximation of the binomial distribution, so the use of the latter is recommended. The relevance of this uncertainty is demonstrated by Monte Carlo simulations of a generic microbial inactivation experiment, where the only source of uncertainty/variability considered is the one generated by serial plating and viable cell enumeration. Due to both the uncertainty of the methodology and the omission of zero-count plates because of the log-transformation, the simulated survival curve can have a tail. Therefore, this phenomenon, which is usually attributed to biological variability, can be to some extent an artefact of the experimental design and/or methodology.

Convergence of Bigelow and Arrhenius models over a wide range of heating temperatures.

The heat resistance of the bacterial spores of Moorella thermoacetica, Clostridium sporogenes, Geobacillus stearothermophilus and Bacillus coagulans was determined over a wide range of temperatures using the capillary method and thermoresistometer Mastia. The results showed that the two experimental methods gave similar heat resistance values excepted for Geobacillus stearothermophilus. The effect of temperature on thermal resistance was evaluated using the Arrhenius and Bigelow models. The fit of the heat sensitivity parameters of the Arrhenius and Bigelow models on the heat resistance parameter values obtained over a wide temperature range was equally good. Despite the apparent mathematical incompatibility of the two equations, it is recognized that they yield the same goodness of fit. This paper finds a mathematical reason for this convergence and explains why inside a temperature range of at least 100 °C, no significant difference in the quality of fit between these two models can be found.

Relevance of the Induced Stress Resistance When Identifying the Critical Microorganism for Microbial Risk Assessment.

Decisions regarding microbial risk assessment usually have to be carried out with incomplete information. This is due to the large number of possible scenarios and the lack of specific data for the problem considered. Consequently, risk assessment studies are based on the information obtained with a small number of bacterial cells which are considered the most heat resistant and/or more capable of multiplying during storage. The identification of the most resistant strains is usually based on D and z-values, normally estimated from isothermal experiments. This procedure omits the potential effect that the shape of the dynamic thermal profile applied in industry has on the microbial inactivation. One example of such effects is stress acclimation, which is related to a physiological response of the cells during sub-lethal treatments that increases their resistance. In this article, we use a recently published mathematical model to compare the development of thermal resistance for Escherichia coli K12 MG1655 and E. coli CECT 515 using inactivation data already published for these strains. Based only on the isothermal experiments, E. coli K12 MG1655 would be identified as more resistant to the thermal treatment than the CECT 515 strain in the 50-65°C temperature range. However, we conclude that stress acclimation is strain (and/or media)-dependent; the CECT 515 strain has a higher capacity for developing a stress acclimation than K12 MG1655 (300% increase of the D-value for CECT 515, 50% for K12 MG1655). It, thus, has the potential to be more resistant to the thermal treatment than the K12 MG1655 strain for some conditions allowing acclimation. A methodology is proposed to identify for which conditions this may be the case. After calibrating the model parameters representing acclimation using real experimental data, the applicability of the proposed approach is demonstrated using numerical simulations, showing how the CECT 515 strain can be more resistant for some heating profiles. Consequently, the most resistant bacterial strain to a dynamic heating profile should not be identified based only on isothermal experiments (D- and z-value). The relevance of stress acclimation for the treatment studied should also be evaluated.

Mathematical quantification of the induced stress resistance of microbial populations during non-isothermal stresses.

This contribution presents a mathematical model to describe non-isothermal microbial inactivation processes taking into account the acclimation of the microbial cell to thermal stress. The model extends the log-linear inactivation model including a variable and model parameters quantifying the induced thermal resistance. The model has been tested on cells of Escherichia coli against two families of non-isothermal profiles with different constant heating rates. One of the families was composed of monophasic profiles, consisting of a non-isothermal heating stage from 35 to 70°C; the other family was composed of biphasic profiles, consisting of a non-isothermal heating stage followed by a holding period at constant temperature of 57.5°C. Lower heating rates resulted in a higher thermal resistance of the bacterial population. This was reflected in a higher D-value. The parameter estimation was performed in two steps. Firstly, the D and z-values were estimated from the isothermal experiments. Next, the parameters describing the acclimation were estimated using one of the biphasic profiles. This set of parameters was able to describe the remaining experimental data. Finally, a methodology for the construction of diagrams illustrating the magnitude of the induced thermal resistance is presented. The methodology has been illustrated by building it for a biphasic temperature profile with a linear heating phase and a holding phase. This diagram provides a visualization of how the shape of the temperature profile (heating rate and holding temperature) affects the acclimation of the cell to the thermal stress. This diagram can be used for the design of inactivation treatments by industry taking into account the acclimation of the cell to the thermal stress.

Nanoemulsified D-Limonene Reduces the Heat Resistance of Salmonella Senftenberg over 50 Times.

Salmonella Senftenberg is a pathogen agent causative of foodborne disease and it is considered the most heat-resistant serovar within this genus. Food industries use heat treatment and chemical antimicrobials in order to eliminate this microorganism in food, but consumers prefer natural antimicrobials as essential oils and their components. This study evaluates the combined effect of thermal treatments and different concentrations of D-limonene nanoemulsion on the inactivation of Salmonella (S.) Senftenberg. The results showed an important effect of the nanoemulsified D-limonene on the heat resistance of S. Senftenberg. The δ50 °C value was reduced by 85%, 96% and 98% when 0.1, 0.5 and 1 mM of nanoemulsified D-limonene was added to the heating medium. The effect was kept along all the heating temperatures researched and the shape of the survival curves did not change with the addition of the antimicrobial. The results obtained in this research could be very useful for food industries for optimizing or improving heat treatments applied to food.

Effect of heating rate on highly heat-resistant spore-forming microorganisms.

Highly heat-resistant spore-forming Bacillus cause nonsterility problems in canned food and reduce the shelf life of many processed foods. The aim of this research was to evaluate the thermal inactivation of Bacillus sporothermodurans IIC65, Bacillus subtilis IC9, and Geobacillus stearothermophilus T26 under isothermal and nonisothermal conditions. The data obtained showed that B. sporothermodurans and B. subtilis were more heat resistant than G. stearothermophilus. The survival curves of B. sporothermodurans and B. subtilis showed shoulders, while the survival curves of G. stearothermophilus showed tails. Under nonisothermal treatment, at heating rates of 1 and 20 ℃/min, time needed to completely inactivate G. stearothermophilus was shorter than that required for B. sporothermodurans and B. subtilis. In complex heat treatments (heating-holding-cooling), the survival curves of B. sporothermodurans and B. subtilis showed the same activation shoulders than those obtained under isothermal treatments and the activation shoulders were again absent in the case of G. stearothermophilus. Predictions fitted quite well the data obtained for B. sporothermodurans. In contrast, the data for B. subtilis showed half a log cycle more survival than expected and in the case of G. stearothermophilus, the survival curve obtained showed much higher inactivation than expected.