Toxin producing Bacillus cereus persist in ready-to-reheat spaghetti Bolognese mainly in vegetative state

The potential of Bacillus cereus to cause a diarrheal toxico-infection is related to its ability to perform de novo enterotoxin production in the small intestine. A prerequisite for this is presence of sufficient numbers of B. cereus that have survived gastro-intestinal passage. It is known that the percentage of survival is much smaller for vegetative cells in comparison to spores and it is therefore important to know the state in which B. cereus is ingested. The results of the current study performed on twelve B. cereus strains, comprising both diarrheal and emetic type, indicate that exposure via contaminated foods mainly concerns vegetative cells. Inoculated vegetative cells grew to high counts, with the growth dynamic depending on the storage temperature. At 28 °C growth to high counts resulted in spore formation, in general, after 1 day of storage. One strain was an exception, producing spores only after 16 days. At 12 °C obtained high counts did not result in spore formation for 11 of 12 tested strains after two weeks of storage. The highest counts and time to sporulation were different between strains, but no difference was observed on the group level of diarrheal and emetic strains. The spore counts were always lower than vegetative cell counts and occurred only when food was obviously sensory spoiled (visual and odor evaluation). Similar observations were made with food inoculated with B. cereus spores instead of vegetative cells. Although the prospect of consuming spores was found very weak, the numbers of vegetative B. cereus cells were high enough, without obvious sensory deviation, to survive in sufficient level to cause diarrheal toxico-infection.     

Persistence strategies of Bacillus cereus spores isolated from dairy silo tanks

Survival of Bacillus cereus spores of dairy silo tank origin was investigated under conditions simulating those in operational dairy silos. Twenty-three strains were selected to represent all B. cereus isolates (n = 457) with genotypes (RAPD-PCR) that frequently colonised the silo tanks of at least two of the sampled eight dairies. The spores were studied for survival when immersed in liquids used for cleaning-in-place (1.0% sodium hydroxide at pH 13.1, 75 °C; 0.9% nitric acid at pH 0.8, 65 °C), for adhesion onto nonliving surfaces at 4 °C and for germination and biofilm formation in milk. Four groups with different strategies for survival were identified. First, high survival (log 15 min kill ≤1.5) in the hot-alkaline wash liquid. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterised by slow germination in rich medium and well preserved viability when exposed to heating at 90 °C. Fourth, spores capable of germinating at 8 °C and possessing the cspA gene. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot-alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus.     

Addition of ethanol to supercritical carbon dioxide enhances the inactivation of bacterial spores in the biofilm of Bacillus cereus

Supercritical carbon dioxide (SC-CO₂) was used to inactivate Bacillus cereus spores inside biofilms, which were grown on stainless steel. SC-CO₂ treatment was tested using various conditions, such as pressure treatment (10–30 MPa), temperature (35–60 °C), and time (10–120 min). B. cereus vegetative cells in the biofilm were completely inactivated by treatment with SC-CO₂ at 10 MPa and at 35 °C for 5 min. However, SC-CO₂ alone did not inactivate spores in biofilm even after the treatment time was extended to 120 min. When ethanol was used as a cosolvent with SC-CO₂ in the SC-CO₂ treatment using only 2–10 ml of ethanol in 100 ml of SC-CO₂ vessel for 60–90 min of treatment time at 10 MPa and 60 °C, B. cereus spores in the biofilm were found to be completely inactivated in the colony-forming test. We also assessed the viability of SC-CO₂-treated bacterial spores and vegetative cells in the biofilm by staining with SYTO 9 and propidium iodide. The membrane integrity of the vegetative cells was completely lost, while the integrity of the membrane was still maintained in most spores. However, when SC-CO₂ along with ethanol was used, both vegetative cells and spores lost their membrane integrity, indicating that the use of ethanol as a cosolvent with SC-CO₂ is efficient in inactivating the bacterial spores in the biofilm.     

Effect of sodium hypochlorite (NaClO) sanitizer-induced stress on growth kinetics and morphological changes in Escherichia coli and Bacillus cereus spores

Sanitizer-induced stress on the growth kinetics of Escherichia coli and Bacillus cereus spores was investigated. Morphologies of B. cereus vegetative cells and spores were also compared. Nonpathogenic E. coli and pathogenic E. coli O157:H7 and B. cereus spores were treated with 100 ppm sodium hypochlorite in inoculated broth and stored at 13, 18, 24, 30, and 36°C. The lag time (LT) of stressed E. coli was extended more at low temperatures, regardless of pathogenicity. E. coli and B. cereus vegetative cells were sensitive to a sanitizer treatment with NaClO. Stressed strains grew more rapidly than unstressed strains. However, sanitizer stress did not significantly (p>0.05) affect the LT and specific growth rate (SGR) of B. cereus spores, regardless of storage temperature. Transmission electron microscopy analysis also revealed that stress induced using the NaClO sanitizer caused morphological damage to B. cereus vegetative cells, but not to B. cereus spores.     

Preconditioning of the stainless steel surface affects the adhesion of Bacillus cereus spores

The adhesion of Bacillus cereus on stainless steel, with and without prior conditioning of the surface (water, skimmed milk, and whole milk) was evaluated. Inocula consisting of a pool of spores of four different B. cereus strains isolated from the dairy industry, and spores of B. cereus ATCC 14579 were used. The pool of B. cereus spores adhered in all conditions evaluated. Higher adhesion of B. cereus spores (4.93 log cfu cm⁻²) was observed when using whole milk as conditioning matrix. However, without prior conditioning, lower adhesion was observed (3.01 log cfu cm⁻²) when the pool of B. cereus spores was inoculated on whole milk, suggesting the interaction between milk fat and microorganism on the stainless steel. The pool of B. cereus spores showed higher adhesion to the surface, possibly due to its greater hydrophobicity (66%) when compared with the B. cereus ATCC 14579 spores (47%).     

Development of oscillation method for reducing foodborne pathogens on lettuce and spinach

In this study, the efficacy of an oscillator for reducing the numbers of foodborne pathogens on lettuce and spinach was tested. A cocktail of three strains each of Salmonella typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes cells and of Bacillus cereus spores was inoculated onto lettuce and spinach leaves and followed by oscillation at 10 Hz and 20 Hz for up to 30 s. After treatment of inoculated lettuce leaf with an oscillator at 20 Hz for 30 s, 2.58, 2.82, 2.21 and 2.22 Log₁₀ CFU/g reductions were obtained with the cells of S. typhimurium, E. coli O157:H7 and L. monocytogenes and the spores of B. cereus, respectively. In the case of the oscillation treatment of spinach leaf, 2.89, 3.73, 2.46 and 2.25 Log₁₀ CFU/g reductions of those pathogens were achieved under the same condition. Statistically significant reductions were observed after oscillation treatment at 20 Hz for 5-10 s. The oscillation treatment at 10 Hz led to slightly less reductions of the pathogens tested as compared to the treatment at 20 Hz. In conclusion, the oscillation method developed shows to be highly efficacious in reducing foodborne pathogens on lettuce and spinach leaves.     

Transcriptional and functional responses of Escherichia coli O157:H7 growing in the lettuce rhizoplane

Lettuce and spinach are increasingly implicated in foodborne illness outbreaks due to contamination by Escherichia coli O157:H7. While this bacterium has been shown to colonize and survive on lettuce leaf surfaces, little is known about its interaction with the roots of growing lettuce plants. In these studies, a microarray analyses, mutant construction and confocal microscopy were used to gain an understanding of structure and function of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots. After three days of interaction with lettuce roots, 94 and 109 E. coli O157:H7 genes were significantly up- and down-regulated at least 1.5 fold, respectively. While genes involved in biofilm modulation (ycfR and ybiM) were significantly up-regulated, 40 of 109 (37%) of genes involved in protein synthesis were significantly repressed. E. coli O157:H7 was 2 logs less efficient in lettuce root colonization than was E. coli K12. We also unambiguously showed that a ΔycfR mutant of E. coli O157:H7 was unable to attach to or colonize lettuce roots. Taken together these results indicate that bacterial genes involved in attachment and biofilm formation are likely important for contamination of lettuce plants with Shiga toxin-producing E. coli strains.     

Role of mechanical vs. chemical action in the removal of adherent Bacillus spores during CIP procedures

This study was designed to evaluate the respective roles of mechanical and chemical effects on the removal of Bacillus spores during cleaning-in-place. This analysis was performed on 12 strains belonging to the Bacillus cereus group (B. cereus, Bacillus anthracis, Bacillus thuringiensis) or to less related Bacillus species (Bacillus pumilus, Bacillus licheniformis, Bacillus sporothermodurans, Bacillus subtilis). Adherent spores were subjected to rinsing-in-place (mechanical action) and cleaning-in-place (mechanical and chemical actions) procedures, the latter involving NaOH 0.5% at 60 °C. Results revealed that mechanical action alone only removed between 53 and 89% of the attached spores at a shear stress of 500 Pa. This resistance to shear was not related to spore surface properties. Conversely, in the presence of NaOH at a shear stress of 4 Pa, spores were readily detached, with between 80 and 99% of the adherent spores detached during CIP and the chemical action greatly depended on the strain. This finding suggests that chemical action plays the major role during CIP, whose efficacy is significantly governed by the spore surface chemistry.     

Novel approach to decontaminate food-packaging from pathogens in non-thermal and not chemical way: Chlorophyllin-based photosensitization

This study was focused on the possibility to inactivate main food pathogens, their spores and biofilms on the surface of packaging material polyolefine by Na-chlorophyllin (Na-Chl)-based photosensitization and to compare efficiency of this treatment with conventional antimicrobials.Data indicate that Bacillus cereus and Listeria monocytogenes were effectively inactivated (7 log) by Na-Chl (7.5 × 10⁻⁷ M)-based photosensitization in vitro and on the surface of packaging. Meanwhile to achieve adequate inactivation of thermo-resistant strains, spores or biofilms the higher Na-Chl concentration and longer illumination times had to be used. Comparison of different surface decontamination treatments reveal that photosensitization is much more effective against B. cereus and L. monocytogenes attached on the surface than washing with water or 200 ppm Na-hypochlorite.Our data support the idea that photosensitization may serve in the future for the development of human and environment friendly, non-thermal surface decontamination technique.     

Minimizing the level of Bacillus cereus spores in farm tank milk

In a year-long survey on 24 Dutch farms, Bacillus cereus spore concentrations were measured in farm tank milk (FTM), feces, bedding material, mixed grass and corn silage, and soil from the pasture. The aim of this study was to determine, in practice, factors affecting the concentration of B. cereus spores in FTM throughout the year. In addition, the results of the survey were used in combination with a previously published modeling study to determine requirements for a strategy to control B. cereus spore concentrations in FTM below the MSL of 3 log₁₀ spores/L. The B. cereus spore concentration in FTM was 1.2 ± 0.05 log₁₀ spores/L and in none of samples was the concentration above the MSL. The spore concentration in soil (4.9 ± 0.04 log₁₀ spores/g) was more than 100-fold higher than the concentration in feces (2.2 ± 0.05 log₁₀ spores/g), bedding material (2.8 ± 0.07 log₁₀ spores/g), and mixed silage (2.4 ± 0.07 log₁₀ spores/g). The spore concentration in FTM increased between July and September compared with the rest of the year (0.5 ± 0.02 log₁₀ spores/L difference). In this period, comparable increases of the concentrations in feces (0.4 ± 0.03 log₁₀ spores/g), bedding material (0.5 ± 0.05 log₁₀ spores/g), and mixed silage (0.4 ± 0.05 log₁₀ spores/g) were found. The increased B. cereus spore concentration in FTM was not related to the grazing of cows. Significant correlations were found between the spore concentrations in FTM and feces (r = 0.51) and in feces and mixed silage (r = 0.43) when the cows grazed. The increased concentrations during summer could be explained by an increased growth of B. cereus due to the higher temperatures. We concluded that year-round B. cereus spores were predominantly transmitted from feeds, via feces, to FTM. Farmers should take measures that minimize the transmission of spores via this route by ensuring low initial contamination levels in the feeds (<3 log₁₀ spores/g) and by preventing growth of B. cereus in the farm environment. In addition, because of the extremely high B. cereus spore concentrations in soil, the contamination of teats with soil needs to be prevented.     

Relevant factors affecting microbial surface decontamination by pulsed light

Pulsed Light (PL) uses intense flashes of white light rich in ultraviolet (UV) light for decontamination. A log-reduction higher than 5 was obtained in one flash and at fluences lower than 1.8 J/cm² on spores of a range of spore-forming bacteria, of vegetative cells of non-spore-forming bacteria and on yeasts spread on agar media. Vegetative cells were more sensitive than spores. The inactivation by PL of Bacillus subtilis, B. atrophaeus, B. cereus, Geobacillus stearothermophilus, and Aspergillus niger spores sprayed on polystyrene was similar. The inactivation by PL of B. subtilis and A. niger spores sprayed on glass was slightly lower than on polystyrene. No alteration of the spore structures was detected by scanning electron microscopy for both PL treated B. subtilis and A. niger spores. The inactivation of spores of B. subtilis, B. atrophaeus, B. cereus and B. pumilus by PL or by continuous UV-C at identical fluences was not different, and was much higher by PL for A. niger spores. The increase in the input voltage of the lamps (which also increases the UV-C %) resulted in a higher inactivation. There was no correlation between the resistance to heat and the resistance to PL. The relative effect of UV-C radiations and light thermal energy on PL inactivation was discussed.     

Inhibition of Bacillus cereus spore outgrowth and multiplication by chitosan

Bacillus cereus is an endospore-forming bacterium able to cause food-associated illness. Different treatment processes are used in the food industry to reduce the number of spores and thereby the potential of foodborne disease. Chitosan is a polysaccharide with well-documented antibacterial activity towards vegetative cells. The activity against bacterial spores, spore germination and subsequent outgrowth and growth (the latter two events hereafter denoted (out)growth), however, is poorly documented. By using six different chitosans with defined macromolecular properties, we evaluated the effect of chitosan on Bacillus cereus spore germination and (out)growth using optical density assays and a dipicolinic acid release assay. (Out)growth was inhibited by chitosan, but germination was not. The action of chitosan was found to be concentration-dependent and also closely related to weight average molecular weight (M_w) and fraction of acetylation (F_A) of the biopolymer. Chitosans of low acetylation (F_A = 0.01 or 0.16) inhibited (out)growth more effectively than higher acetylated chitosans (F_A = 0.48). For the F_A = 0.16 chitosans with medium (56.8 kDa) and higher M_w (98.3 kDa), a better (out)growth inhibition was observed compared to low M_w (10.6 kDa) chitosan. The same trend was not evident with chitosans of 0.48 acetylation, where the difference in activity between the low (19.6 kDa) and high M_w (163.0 kDa) chitosans was only minor. In a spore test concentration corresponding to 10²-10³ CFU/ml (spore numbers relevant to food), less chitosan was needed to suppress (out)growth compared to higher spore numbers (equivalent to 10⁸ CFU/ml), as expected. No major differences in chitosan susceptibility between three different strains of B. cereus were detected. Our results contribute to a better understanding of chitosan activity towards bacterial spore germination and (out)growth.     

Comparison of adhesion characteristics of common dairy sporeformers and their spores on unmodified and modified stainless steel contact surfaces

The attachment of aerobic spore-forming bacteria and their spores to the surfaces of dairy processing equipment leads to biofilm formation. Although sporeformers may differ in the degree of attachment, various surface modifications are being studied in order to develop a surface that is least vulnerable to attachment. This study was conducted to compare the extent of adhesion of spores and vegetative cells of the thermotolerant sporeformer Bacillus licheniformis and the high-heat-resistant sporeformers Geobacillus stearothermophilus and Bacillus sporothermodurans on both native and modified stainless steel surfaces. We studied the effect of contact surface and cell surface properties (including surface energy, surface hydrophobicity, cell surface hydrophobicity, and zeta potential) on the adhesion tendency of both types of sporeformers and their spores. Attachment to native and modified (Ni-P-polytetrafluoroethylene, Ni-P-PTFE) stainless steel surfaces was determined by allowing interaction between the respective contact surface and vegetative cells or spores for 1 h at ambient temperature. The hydrophobicity of vegetative cells and spores of aerobic spore-forming bacteria was determined using the hexadecane assay, and zeta potential was determined using the Zeta sizer Nano series instrument (Malvern Panalytical, Malvern, UK). The results indicated a higher adhesion tendency of spores over vegetative cells for both thermotolerant and high-heat-resistant sporeformers. On comparing the sporeformers, B. sporothermodurans demonstrated the highest adhesion tendency followed by G. stearothermophilus; B. licheniformis exhibited minimal attachment on both surfaces. The tendency to adhere varied with cell surface properties, decreasing with lower cell surface hydrophobicity and higher cell surface charge. On the other hand, modifying contact surface properties for higher surface hydrophobicity and lower surface energy decreased attachment.     

Efficiency of fractionated γ-irradiation doses to eliminate vegetative cells and spores of Bacillus cereus from raw rice

Efficacy of fractionated γ-irradiation to eliminate vegetative and spore forms of Bacillus cereus from raw rice was studied. Viable bacteria and spores count performed after irradiation treatment revealed that vegetative cells and spores (7.9 and 7.7 log CFU/g) of B. cereus in raw rice tolerated γ-irradiation up to 10 and 20 kGy, respectively and were eliminated at 15 and 25 kGy respectively on single treatment. Exactly 2 times of 5 kGy irradiation treatment eliminated all vegetative B. cereus (7.9 log CFU/g). A treatment with fractionated doses of γ-irradiation effectively eliminated vegetative bacteria but not spores of B. cereus. Field emission SEM images revealed the damage by γ-irradiation to the spore exosporium. This study suggests new approach of using fractionated doses of γ-irradiation to eliminate foodborne pathogens in food which are affected by high doses of γ-irradiation.     

Morphology and physico-chemical properties of Bacillus spores surrounded or not with an exosporium: Consequences on their ability to adhere to stainless steel

This study was designed to elucidate the influence of spore properties such as the presence of an exosporium, on their ability to adhere to materials. This analysis was performed on 17 strains belonging to the B. cereus group and to less related Bacillus species. We first demonstrated that spores of the B. cereus group, surrounded by an exosporium, differed in their morphological features such as exosporium size, number of appendages or hair-like nap length. We also found that the saccharidic composition of exosporium differed among strains, e.g. concerning a newly identified rhamnose derivative: the 2,4-O-dimethyl-rhamnose. Conversely, spores of distant Bacillus species shared morphological and physico-chemical properties with B. cereus spores. Some external features were also observed on these spores, such as a thin loose-fitting layer, whose nature is still to be determined, or a thick saccharidic layer (mainly composed of rhamnose and quinovose). The ability of spores to adhere to stainless steel varied among strains, those belonging to the B. cereus group generally being the most adherent. However, the presence of an exosporium is not sufficient to explain the ability of spores to adhere to inanimate surfaces. Indeed, when the 17 strains were compared, hydrophobicity and the number of appendages were the only significant adhesion parameters. Furthermore, the differences in spore adhesion observed within the B. cereus group were related to differences in the number of appendages, the exosporium length and to a lesser extent, the zeta potential.     

Inactivation of Bacillus cereus spores using a combined treatment of UV-TiO2 photocatalysis and high hydrostatic pressure

Bacillus cereus spores are resistant to high hydrostatic pressure (HHP) processing treatment. A combination of UV-TiO₂ photocatalysis (UVTP for 10 min) and two cycles of 600 MPa HHP treatment for 10 min for the first cycle and 1 min for the second cycle (UVTP-2HHP) at ambient temperature was applied to inactivate B. cereus spores inoculated on a solidified agar matrix (SAM) used as a model matrix. Two cycles of HHP treatment were used as a strategy for induction of spore germination, followed by inactivation. UVTP and 2 cycles of HHP resulted in a 5.0-log CFU/cm² spore reduction (initial spore count was 6.6 log CFU/cm²), including an approximate 0.8-log CFU/cm² reduction due to a synergistic effect. The inactivation mechanism of UVTP pretreatment was related to lipid peroxidation of the spore membrane based on the level of malondialdehyde (MDA) making spores susceptible to the HHP treatment. Flow cytometry and transmission electron microscopic (TEM) analyses showed severe physiological alteration and structural damage to spores after the combined treatment. UVTP and 2 cycles of HHP showed potential for effective inactivation of B. cereus to ensure food safety from B. cereus spores on food products.Practical applicationsInactivation of bacterial spores remains a technical challenge for HHP and other interventions because spores are highly resistant to high pressure. Pretreatment with UVTP followed by two cycles of HHP resulted in reduction in B. cereus spores due to a synergistic effect. This hurdle technology of UVTP and HHP can help food industry in ensuring food safety against the Bacillus spores.     

Biofilm formation of Salmonella Typhimurium on stainless steel and acrylic surfaces as affected by temperature and pH level

The effect of temperature (28, 37 and 42 °C) and pH (6 and 7) on the biofilm formation capability of Salmonella Typhimurium on stainless steel and acrylic was investigated. The rate of biofilm formation increased with increasing temperature and pH, while the number of attached cells after 240 h decreased with increasing temperature and was not different between pH 6 and 7. The surface hydrophobicity of bacterial cells was not significantly (p > 0.05) different among tested conditions. Electron-donating/accepting properties changed with pH and temperature, although these changes did not correlate with the ability to form biofilms under respective conditions. Attachment of S. Typhimurium showed a preference for stainless steel compared to acrylic surfaces under all conditions tested. The results suggest that salmonellae were less adherent to acrylic than to stainless steel surfaces; thus, acrylic-type surfaces should be considered for use in the food industry over stainless steel where applicable. The rate of biofilm formation increased at higher temperatures and pH levels within the tested ranges. Hurdle technology using lower temperatures reduced pH may help delay biofilm formation on food contact surfaces contaminated with S. Typhimurium.     

Combination of vaporized ethyl pyruvate and non-thermal atmospheric pressure plasma for the inactivation of bacteria on lettuce surfaces

The effect of vaporized ethyl pyruvate (EP) and atmospheric pressure plasma (APP) treatments on the inactivation of total viable counts of bacteria on fresh lettuce leaves samples after treatment and during storage (1 and 3 days) at 6 and 25 °C was studied. For this purpose, freshly grown B. cereus and Escherichia coli were inoculated on the lettuce leaves prior to treatments. Combination of EP (at a concentration of around 10 μL dm⁻³) with APP was more effective on inactivation of bacteria when compared to EP and APP treatments separately, reducing the total viable counts nearly 5 CFU cm⁻² compared to control. The use of EP and APP together led to around 2.5 more log (CFU cm⁻²) reductions when compared to the sole use of EP and APP separately. B. cereus cells were almost three times more susceptible to APP treatment than E. coli. Growth inhibition increased after storing the treated samples at 25 °C by around 2.5 and 1.5 logs for E. coli and B. cereus, respectively, compared to 6 °C. APP treatment time (30 and 60 s) and storage time (1 and 3 days) did not significantly affect the inactivation levels. E. coli was more effectively inactivated after EP + O₂ treatment followed by storage at 25 °C. The highest level of inactivation was noted as nearly 5 log reductions. Slight differences in the peak intensities of FTIR spectra of treated lettuce samples were observed compared to control, indicating slight modifications on the chemical structure on the lettuce leaves.Industrial relevanceThe microbial load influences the quality, safety, and shelf-life of fresh produce such as lettuce. Current decontamination techniques may cause some unwanted effects such as odor, discoloration, and decreased nutritional value. This study shows that the use of the APP-EP hurdle represents a promising strategy to improve the decontamination efficiency and hence, enhance the shelf-life of freshly cut vegetables. The data obtained contribute to a better understanding of APP-EP-induced effects on the quality and shelf-life of fresh-cut lettuce and provide a scientific basis for industrial implementation.     

Microbiological quality of fresh lettuce from organic and conventional production

Previously there was no available information on the levels of indicator bacteria and the prevalence of pathogens in fresh lettuce grown in organic and conventional farms in Spain. A total of 72 lettuce samples (18 farms for 4 repetitions each) for each type of the agriculture were examined in order to assess the bacteriological quality of the lettuces, in particular the prevalence of selected pathogens. The lettuce samples were analyzed for the presence of aerobic mesophilic, psychrotrophic microorganisms, yeasts and moulds, Enterobacteriaceae, mesophilic lactic acid bacteria, Pseudomonas spp. and presumptive Escherichia coli, Salmonella spp. and Listeria monocytogenes. The mean aerobic mesophilic counts (AM) were 6.35 ± 0.69 log₁₀ cfu g⁻¹ and 5.67 ± 0.80 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. The mean counts of psychrotrophic microorganisms were 5.82 ± 1.01 log₁₀ cfu g⁻¹ and 5.41 ± 0.92 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Yeasts and moulds (YM) mean counts were 4.74 ± 0.83 log₁₀ cfu g⁻¹ and 4.21 ± 0.96 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Lactic acid bacteria (LAB) were present in low numbers and the mean counts were 2.41 ± 1.10 log₁₀ cfu g⁻¹ and 1.99 ± 0.91 log₁₀ cfu g⁻¹ from organic and conventional lettuce, respectively. Pseudomonas spp. mean counts were 5.49 ± 1.37 log₁₀ cfu g⁻¹ and 4.98 ± 1.26 log₁₀ cfu g⁻¹ in organic and conventional lettuce, respectively. The mean counts for Enterobacteriaceae were 5.16 ± 1.01 log₁₀ cfu g⁻¹ and 3.80 ± 1.53 log₁₀ cfu g⁻¹ in organic and conventional lettuce, respectively. E. coli was detected in 22.2% (16 samples) of organic lettuce and in 12.5% (9 samples) of conventional lettuce. None of the lettuce samples was positive for E. coli O157:H7, L. monocytogenes and Salmonella spp. From the samples analyzed by principal component analysis (PCA) a pattern with two different groups (conventional and organic) can be observed, being the highest difference between both kinds of samples the Enterobacteriaceae count.     

The potential of atmospheric air cold plasma for control of bacterial contaminants relevant to cereal grain production

The aim of this work was to investigate the efficacy of dielectric barrier discharge atmospheric cold plasma (DBD ACP) against bacteria associated with grains quality and safety. ACP inactivation efficacy was tested against biofilms formed by different strains of E. coli, Bacillus and Lactobacillus in grain model media and against B. atrophaeus endospores either in grain media or attached on abiotic surfaces. Effects were dependent on bacterial strain, media composition and mode of ACP exposure. ACP treatment for 5 min reduced E. coli spp., B. subtilis and Lactobacillus spp. biofilms by > 3 log₁₀, whereas insignificant reductions were achieved for B. atrophaeus. ACP treatment of 5–20 min reduced B. atrophaeus spores in liquids by > 5 log₁₀. Treatment for 30 min reduced spores on hydrophobic surface by > 6 log₁₀, whereas maximum of 4.4 log reductions were achieved with spores attached to hydrophilic surface. Microscopy demonstrated that ACP caused significant damage to spores. In package ACP treatment has potential to inactivate grain contaminants in the form of biofilms, as well as spores and vegetative cells.Industrial relevanceThis study demonstrates that ACP technology is a promising tool for effective bio-decontamination which offers a wide range of possible applications including inactivation of microorganisms on cereal grains. However, due to the nature of the microbial contamination of grains and complex grain structures it may be necessary to optimise the potential for surface inactivation at several stages of grain processing and storage to enhance ACP efficacy against bacterial endospores.