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.

A Novel Technique for Sterilization Using a Power Self-Regulated Single-Mode Microwave Cavity.

In this paper, a novel technique to achieve precise temperatures in food sterilization has been proposed. An accurate temperature profile is needed in order to reach a commitment between the total removal of pathogens inside the product and the preservation of nutritional and organoleptic characteristics. The minimal variation of the target temperature in the sample by means of a monitoring and control software platform, allowing temperature stabilization over 100 °C, is the main goal of this work. A cylindrical microwave oven, under pressure conditions and continuous control of the microwave supply power as function of the final temperature inside the sample, has been designed and developed with conditions of single-mode resonance. The uniform heating in the product is achieved by means of sample movement and the self-regulated power control using the measured temperature. Finally, for testing the sterilization of food with this technology, specific biological validation based on Bacillus cereus as a biosensor of heat inactivation has been incorporated as a distribution along the sample in the experimental process to measure the colony-forming units (CFUs) for different food samples (laboratory medium, soup, or fish-based animal by-products). The obtained results allow the validation of this new technology for food sterilization with precise control of the microwave system to ensure the uniform elimination of pathogens using high temperatures.

Microwave Treatment vs. Conventional Pasteurization: The Effect on Phytochemical and Microbiological Quality for Citrus-Maqui Beverages.

This study uncovered the impacts of microwave (MW) treatments compared to conventional pasteurization (TP) on the quality of functional citrus-maqui beverages, with added sucrose or stevia. The influence of these thermal treatments on the microbiological burden and phytochemical composition was determined by processing under two MW power levels (600 W and 800 W) and TP at 85 °C for 15 s for 60 days at room temperature (20 °C). The results indicated that, beyond the microbiological quality achieved in the juices treated by both MW and TP technology, there were no differences among the treatments regarding the stability of vitamin C, anthocyanin, and flavanone concentrations. However, anthocyanins were more stable in those beverages with sucrose added, rendering a better red color. Besides, all treatments ensured microbiological stability throughout the entire storage time. In conclusion, MW treatment could be considered as an alternative to TP, which ensures microbial safety, protecting functional compounds associated with health effects.

Development of a high-resolution melting-based approach for efficient differentiation among Bacillus cereus group isolates.

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.

A New Food Ingredient Rich in Bioaccessible (Poly)Phenols (and Glucosinolates) Obtained from Stabilized Broccoli Stalks.

Broccoli (Brassica oleracea var. italica) stalks account for up to 35% of the broccoli harvest remains with the concomitant generation of unused waste that needs recovery to contribute to the sustainability of the system. However, due to its phytochemical composition, rich in bioactive (poly)phenols and glucosinolates, as well as other nutrients, the development of valorization alternatives as a source of functional ingredients must be considered. In this situation, the present work aims to develop/obtain a new ingredient rich in bioactive compounds from broccoli, stabilizing them and reducing their degradation to further guarantee a high bioaccessibility, which has also been studied. The phytochemical profile of lyophilized and thermally treated (low-temperature and descending gradient temperature treatments), together with the digested materials (simulated static in vitro digestion) were analysed by HPLC-PDA-ESI-MSn and UHPLC-3Q-MS/MS. Broccoli stalks and co-products were featured by containing phenolic compounds (mainly hydroxycinnamic acid derivatives and glycosylated flavonols) and glucosinolates. The highest content of organosulfur compounds corresponding to the cores of the broccoli stalks treated by applying a drying descendant temperature gradient (aliphatic 18.05 g/kg dw and indolic 1.61 g/kg dw, on average, while the breakdown products were more abundant in the bark ongoing low temperature drying 11.29 g/kg dw, on average). On the other hand, for phenolics, feruloylquinic, and sinapoylquinic acid derivatives of complete broccoli stalk and bark, were more abundant when applying low-temperature drying (14.48 and 28.22 g/kg dw, on average, respectively), while higher concentrations were found in the core treated with decreasing temperature gradients (9.99 and 26.26 g/kg dw, on average, respectively). When analysing the bioaccessibility of these compounds, it was found that low-temperature stabilization of the core samples provided the material with the highest content of bioactives including antioxidant phenolics (13.6 and 33.9 g/kg dw of feruloylquinic and sinapoylquinic acids, on average, respectively) and sulforaphane (4.1 g/kg dw, on average). These processing options enabled us to obtain a new product or ingredient rich in bioactive and bioaccessible compounds based on broccoli stalks with the potential for antioxidant and anti-inflammatory capacities of interest.

Combined effect of lysozyme and nisin at different incubation temperature and mild heat treatment on the probability of time to growth of Bacillus cereus.

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.

Influence of microwave bag vs. conventional microwave cooking on phytochemicals of industrially and domestically processed broccoli.

Cooking vegetables in microwave bags is becoming a popular domestic cooking method, being relevant to know how this cooking method affects health-promoting phytochemicals of staples such as broccoli. The aim of this work was to study the effect of microwave bag cooking versus conventional microwaving on bioactive compound content (glucosinolates and hydroxycinnamic acid derivatives) and other quality parameters (such as antioxidant capacity, mineral content and microbial load) of broccoli florets. The influence of cooking time on bioactive compounds content was also evaluated. The study was carried out in two independent experiments; using intact broccoli and broccoli preprocessed in industry. Microwave bag cooked broccoli for 5 min (following label recommendation) showed higher glucosinolate content retention compared to conventional microwaving. Results suggest that volatilization could be an important phenomenon in reduction of glucosinolates during microwave cooking of broccoli florets. Glucosinolate profile did not change after cooking, regardless of cooking method applied. Furthermore, microwave bag cooked broccoli presented higher antioxidant capacity (by DPPH assay) than conventional microwaved broccoli. Hydroxycinnamic acid derivatives content was reduced in microwave cooking, regardless of method applied. Altogether, the use of microwave bags for microwaving is a novel method that retains main bioactive components of broccoli. This option is a fast, easy and considerably clean cooking option to fulfill modern consumer needs.

High Hydrostatic Pressure vs. Thermal Pasteurization: The Effect on the Bioactive Compound Profile of a Citrus Maqui Beverage.

The effects of high hydrostatic pressure (HHP) compared to thermal pasteurization (TP) were studied in healthy citrus-maqui beverages. The impact of the processing technologies on the microbiological and phytochemical profile was assessed by applying two HHP treatments at 450 and 600 MPa for 180 s and TP at 85 °C for 15 s. The shelf life under refrigeration (4 °C) and room temperature (20 °C) was monitored for 90 days. All treatments ensured microbiological stability at both storage temperatures. Aside from that, the physicochemical parameters were not significantly different after processing or throughout the storage period. Regarding color parameters, an increase in the reddish coloration was observed during storage for those beverages treated by HHP. In general, phenolic compounds were little affected by the processing technique, even when treatment under HHP was more stable than by TP during storage. On the other hand, vitamin C showed great degradation after processing under any condition. It can be concluded that HHP is an effective alternative to thermal treatments, achieving effective microbial inactivation and extending the shelf life of the juices by contributing to a better preservation of color and bioactive compounds.

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.

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.

Microbiological and process variability using biological indicators of inactivation (BIIs) based on Bacillus cereus spores of food and fish-based animal by-products to evaluate microwave heating in a pilot plant.

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.

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.

Combined effect of a nanoemulsion of D-limonene and nisin on Listeria monocytogenes growth and viability in culture media and foods.

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.

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.

Antimicrobial activity of nisin, thymol, carvacrol and cymene against growth of Candida lusitaniae.

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.

A PAS domain-containing regulator controls flagella-flagella interactions in Campylobacter jejuni.

The bipolar flagella of the foodborne bacterial pathogen Campylobacter jejuni confer motility, which is essential for virulence. The flagella of C. jejuni are post-translationally modified, but how this process is controlled is not well understood. In this work, we have identified a novel PAS-domain containing regulatory system, which modulates flagella-flagella interactions in C. jejuni. Inactivation of the cj1387c gene, encoding a YheO-like PAS6 domain linked to a helix-turn-helix domain, resulted in the generation of a tightly associated "cell-train" morphotype, where up to four cells were connected by their flagella. The morphotype was fully motile, resistant to vortexing, accompanied by increased autoagglutination, and was not observed in aflagellated cells. The Δcj1387c mutant displayed increased expression of the adjacent Cj1388 protein, which comprises of a single endoribonuclease L-PSP domain. Comparative genomics showed that cj1387c (yheO) orthologs in bacterial genomes are commonly linked to an adjacent cj1388 ortholog, with some bacteria, including C. jejuni, containing another cj1388-like gene (cj0327). Inactivation of the cj1388 and cj0327 genes resulted in decreased autoagglutination in Tween-20-supplemented media. The Δcj1388 and Δcj0327 mutants were also attenuated in a Galleria larvae-based infection model. Finally, substituting the sole cysteine in Cj1388 for serine prevented Cj1388 dimerization in non-reducing conditions, and resulted in decreased autoagglutination in the presence of Tween-20. We hypothesize that Cj1388 and Cj0327 modulate post-translational modification of the flagella through yet unidentified mechanisms, and propose naming Cj1387 the Campylobacter Flagella Interaction Regulator CfiR, and the Cj1388 and Cj0327 protein as CfiP and CfiQ, respectively.

Analysis of the heat-adaptive response of psychrotrophic Bacillus weihenstephanensis.

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.

Use of carvacrol and cymene to control growth and viability of Listeria monocytogenes cells and predictions of survivors using frequency distribution functions.

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.

Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579.

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 degrees C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50 degrees C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42 degrees 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.