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.

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.

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.

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.

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.

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.

High Heating Rates Affect Greatly the Inactivation Rate of Escherichia coli.

Heat resistance of microorganisms can be affected by different influencing factors. Although, the effect of heating rates has been scarcely explored by the scientific community, recent researches have unraveled its important effect on the thermal resistance of different species of vegetative bacteria. Typically heating rates described in the literature ranged from 1 to 20°C/min but the impact of much higher heating rates is unclear. The aim of this research was to explore the effect of different heating rates, such as those currently achieved in the heat exchangers used in the food industry, on the heat resistance of Escherichia coli. A pilot plant tubular heat exchanger and a thermoresistometer Mastia were used for this purpose. Results showed that fast heating rates had a deep impact on the thermal resistance of E. coli. Heating rates between 20 and 50°C/min were achieved in the heat exchanger, which were much slower than those around 20°C/s achieved in the thermoresistometer. In all cases, these high heating rates led to higher inactivation than expected: in the heat exchanger, for all the experiments performed, when the observed inactivation had reached about seven log cycles, the predictions estimated about 1 log cycle of inactivation; in the thermoresistometer these differences between observed and predicted values were even more than 10 times higher, from 4.07 log cycles observed to 0.34 predicted at a flow rate of 70 mL/min and a maximum heating rate of 14.7°C/s. A quantification of the impact of the heating rates on the level of inactivation achieved was established. These results point out the important effect that the heating rate has on the thermal resistance of E. coli, with high heating rates resulting in an additional sensitization to heat and therefore an effective food safety strategy in terms of food processing.

Quality and microbial safety evaluation of new isotonic beverages upon thermal treatments.

In the present study, it was evaluated how two different thermal treatments (Mild and Severe) may affect the anthocyanin content, antioxidant capacity (ABTS(+), DPPH, and FRAP), quality (CIELAB colour parameters), and microbiological safety of a new isotonic drink made of lemon and maqui berry over a commercial storage simulation using a shelf life of 56days at two preservation temperature (7°C and 37°C). Both heat treatments did not affect drastically the anthocyanins content and their percentage of retention. The antioxidant capacity, probably because of the short time, was also not affected. The CIELAB colour parameters were affected by the heat, although the isotonic drinks remained with attractive red colour during shelf life. From a microbiological point of view, the Mild heat treatment with storage at 7°C is the ideal for the preservation of microbial growth, being useful for keeping the quality and safety of beverages in commercial life.

Effect of Nisin and Thermal Treatments on the Heat Resistance of Clostridium sporogenes Spores.

The aim of this research was to evaluate the effect of thermal treatments (isothermal or nonisothermal) combined with nisin, a natural antimicrobial, on the survival and recovery of Clostridium sporogenes spores. The addition of nisin to the heating medium at concentrations up to 0.1 mg liter(-1) did not reduce the heat resistance of C. sporogenes. Without a thermal treatment, nisin added at concentrations up to 0.1 mg liter(-1) did not reduce the viable counts of C. sporogenes when added to the recovery medium, but inactivation of more than 4 log cycles was achieved after only 3 s at 100°C. At 100°C, the time needed to reduce viable counts by more than 3 log cycles was nine times shorter when 0.01 mg liter(-1) nisin was added to the recovery medium than without it. The heat resistance values calculated under isothermal conditions were used to predict the survival in the nonisothermal experiments, and the predicted values accurately fit the experimental data. The combination of nisin with a thermal treatment can help control C. sporogenes.

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.

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.