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Cruciferous sprouts (e.g. broccoli and red radish) are rich source of health-promoting phytochemicals that are more concentrated than in the adult plant edible organs; however, these tiny microgreens need cold storage conditions to preserve their quality to reach the consumers in microbiologically safe conditions, maintaining their composition and acceptability. In this work, the microbiological status and phytochemical composition of broccoli and radish sprouts were evaluated at harvest (Day 0), and after seven and fourteen days of storage at 5 and 10 °C. Pathogenic microorganisms were absent during shelf-life; nevertheless, the slight growth of Enterobacteriaceae organisms, aerobic mesophilic and psychotropic bacteria, molds and yeasts was assessed. The storage temperature influenced the quality and content of bioactives in the sprouts, and for practical applications, storage at 5 °C is the most suitable option. Moreover, these fresh crucifers remain acceptable for consumers after 14 d storage period, being an interesting option for consuming fresh and naturally-functional foods.

Microwave processing can be a valid alternative to conventional heating for different types of products. It enables a more efficient heat transfer in the food matrix, resulting in higher quality products. However, for many food products a uniform temperature distribution is not possible because of heterogeneities in their physical properties and non-uniformtiy in the electric field pattern. Hence, the effectiveness of microwave inactivation treatments is influenced by both intrinsic (differences between cells) and extrinsic variability (non-uniform temperature). Interpreting the results of the process and considering its impact on microbial inactivation is essential to ensure effective and efficient processing. In this work, we quantified the variability in microbial inactivation attained in a microwave pasteurization treatment with a tunnel configuration at pilot-plant scale. The configuration of the equipment makes it impossible to measure the product temperature during treatment. For that reason, variability in microbial counts was measured using Biological Inactivation Indicators (BIIs) based on spherical particles of alginate inoculated with spores of Bacillus spp. The stability of the BIIs and the uncertainty associated to them was assessed using preliminary experiments in a thermoresistometer. Then, they were introduced in the food product to analyse the microbial inactivation in different points of the products during the microwave treatment. Experiments were made in a vegetable soup and a fish-based animal by-product (F-BP). The results show that the variation in the microbial counts was higher than expected based on the biological variability estimated in the thermoresistometer and the uncertainty of the BIIs. This is due to heterogeneities in the temperature field (measured using a thermographic camera), which were higher in the F-BP than in the vegetable soup. Therefore, for the process studied, extrinsic variability was more relevant than intrinsic variability. The methodology presented in this work can be a valid method to evaluate pasteurization treatments of foods processed by heating, providing valuable information of the microbial inactivation achieved. It can contribute to design microwave processes for different types of products and for product optimization.

The present work studies the effect of acid shock on cell viability of Bacillus cereus and Bacillus weihenstephanensis. Experiments were performed by means of flow cytometry (FCM) combined with fluorescent labelling. Propidium iodide, for membrane evaluation, and carboxyfluorescein diacetate, for esterase activity were used for differential staining of both strains. pH values of 3.4, 3.8 and 4.2 were selected on the basis of previous screening tests. FCM analysis showed to clearly discriminate between different populations, viable and damage cells, leading to successful assessment of acid shock effect on B. cereus and B. weihenstephanensis vegetative cell viability. Cellular viability decreased at lower pH values and longer exposure times, being B. weihenstephanensis more acid resistant than B. cereus. Reliability of FCM analysis for detection of B. cereus and B. weihenstephanensis vegetative cells was compared with classical viable plate count techniques (VPC). Although FCM and VPC data were not directly correlated and classical analytical methods show higher reliability, FCM analysis provides high-speed information at real time on damage at single cell level, whereas VPC only gives an indication of cells able to grow at a certain time. These data could help to establish more accurately the potential risk associated to foods or processing conditions where pH is involved, taking damage into consideration.

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.

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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.

Spore-forming bacteria with high heat resistance increasingly challenge industrial sterilisation processes in foods. To ensure stability of manufactured foods, generally worst case scenarios are applied often leading to unwanted over processing of foods. This means bigger requirements of energy and larger emissions of CO2 into the atmosphere. A heat-resistant spore-forming bacterium, Bacillus sporothermodurans, was tested for its ability to produce consistently highly heat-resistant spore crops to be used to more precisely determine the minimal thermal process that is compatible with microbial stability, thus avoiding the indicated overprocessing. Isothermal survival curves of B. sporothermodurans IC4 spores were determined in distilled water at temperatures up to 125 °C using the capillary tube technique and a thermoresistometer Mastia. B. sporothermodurans consistently produced crops of heat-resistant spores with kmax values at 121 °C up to 0.46 min−1. After consecutive sporulation cycles, the measured heat resistance of B. sporothermodurans was not reduced. Survival curves showed shoulders that were characterized by means of existing models (Geeraerd, Weibull) that were compared with the classical, log-linear one. Shoulders are a common phenomenon in heat-resistant spore-forming bacteria and may be related to the need of more stringent processing conditions. B. sporothermodurans spores were considered adequate to optimize sterilisation processes of low acid foods under a wide variety of conditions. Establishing precise heat treatments can be a way to guarantee food safety and stability while reducing the use of energy and contaminating emissions.

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 bactericidal action of thymol and cymene on two Bacillus cereus strains (INRA-AVTZ415 and INRA-AVZ421) was studied. Increasing concentrations of thymol (0.2–1.0 mmol l−1) or cymene (0.2–2.0 mmol l−1) showed higher bactericidal effect on exponential growth phase B. cereus cells suspended in HEPES buffer (pH 7), at 30°C. The two strains tested presented different sensitivity to these natural antimicrobials. When thymol and cymene were combined, it resulted in a greater bactericidal effect on B. cereus cells than when these compounds in the essential oil fraction of aromatic plants were applied separately. There was a synergistic effect of both natural antimicrobials on the viability of exponential growth phase B. cereus cells in pH 7 HEPES buffer, at 30°C. Survivor curves did not follow a first-order kinetics, which makes it difficult to establish processing conditions. Experimental data were modeled using a frequency distribution function (Weibull). This model guaranteed a good description of the experimental results and allowed predictions of time to a specific decrease in bacterial populations. This study indicates the potential use of thymol and cymene applied separately or simultaneously for preservation of minimally processed foods, as well as the validity of the Weibull distribution to describe the resulting nonlinear data and establish effective treatment conditions with these compounds.

The antibacterial action of thymol and cymene on two Bacillus cereus strains (INRA-AVTZ415 and INRA-AVZ421) was studied. Thymol and cymene alone showed higher antibacterial effect on the growth of B. cereus in BHI at 30 °C, both in lag and in exponential growth phases, with increasing added concentrations. However, the two strains tested showed different sensitivity to these natural compounds. When thymol and cymene were applied simultaneously, it resulted in a greater antibacterial effect than when the compounds were applied separately. The synergistic behaviour of both compounds was also shown for lag phase B. cereus INRA-AVZ421 cells in food substrates (carrot juice, chicken soup and pumpkin cream) at 30 °C, though a clear food-related effect was observed. This study indicates the potential use of thymol and cymene applied simultaneously for preservation of minimally processed foods.

The effect of irradiation with accelerated electrons on Bacillus cereus and Bacillus subtilis spore counts and spore heat resistance was investigated. Spore counts reduced approximately two log cycles for B. cereus and up to five log cycles for B. subtilis after a radiation dose of 7.6 kGy, with D10 values ranging from 1.5 to 3.8 kGy. Radiation had an important heat-sensitising effect on bacterial spores. After a 3.3 kGy treatment DT values were reduced more than three times for both B. cereus and B. subtilis spores. This study indicates the potential use of accelerated electrons, alone or followed by a heat treatment, for food preservation.

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.

The influence of mild heat pretreatment on the bactericidal action of nisin, carvacrol and thymol on stationary and exponential phase cells of two Bacillus cereus strains was studied. Carvacrol or thymol concentrations of 0.3 mmol/L had no bactericidal effect on unheated, and a minor effect on heated B. cereus cells either in stationary or exponential growth phases. Nisin (0.15 mg/mL) alone decreased the viable counts in all cases (unheated and heated cells in stationary and exponential phase) although the two strains tested showed different sensitivity to this natural antimicrobial between them. When carvacrol or thymol were combined with nisin, it resulted in a greater loss of viability of heated cells than when nisin was applied alone. There was a synergistic effect of nisin and both essential oils with a mild heat pretreatment on the viability of B. cereus cells. This study pointed out the potential use of nisin alone and in combination with carvacrol or thymol applied after a mild heat pretreatment for preservation of minimally processed 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.

Thermobacteriological studies using Bacillus stearotherrnophilus spores were carried out by heating the spores under isothermal and non-isothermal conditions followed by an isothermal period. Ex perimental data obtained after isothermal heating were analyzed using a two-step linear regression procedure and a one-step nonlinear regression method. Results obtained using both analytical tech niques were close, but the 90% interval of confidence for predictions was lower when the one-step nonlinear regression was used. These results indicated the convenience of using the one-step nonlin ear regression method to obtain thermal kinetic parameters for bacterial spores. Also, the parameters obtained by a non-isothermal program followed by an isothermal heating period were similar to those obtained after isothermal heating. Nevertheless, the joint confidence regions at 90% did not overlap, indicating that there are significant differences. The z values obtained under isothermal (7.0 °C) and non-isothermal (8.5 °C) conditions are close and characteristic for this microorganisms.

The effect of different concentrations of sodium chloride (NaCl) in the recovery medium on the apparent thermal resistance of Bacillus stearothermophilus spores (strain ATCC 12980) was studied. Spores were heated at different temperatures (115–125°C) and subcultured in medium with different NaCl concentrations [1, 2, 3 and 3.5% (w/v)]. The results indicate that increasing NaCl levels decreases the apparent heat resistance of the spores, over a temperature range of 115–125°C. No significant difference was found between 3 and 3.5% (w/v) NaCl although both reduced B. stearothermophilus heat resistance considerably. A second-order polynomial equation provided a good description of the spores’ D value, as influenced by the temperature and NaCl concentration. Predictions from the model compared well with published data, and therefore the model is suitable for predicting the effect of NaCl concentration on thermal inactivation of B. stearothermophilus.

The effect of recovering Bacillus stearothermophilus spores under anaerobic conditions on their apparent thermal resistance was studied. Spores were suspended in bidistilled water as a reference medium, heated at 115, 117, 119, 121, 123 and 125°C and recovered under aerobic and anaerobic conditions. D values (decimal reduction time) obtained following recovery under anaerobic conditions were lower than those obtained under aerobic conditions. Reductions of between 31 and 48% were found for all the temperatures studied. When spores were suspended in mushroom extract and recovered under anaerobic conditions the apparent heat resistance was much lower than that obtained under aerobic conditions (D121°C was 4.3 min and 1.7 min, under aerobic and anaerobic conditions, respectively). Heating the spores in mushroom extract and recovering the spores under anaerobic conditions produced an additive effect, decreasing the apparent heat resistance of the B. stearothermophilus spores.

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.

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