Inactivation of Bacillus cereus by Na‐chlorophyllin‐based photosensitization on the surface of packaging

Aims: This study was focused on the possibility to inactivate food‐borne pathogen Bacillus cereus by Na‐chlorophyllin (Na‐Chl)‐based photosensitization in vitro and after attachment to the surface of packaging material. Methods and Results: Bacillus cereus in vitro or attached to the packaging was incubated with Na‐Chl (7·5 × 10^?8 to 7·5 × 10^?5 mol l^?1) for 2–60 min in phosphate buffer saline. Photosensitization was performed by illuminating cells under a light with a λ of 400 nm and an energy density of 20 mW cm^?2. The illumination time varied 0–5 min and subsequently the total energy dose was 0–6 J cm^?2. The results show that B. cereus vegetative cells in vitro or attached to the surface of packaging after incubation with 7·5 × 10^?7 mol l^?1 Na‐Chl and following illumination were inactivated by 7 log. The photoinactivation of B. cereus spores in vitro by 4 log required higher (7·5 × 10^?6 mol l^?1) Na‐Chl concentration. Decontamination of packaging material from attached spores by photosensitization reached 5 log at 7·5 × 10^?5 mol l^?1 Na‐Chl concentration. Comparative analysis of different packaging decontamination treatments indicates that washing with water can diminish pathogen population on the surface by <1 log, 100 ppm Na‐hypochlorite reduces the pathogens about 1·7 log and 200 ppm Na‐hypochlorite by 2·2 log. Meanwhile, Na‐Chl‐based photosensitization reduces bacteria on the surface by 4·2 orders of magnitude. Conclusions: Food‐borne pathogen B. cereus could be effectively inactivated (7 log) by Na‐Chl‐based photosensitization in vitro and on the surface of packaging material. Spores are more resistant than vegetative cells to photosensitization‐based inactivation. Comparison of different surface decontamination treatments indicates that Na‐Chl‐based photosensitization is much more effective antibacterial tool than washing with water or 200 ppm Na‐hypochlorite. Significance and Impact of the Study: Our data support the idea that Na‐Chl‐based photosensitization has great potential for future application as an environment‐friendly, nonthermal surface decontamination technique.     

The preparation,germination properties and stability of superdormant spores of Bacillus cereus

Aims: To determine yields, germination and stability of superdormant Bacillus cereus spores. Methods and Results: Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or l ‐alanine in 2–5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca‐DPA, dodecylamine, l ‐alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat‐activated spores were germinated. Superdormant spores stored at 4°C slowly recovered some germination capacity, but recovery was slowed significantly at ?20°C and ?80°C. Conclusions: Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores. Significance: Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.     

Inactivation of food pathogen Bacillus cereus by photosensitization in vitro and on the surface of packaging material

Aims: The study was focused on the possibility to inactivate food pathogen Bacillus cereus by 5‐aminolevulinic acid (ALA) – based photosensitization in vitro and after adhesion on the surface of packaging material. Methods and Results: Bacillus cereus was incubated with ALA (3–7·5 mmol l^?1) for 5–60 min in different environment (PBS, packaging material and wheat grains) and afterwards illuminated with visible light. The light source used for illumination emitted light at λ = 400 nm with energy density at the position of the cells, 20 mW cm^?2. The illumination time varied from 0 to 20 min, and subsequently a total energy dose was between 0 and 24 J cm^?2. The obtained results indicate that B. cereus after the incubation with 3–7·5 mmol l^?1 ALA produces suitable amounts of endogenous photosensitizers. Following illumination, micro‐organism inactivated even by 6·3 log. The inactivation of B. cereus after adhesion on the surface of food packaging by photosensitization reached 4 log. It is important to note that spores of B. cereus were susceptible to this treatment as well; 3·7‐log inactivation in vitro and 2·7‐log inactivation on the surface of packaging material were achieved at certain experimental conditions. Conclusions: Vegetative cells and spores of Gram‐positive food pathogen B. cereus were effectively inactivated by ALA‐based photosensitization in vitro. Moreover, the significant inactivation of B. cereus adhered on the surface of packaging material was observed. It was shown that photosensitization‐based inactivation of B. cereus depended on the total light dose (illumination time) as well as on the amount of endogenous porphyrins (initial ALA concentration, time of incubation with ALA). Significance and Impact of the Study: Our previous data, as well as the one obtained in this study, support the idea that photosensitization with its high selectivity, antimicrobial efficiency and nonthermal nature could serve in the future for the development of completely safe, nonthermal surface decontamination and food preservation techniques.     

Potential application of the nisin Z preparation of Lactococcus lactis W8 in preservation of milk

Aims: The aim of the study is to evaluate the effectiveness of the preparation of nisin Z from Lactococcus lactis W8‐fermented milk in controlling the growth of spoilage bacteria in pasteurized milk. Methods and Results: Spoilage bacteria isolated from pasteurized milk at 8 and 15°C were identified as Enterococcus italicus, Enterococcus mundtii, Enterococcus faecalis, Bacillus thuringiensis, Bacillus cereus, Lactobacillus paracasei, Acinetobacter sp., Pseudomonas fluorescens and Enterobacter aerogenes. These bacteria were found to have the ability to survive pasteurization temperature. Except Enterobacter aerogenes, the spoilage bacteria were sensitive to the nisin Z preparation of the L. lactis W8. Addition of the nisin Z preparation to either the skim milk or fat milk inoculated with each of the spoilage bacteria reduced the initial counts (about 5 log CFU ml^?1) to an undetectable level within 8–20 h. The nisin Z preparation extended the shelf life of milk to 2 months under refrigeration. Conclusions: The nisin Z preparation from L. lactis W8‐fermented milk was found to be effective as a backup preservative to counteract postpasteurization contamination in milk. Significance and Impact of the Study: A rapid inhibition of spoilage bacteria in pasteurized skim and fat milk with the nisin Z preparation of L. lactis W8 is more significant in comparison with the commercially available nisin (nisin A). The nisin Z preparation can be used instead of commercial nisin, which is not effective in fat milk.     

Isolation and characterization of phages infecting Bacillus cereus

Aim: To isolate and characterize bacteriophages (phages) that infect the foodborne pathogen Bacillus cereus. Methods and Results: Two phages were isolated from soil based on their ability to form plaques on four indicator hosts including Bacillus thuringiensis subsp. israelensis, and three isolates of B. cereus. The purified phages were characterized by morphology, host range, single‐step growth curves and restriction enzyme digestion profiles. The phages appeared to be of the Myoviridae family based on their structure in electron micrographs. The phages lysed bacteria of several species, produced average burst sizes of 322 and 300 phages per infected cell, and both had genomes over 90 kb. The phages were chloroform‐resistant and stable at 4°C. They reduced the concentration of B. cereus in mashed potatoes by >6 log₁₀ CFU ml^?1 within 24 h at room temperature, when applied at a high concentration. Conclusions: The relatively narrow host range within B. cereus might mean that these phages need to be used as part of a ‘cocktail’ of phages for biocontrol, but their efficacy for the control of their host in food was demonstrated. Significance and Impact of the Study: This is the first report of biocontrol by phages of B. cereus in food.     

Prevalence, phenotypic traits and molecular characterization of emetic toxin-producing Bacillus cereus strains isolated from human stools in Korea

Aims: To investigate the prevalence and genotypic/phenotypic characters of emetic toxin‐producing Bacillus cereus strains isolated from sporadic food poisoning cases in Korea. Methods and Results: The prevalence of emetic B. cereus was determined in 56 899 stool samples from sporadic food poisoning cases in Korea between 2004 and 2006. We assessed toxin profiles, phenotypic traits and antibiotic resistance. The molecular subtyping was ascertained using an automated repetitive sequence‐based PCR (rep‐PCR) system, DiversiLab?, with these emetic strains isolated from sporadic food poisoning cases and other emetic strains isolated from an outbreak and food samples. Emetic B. cereus was present in 0·012% of sporadic food poisoning cases. The prevalence of nheABC, hblCDA, cytK and entFM enterotoxin genes among emetic strains was 100, 14·3, 14·3 and 100%, respectively. Most emetic strains were negative for salicin hydrolysis (100%), starch fermentation (85·7%) and haemolysis (85·7%). One emetic isolate, VK7, exhibited several unique traits, such as harbouring the hbl gene and ability to hydrolyse starch. All isolated strains were highly resistant to β‐lactam antibiotics. All emetic strains except VK7 exhibited an identical rep‐PCR banding pattern, while nonemetic strains were classified into various pulsotypes. Conclusions: Most emetic strains except one isolate exhibited similar genotypic/phenotypic traits and subtyping pattern. Automatic rep‐PCR (DiversiLab?) may be used to discriminate emetic strains from nonemetic strains, although we could not distinguish between most emetic strains using that. Significance and Impact of the Study: Result of this study may contribute an extended database on the prevalence and toxigenic traits of emetic B. cereus strains isolated from Korea.     

Optimization of Inactivation of Endospores of <Emphasis Type="Italic">Bacillus cereus</Emphasis> in Milk by Surfactin and Fengycin Using a Response Surface Method

In this paper, the sterilization of surfactin and fengycin to Bacillus cereus was observed, and the optimization of the inactivation of surfactin and fengycin to spores of B. cereus by a response surface methodology was studied. Results showed that surfactin and fengycin had high sterilization to B. cereus, whose minimal inhibitory concentration was 31.25 μM and 62.5 μM respectively. The optimization result indicated that spores of B. cereus could be inactivated by two orders of magnitude when the temperature was 20.41°C, the action time was 21.13 h, and the concentration (surfactin/fengycin molar ratio 1:1) was 54.20 μM.     

Efficient decomposition of shrimp shell waste using <Emphasis Type="Italic">Bacillus cereus</Emphasis> and <Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis>

Two bacterial cultures were isolated and tested for degradation of shrimp shell waste. According to morphological examination, physiological tests, and applied molecular techniques, isolates were identified as Bacillus cereus and Exiguobacterium acetylicum. Both strains were cultivated separately in flasks with 100 mL of shrimp shell waste broth (3% of washed, dried and ground shrimp shell waste in tap water, pH 7.0) at 37°C. At determined periods of time, deproteinization and demineralization of residuals were measured. Fermentation of 3% shell waste with B. cereus indicated 97.1% deproteinization and 95% demineralization. For E. acetylicum, the level of deproteinization and demineralization was 92.8 and 92%, respectively. Protein content was reduced from 18.7 to 5.3% with B. cereus and to 7.3% with E. acetylicum. No additional supplements were used during the fermentation of shell waste. B. cereus strain showed higher efficacy in decomposition of shell waste and was used for large-scale fermentation in 12 L of 10% shrimp shell waste broth. Incubation of bacteria with shell waste during 14 days at 37°C resulted in 78.6% deproteinization and 73% demineralization. High activity of isolated cultures in decomposition of shrimp shell waste suggests broad potential for application of these bacteria in environmentally friendly approaches to chitin extraction from chitin-rich wastes.     

Isolation of glucosidase and phospholipase positive Bacillus circulans on ALOA medium

Aim: To report the growth of glucosidase and phospholipase positive bacteria on agar Listeria according to Ottaviani and Agosti (ALOA) different from Listeria monocytogenes, Listeria ivanovii and Bacillus cereus. Methods and Results: Raw water‐buffalo milk was analysed according to EN ISO 11290. Streaking of Fraser broth on ALOA resulted in green colonies with an opaque halo after 48 h at 30°C. Colonies were transferred onto Tryptone soya yeast extract agar and showed cultural characteristics atypical for L. monocytogenes. Results of confirmation tests according to EN ISO 11290 method: negative haemolysis test, weak positive camp test in correspondence with Staphylococcus aureus, no fermentation of rhamnose, fermentation of xylose. Gram staining showed tapered, curved, Gram‐positive rods with subterminal to terminal ellipsoidal spores, 0·5–0·7 μm diameter 4–5 μm. API 50CH CHB kit (99·9% percentage of identification) and the sequence of the 833 bp PCR product (portion of 16S rRNA, generic primers 1492‐r; p27‐f) showed 97% identity with Bacillus circulans ATCC 4513 (GenBank AY724690 ). Conclusions: Some B. circulans strains can grow on ALOA, according to ISO 11290, confirmation tests readily differentiate B. circulans from L. monocytogenes. Significance and Impact of the Study: The different morphology of the colonies must be kept in mind to select true L. monocytogenes for confirmation test and to avoid overestimation of L. monocytogenes count.     

Nisin-activated hydrophobic and hydrophilic surfaces: assessment of peptide adsorption and antibacterial activity against some food pathogens

An effective antimicrobial packaging or food contact surface should be able to kill or inhibit micro-organisms that cause food-borne illnesses. Setting up such systems, by nisin adsorption on hydrophilic and hydrophobic surfaces, is still a matter of debate. For this purpose, nisin was adsorbed on two types of low-density polyethylene: the hydrophobic native film and the hydrophilic acrylic acid-treated surface. The antibacterial activity was compared for those two films and it was highly dependent on the nature of the surface and the nisin-adsorbed amount. The hydrophilic surfaces presented higher antibacterial activity and higher amount of nisin than the hydrophobic surfaces. The effectiveness of the activated surfaces was assessed against Listeria innocua and the food pathogens Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus. S. aureus was more sensitive than the three other test bacteria toward both nisin-functionalized films. Simulation tests to mimic refrigerated temperature showed that the films were effective at 20 and 4 °C with no significant difference between the two temperatures after 30 min of exposure to culture media.     

Competitive exclusion as a mode of action of a novel Bacillus cereus aquaculture biological agent

Aims: To determine the contribution of potential modes of action of a Bacillus cereus aquaculture biological control agent in inhibition of the fish pathogen, Aeromonas hydrophila. Methods and Results: When B. cereus was tested in plate well inhibition studies, no production of antimicrobial compounds was detected. Bacillus cereus had a high growth rate (0·96 h^?1), whereas Aer. hydrophila concentration decreased by c. 70% in co‐culture experiments. In nutrient limitation studies, B. cereus had a significantly higher growth rate when cultured under glucose (P < 0·05) and iron (P < 0·01) limitation in comparison with Aer. hydrophila. Bacillus cereus glucose (0·30 g l^?1 h^?1) and iron (0·60 mg l^?1 h^?1) uptake rates were also significantly higher (P < 0·01) than the Aer. hydrophila glucose (0·14 g l^?1 h^?1) and iron (0·43 mg l^?1 h^?1) uptake rates. Iron uptake was facilitated by siderophore production shown in time profile studies where relative siderophore production was c. 60% through the late exponential and sporulation phases. Conclusions: Competitive exclusion by higher growth rate, competition for organic carbon and iron, facilitated by siderophore production, could be identified as mechanisms of pathogen growth inhibition by B. cereus. Significance and Impact of the Study: This study is the first elucidation of the mechanism of action of our novel B. cereus biological agent in growth attenuation of pathogenic Aer. hydrophila. This study enhances the application knowledge and attractiveness for adoption of B. cereus NRRL 100132 for exploitation in aquaculture.     

Kinetic resolution of N‐acetyl‐DL‐alanine methyl ester using immobilized Escherichia coli cells bearing recombinant esterase from Bacillus cereus

D‐alanine is widely used in medicine, food, additives, cosmetics, and other consumer items. Esterase derived from Bacillus cereus WZZ001 exhibits high hydrolytic activity and stereoselectivity. In this study, we expressed the esterase gene in Escherichia coli BL21 (DE3). We analyzed the biocatalytic resolution of N‐acetyl‐DL‐alanine methyl ester by immobilized whole E. coli BL21 (DE3) cells, which were prepared through embedding and cross‐linking. We analyzed biocatalytic resolution under the optimal conditions of pH of 7.0, temperature of 40°C and substrate concentration of at 700 mM with an enantiomeric excess of 99.99% and e.e._p of 99.50%. The immobilized recombinant B. cereus esterase E. coli BL21 (DE3) cells exhibited excellent reusability and retained 86.04% of their initial activity after 15 cycles of repeated reactions. The immobilized cells are efficient and stable biocatalysts for the preparation of N‐acetyl‐D‐alanine methyl esters.     

Using temperature and time criteria to control the effectiveness of continuous thermal sanitation of piggery effluent in terms of set microbial indicators

Aim: To determine the minimal conditions (temperature–time), necessary to achieve set sanitation targets for selected microbial indicators during the continuous thermal treatment of pig slurry. Methods and Results: The effectiveness of thermal treatment between 55 and 96°C was studied using Escherichia coli, enterococci, sulfite‐reducing Clostridia (SRC), mesophilic culturable bacteria (MCB), F+‐specific and somatic phages. Identification of SRC and MCB was performed using 16S rRNA gene analysis. Ten minutes at 70°C or 1 h at 60°C was sufficient to reduce the vegetative bacteria by 4–5 log₁₀, but it had little effect on somatic phages nor on spore formers, dominated by Clostridium sp. At 96°C, somatic phages were still detected, but there was a reduction of 3·1 log₁₀ for SRC and of 1·4 log₁₀ for MCB. At 96°C, Clostridium botulinum was identified among the thermotolerant MCB. Conclusion: Only those hygienic risks relating to mesophilic vegetative bacteria can be totally eliminated from pig slurry treated at 60°C (60 min) or 70°C (<10 min). Significance and Impact of the Study: Hygiene standards based on the removal of the indicators E. coli and enterococci can easily be met by treatment as low as 60°C (enabling, a low‐cost treatment using heat recovery). However, even at 96°C, certain pathogens may persist.     

Chemical Composition and Biological Studies of the Essential Oil from Aerial Parts of Beta vulgaris subsp. maritima (L.) Arcang. Growing in Tunisia

The chemical composition, antioxidant, cytotoxic, anticholinesterase and anti‐tyrosinase activities of the hydrodistilled essential oil of the aerial parts of Beta vulgaris subsp. maritime (L.) Arcang . from Tunisia have been evaluated. The chemical composition of the oil (yield 0.037% [w/w]), determined by GC‐FID and GC/MS is reported for the first time. Twenty five components, accounting for 98.1% of the total oil have been identified. The oil was characterized by a high proportion of oxygenated sesquiterpenes (39.2%), followed by sesquiterpene hydrocarbons (30.3%) and one apocarotenoids (26.3%). The main compounds were γ‐irone (26.3%), α‐cadinol (12.1%), T‐cadinol (10.6%), bicyclogermacrene (10.4%) and δ‐cadinene (6.0%). The isolated oil was tested for its antioxidant activity using the DPPH^·, ABTS^+·, catalase, and paraoxonase assays and also for its cytotoxic, anticholinesterase, and anti‐tyrosinase activities. The essential oil exhibited high antioxidant activity (IC₅₀ = 0.055 ± 0.006 mg/ml) and important result oncatalase (524.447 ± 2.58 Units/mg protein). Furthermore, it exerted a significant cytotoxic effect against A549 cell line, with IC₅₀ = 42.44 ± 1.40 μg/ml. The results indicate that the essential oil of B. vulgaris subsp. maritima (L.) Arcang . aerial parts may be used in future as an alternative to synthetic antioxidant agents, with potential application in the food and pharmaceutical industries.     

Postprocessing microflora of commercial attieke (a fermented cassava product) produced in the south of Côte d'Ivoire

The distribution and presence of hygiene indicator and pathogenic micro‐organisms in 375 samples of attieke marketed in Côte d'Ivoire, and their roles in the food poisoning were evaluated. Microbiological analyses were carried out, which included the total viable bacteria, coliforms, Escherichia coli, Staphylococcus aureus, Salmonella, Bacillus spores, fungi and Clostridium perfringens. The results revealed that the viable bacteria counts ranged from 2·2 ± 1·2 × 10⁵ to 3·4 ± 1·4 × 10⁶ CFU g^?1, while the yeasts and the moulds counts ranged, respectively, from 2·4 ± 0·12 × 10⁴ to 9·8 ± 0·4 × 10⁵ CFU g^?1 and 1·3 ± 0·7 × 10¹ to 1·7 ± 0·7 × 10² CFU g^?1. Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Citrobacter freundi, Enterobacter amnigenus, Citrobacter youngae, Enterobacter aerogenes, Klebsiella pneumoniae, Serratia marcescens, Enterobacter agglomerans and Klebsiella oxytoca were the bacteria isolated, and Rhizopus spp., Mucor spp., Thamnidium spp., Fusarium spp., Moniliella spp. the fungi. Escherichia coli, Clostridium perfringens and Salmonella spp. were not found. The occurrence of some bacteria and fungi illustrate that attieke collected in Côte d'Ivoire markets may act as a reservoir of pathogenic micro‐organisms for human.

Significance and Impact of Study

This study demonstrates the great need to carry out microbiological tests frequently on attieke and even more the need to apply correct HACCP system during the production. Attieke is especially a well‐known product in West Africa; hence, it is extremely important to ensure an adequate microbiological quality to guarantee consumers health. Overall, the study highlighted the need for effective communication on microbiological food risks, proper instruction and supervision in food‐handling procedures, greater education on food safety risks.     

Microbial contamination of food products consumed by infants and babies in Korea

Aims: The objectives of this study were to investigate the microbiological safety of various foods intended for consumption by infants and babies. Methods and Results: The incidence of Cronobacter spp. and Enterobacteriaceae from powdered infant formula (PIF, n = 75) and baby soy milk (n = 10) was examined. Additionally, aerobic plate count, coliforms and the prevalence of foodborne pathogens were investigated in 230 samples from a variety of infant and baby foods, including cereal‐based follow‐up formulas (FUF), liquid FUF and other infant foods. High APCs were observed in nutrient supplements and cereal‐based FUF. Coliforms were found in 6 (2·6%) products, and Cronobacter spp. was isolated in 10 (4·4%) samples, including four PIF and six cereal‐based FUF. Bacillus cereus was detected in 48 (20·9%) samples: cereal‐based FUF items (23·0%), rice soups (20·6%), honey samples (40·0%), biscuits (40·0%) and liquid FUF (7·4%). Conclusions: New safety criteria, along with hygienic control measures and consumer education strategies, are essential to improve the microbiological safety of infant or baby foods. Significance and Impact of the Study: This study provides comprehensive information about the prevalence and level of contamination of infant and baby food products by Cronobacter spp. and other major foodborne pathogens.     

A D-Amidase Constitutive Mutant from Arthrobacter sp. NJ-26

Foods contaminated with Salmonella enterica serovar Typhi are a mojor cause of typhoid fever, leading to public health problems and economic losses worldwide. Nisin and ρ-cymene were tested in this study for their antimicrobial activity against S. Typhi at 4 °C and 37 °C. Nisin and ρ-cymene, when used separately, did not inhibit the bacterium at either temperature. A synergistic antimicrobial effect between both compounds was observed when they were used simultaneously. This synergism was greater at 37 °C than at 4 °C. The lowest concentrations of nisin and ρ-cymene required for complete inhibition of S. Typhi at 37 °C were 0.3 ppm and 1.5 ppm, respectively, and 0.3 ppm and 2.5 ppm at 4 °C. The potential of nisin and ρ-cymene to control an S. Typhi population on ready-to-eat Thai-style pork sausage was also examined. The compounds were able to eliminate the contaminating bacterium with concentrations depending on the bacterial cell number on the food.     

Effect of culturing parameters on the vegetative growth of Haematococcus alpinus (strain lcr‐cc‐261f) and modeling of its growth kinetics

The present study investigated the effect of different culture conditions on the vegetative growth of a new species, Haematococcus alpinus (strain LCR‐CC‐261f) using airlift photobioreactors. The influence of culture medium, aeration rates, CO₂ concentration in air‐gas mixture, temperature, light intensities, and wavelengths were investigated to achieve sustainable high cell density cultures. Growth parameters were determined by fitting the data to a form of the logistic equation that included a lag phase. The shear‐sensitive vegetative cells favored lower aeration rates in the photobioreactors. MLA medium increased to 40 mM nitrate produced high density cultures. Temperatures between 12°C and 18°C, 3% (v/v) CO₂ concentration and a narrow photon flux density ranging between 37 and 48 μmol photons · m^?2 · s^?1 were best suited for growth. The wavelength of the light source also impacted growth and a high cell density of 9.6 × 10⁵ cells · mL^?1 was achieved using a mixture of red and blue compared to warm white, red, or blue LEDs.     

Reduction of Bacillus cereus spores in sikhye, a traditional Korean rice beverage, by modified tyndallization processes with and without carbon dioxide injection

Aims: The objective of this study was to inactivate Bacillus cereus spores in sikhye using a modified tyndallization process involving injection with carbon dioxide (CO₂). Methods and Results: Heat tolerance of B. cereus spores in tryptic soy broth and sikhye was evaluated. The D_95°C values of the B. cereus spores were 2·8–4·9 min, dependent of type of heating medium or inoculum level. The lethality of conventional heat treatment and modified tyndallization with or without CO₂ injection against B. cereus spores in sikhye was determined. The order of effectiveness was modified tyndallization with CO₂ > modified tyndallization without CO₂ > conventional heat treatment. Modified tyndallization with CO₂ reduced the number of B. cereus spores in sikhye by 5·8 log CFU ml^?1. The increased CO₂ concentration and decreased pH of sikhye resulting from CO₂ injection rapidly reverted to near‐normal values after heat treatment. Conclusions: Modified tyndallization with CO₂ was more effective than conventional heat treatment or modified tyndallization without CO₂ in reducing B. cereus spores in sikhye. Significance and Impact of the Study: Results of this study will be useful when developing strategies to control B. cereus spores in sikhye and may have application to other beverages.     

High level expression of a recombinant phospholipase C from <Emphasis Type="Italic">Bacillus cereus</Emphasis> in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Twenty-two Bacillus cereus strains were screened for phospholipase C (PLC, EC 3.1.4.3) activity using p-nitrophenyl phosphorylcholine as a substrate. Two strains (B. cereus SBUG 318 and SBUG 516) showed high activity at elevated temperatures (>70°C) at acidic pH (pH 3.5–6) and were selected for cloning and functional expression using Bacillus subtilis. The genes were amplified from B. cereus DNA using primers based on a known PLC sequence and cloned into the expression vector pMSE3 followed by transformation into B. subtilis WB800. On the amino acid level, one protein (PLC318) was identical to a PLC described from B. cereus, whereas PLC516 contained an amino acid substitution (E173D). PLC production using the recombinant strains was performed by an acetoin-controlled expression system. For PLC516, 13.7 U g⁻¹ wet cell weight was determined in the culture supernatant after 30 h cultivation time. Three purification steps resulted in pure PLC516 with a specific activity of 13,190 U mg⁻¹ protein.