Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus

In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacilluscereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to >10.0), and the substance was thermally stable up to 60 °C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5 μg/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254 nm.     

Unsaturated fatty acids from food and in the growth medium improve growth of Bacillus cereus under cold and anaerobic conditions

In a chemically defined medium and in Luria broth, cold strongly reduced maximal population density of Bacillus cereus ATCC 14579 in anaerobiosis and caused formation of filaments. In cooked spinach, maximal population density of B. cereus in anaerobiosis was the same at cold and optimal temperatures, with normal cell divisions. The lipid containing fraction of spinach, but not the hydrophilic fraction, restored growth of B. cereus under cold and anaerobiosis when added to the chemically defined medium. This fraction was rich in unsaturated, low melting point fatty acids. Addition of phosphatidylcholine containing unsaturated, low melting point, fatty acids similarly improved B. cereus anaerobic growth at cold temperature. Addition of hydrogenated phosphatidylcholine containing saturated, high melting point, fatty acids did not modify growth. Fatty acids from phospholipids, from spinach and from hydrogenated phosphatidylcholine, although normally very rare in B. cereus, were inserted in the bacterium membrane. Addition of phospholipids rich in unsaturated fatty acids to cold and anaerobic cultures, increased fluidity of B. cereus membrane lipids, to the same level as those from B. cereus normally cold adapted, i.e. grown aerobically at 15 °C. B. cereus is therefore able to use external fatty acids from foods or from the growth medium to adapt its membrane to cold temperature under anaerobiosis, and to recover the maximal population density achieved at optimal temperature.     

Formation of pyrazines and 2-acetyl-1-pyrroline by Bacillus cereus

The production of alkylpyrazines and 2-acetyl-1-pyrroline by different Bacillus cereus strains, which has been previously reported, was studied in detail. B. cereus ATCC 27522 produced the highest amounts of flavour compounds when grown as surface cultures on plate count agar. Pyrazine, methylpyrazine, 2,5-dimethylpyrazine, trimethylpyrazine and 3-ethyl-2,5-dimethylpyrazine were produced in low amounts. Since they were also detected in control flasks, an enzymatic formation was concluded to be unlikely. Only the production of 3-ethyl-2,5-dimethylpyrazine was in all cases significantly different from the control. Detailed precursor studies revealed that the production of 2-acetyl-1-pyrroline by B. cereus ATCC 27522 proceeds via acetylation of 1-pyrroline, a metabolic degradation product of proline and ornithine. Comparison of results obtained from dynamic headspace and simultaneous steam distillation – solvent extraction showed that the use of a non-thermal extraction method is essential to obtain reliable results on the biological formation of these Maillard flavour compounds.     

Short communication: Presence of neutral metallopeptidase (npr) gene and proteolytic activity of Bacillus cereus isolated from dairy products

The control of proteolytic microorganisms is one of the main challenges of the dairy industry, due to their spoilage activity that jeopardizes the quality of their products. Seventy-four Bacillus cereus strains isolated from powdered, UHT, and pasteurized milks were tested for the presence of the neutral metallopeptidase (npr) gene and proteolytic activity at 7, 10, 25, 30, and 37°C. All strains had the npr gene, and proteolytic activity increased with the incubation temperature. The obtained results highlight the relevance of B. cereus as a spoiling agent in the dairy industry in terms of its genetic predisposition for proteolytic capacity, especially at room temperature.     

Low temperatures and fermentative metabolism limit peptidoglycan digestion of Bacillus cereus. Impact on colony forming unit counts

The impact of fermentative metabolism at low temperature on cell division of Bacillus cereus was studied. Fermentation at 37 °C had no influence on the division of bacteria. Aerobic cultures at 15 °C produced larger cells than at 37 °C, but cell division was normal. In fermentative cultures at 15 °C, no increase in CFU ml⁻¹ was observed. However, A₆₀₀ increased, due to formation of long filaments. Transmission electronic microscopy and light microscopy with fluorescent staining showed several nucleic acid entities separated by a hydrophobic membrane, indicating that each filament contained several individual cells attached by peptidoglycan. When left in air at room temperature, one filament gave several daughter cells, this means that one CFU formed by one filament may represent a greater contamination potential than one CFU formed by a single cell. Division was observed in cultures at 15 °C with anaerobic respiration in the presence of nitrates. Possible filamentous growth must thus be taken into account to avoid underestimating B. cereus growth in vacuum or modified atmosphere packaged foods stored at low temperature.     

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.     

A new method for rapid and quantitative detection of the Bacillus cereus emetic toxin cereulide in food products by liquid chromatography-tandem mass spectrometry analysis

The Bacillus cereus emetic toxin cereulide causes foodborne intoxication, which may occasionally result in severe disease, and even death. To differentially diagnose the emetic-type of foodborne disease caused by B. cereus and assess the safety of commercial food, we developed a rapid method to quantitate cereulide. This method was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for the extraction of cereulide from food using a normal-phase silica gel cartridge. The limits of detection and quantification were 0.1 and 0.5 ng of cereulide ml⁻¹, respectively. Spiked cereulide was reproducibly recovered with over 67% efficiency from nine diverse foods implicated in cereulide food poisoning. The recovery rate, reproducibility, and intermediate precision for this single laboratory validation using boiled rice were 87.1%, 4.4%, and 7.0%, respectively. Further, we detected a wide range of cereulide concentrations in leftover food and vomitus samples from two emetic foodborne outbreaks. LC-MS/MS analysis correlated closely with those acquired using the HEp-2 cell assay, and quantitated cereulide from 10 food samples at least five times faster than the bioassay. This new method will provide clinicians with an improved tool for more rapidly and quantitatively determining the presence of cereulide in food and diagnosing food poisoning caused by cereulide.     

Predictive microbiology models vs. modeling microbial growth within Listeria monocytogenes risk assessment: what parameters matter and why

Predictive microbiology models are essential tools to model bacterial growth in quantitative microbial risk assessments. Various predictive microbiology models and sets of parameters are available: it is of interest to understand the consequences of the choice of the growth model on the risk assessment outputs. Thus, an exercise was conducted to explore the impact of the use of several published models to predict Listeria monocytogenes growth during food storage in a product that permits growth. Results underline a gap between the most studied factors in predictive microbiology modeling (lag, growth rate) and the most influential parameters on the estimated risk of listeriosis in this scenario (maximum population density, bacterial competition). The mathematical properties of an exponential dose-response model for Listeria accounts for the fact that the mean number of bacteria per serving and, as a consequence, the highest achievable concentrations in the product under study, has a strong influence on the estimated expected number of listeriosis cases in this context.     

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.     

Response of Bacillus cereus spores to high hydrostatic pressure and moderate heat

The effects of high pressure (400-600 MPa) and moderate heat (60-80 °C) treatments at various process times (10-20 min) on the reduction of Bacillus cereus As 1.1846 spores, suspended in milk buffer were investigated. In the present work, response surface methodology (RSM) was employed, and a quadratic equation of high hydrostatic pressure inactivation was built with RSM. By analyzing response surface plots and corresponding contour plots and by solving the quadratic equation, experimental values were shown to be significantly in agreement with predicted values, since the adjusted determination coefficient was 0.9752 and the level of significance was P < 0.0001. Optimum process parameters for a six-log cycle reduction of B. cereus spores were obtained: pressure, 540.0 MPa; temperature, 71 °C; and pressure-holding time, 16.8 min. The adequacy of the model equation in predicting optimum response values was verified effectively using experimental test data that was not used in the development of the model.     

Sensitivity of Bacillus weihenstephanensis to acidic changes of the medium is not dependant on physiological state

This study aims to quantify the effect of salt and acid preliminary exposure on acid resistance of vegetative cells of Bacillus weihenstephanensis. The psychrotolerant strain KBAB4 was cultured until the mid-exponentially phase (i) in BHI, (ii) in BHI supplemented with 2.5% salt or (iii) in BHI acidified at pH 5.5 with HCl. The growing cells were subsequently inactivated in lethal acid conditions ranging from 4.45 to 4.70. Based on statistical criteria, a primary mixed-Weibull model was used to fit the acid inactivation kinetics. The acid resistance was enhanced for acid-adapted cells and decreased for salt-adapted cells. The secondary modelling of the bacterial resistance allowed the quantification of the change in pH leading to a ten folds variation of the bacterial resistance, i.e. cells sensitivity (z_pH). This sensitivity was not significantly affected whatever the preliminary mild exposure and the presence of sub-populations with different acid resistances. These results highlighted that pre-incubation conditions influence bacterial acid resistance without affecting the sensitivity to acidic modifications, with a 10 fold reduction of Bacillus acid resistance observed for a reduction of 0.37 pH unit. Quantification of such adaptive stress response might be instrumental in quantitative risk assessment more particularly in food formulation, particularly for low-acid minimally processed foods.     

食品中蜡样芽孢杆菌污染的预测模型及风险评估进展

蜡样芽孢杆菌（Bacillus cereus）是一种食品中常见的食源性条件致病菌,其引发的食源性疾病不仅对食用者健康造成严重损伤,也对社会经济造成严重损失。本文综述了近年来国内外对食品中Bacillus cereus污染预测模型的研究及其在风险评估中的应用进展。将影响因素、食品基质、生产链环节、初级模型和次级模型常用拟合模型等进行总结,发现当前预测模型研究最多的是温度、食品成分、水分活度、pH值等因素对Bacillus cereus增殖影响,而温度是主要影响因素;食品主要集中于动物性食品和大米及其制品。Gompertz模型、Logistic模型、Baranyi模型、Weibull等模型常用于拟合生长或失活曲线,建立初级模型;二次多项式模型和平方根模型常用来拟合生长速率或延滞期的变化,建立次级模型;在此基础上,用软件系统建立三级模型。当前的风险评估研究仅涉及到生产环节或从销售到消费环节,尚未见到某类食品从原料环节到消费环节Bacillus cereus预测模型研究的报道。本文展望了食品中Bacillus cereus污染的风险评估研究,以期为今后的研究提供参考。     

Use of a D-optimal mixture design to estimate the effects of diverse chloride salts on the growth parameters of Lactobacillus pentosus

The effects of NaCl, KCl, CaCl₂, and MgCl₂ and their mixtures on the ionic strength (IS) of the medium and the growth parameters of Lactobacillus pentosus were studied by means of a D-optimal mixture experimental design with constrains (total salt concentration ≤ 9.0%, wt/vol) and the generalized z-value. The IS was linearly related to the concentrations of the diverse salts and its increase, for similar concentrations of salts, followed the order MgCl₂ > CaCl₂ > NaCl > KCl. Within the experimental region, the lag phase duration (λ) was mainly affected by NaCl and CaCl₂ and the interaction KCl with MgCl₂. The maximum specific growth rate (μ_max) decreased as NaCl (the highest effect), CaCl₂, and MgCl₂ increased (regardless of the presence or not of previous NaCl); low KCl concentrations had a stimulating effect on μ_max, but its overall effect showed a similar trend to the other salts. The maximum population reached (N_max) was the least affected parameter and decreased as NaCl and CaCl₂ concentrations increased regardless of the presence of the other salts. The equations that expressed the growth parameters as a function of the diverse chloride salts, within the limits assayed, were developed and the corresponding z- and harmonic Z-values were estimated.     

Effect of the medium characteristics and the heating and cooling rates on the nonisothermal heat resistance of Bacillus sporothermodurans IC4 spores

In recent years, highly thermo-resistant mesophilic spore-forming bacteria belonging to the species Bacillus sporothermodurans have caused non-sterility problems in industrial sterilization processes. The aim of this research was to evaluate the effect of the heating medium characteristics (pH and buffer/food) on the thermal inactivation of B. sporothermodurans spores when exposed to isothermal and non-isothermal heating and cooling treatments and the suitability of non-linear Weibull and Geeraaerd models to predict the survivors of these thermal treatments. Thermal treatments were carried out in pH 3, 5 and 7 McIlvaine buffer and in a courgette soup. Isothermal survival curves showed shoulders that were accurately characterized by means of both models. A clear effect of the pH of the heating medium was observed, decreasing the D₁₂₀ value from pH 7 to pH 3 buffer down to one third. Differences in heat resistance were similar, regardless of the model used and were kept at all temperatures tested. The heat resistance in courgette soup was similar to that shown in pH 7 buffer. When the heat resistance values obtained under isothermal conditions were used to predict the survival in the non-isothermical experiments, the predictions estimated the experimental data quite accurately, both with Weibull and Geeraerd models.     

High prevalence,low counts and uncommon serotypes of Listeria monocytogenes in linguiça, a Brazilian fresh pork sausage

Linguiça is a highly popular and appreciated pork product in Brazil, frequently consumed undercooked. Aiming at collection of data for a future risk assessment, this study evaluated the prevalence and counts of Listeria monocytogenes in linguiça samples collected at retail level in Sao Paulo, SP, Brazil. ISO methods were used for detection and enumeration of the pathogen (11290-1 and 11290-2, respectively). Isolates were submitted to Simplex-PCR for hlyA gene and those with biochemical features of L. monocytogenes and hlyA positive were serotyped using a Multiplex PCR. Ninety percent of the samples were positive for Listeria spp., and L. monocytogenes was detected in 42% of the samples, with counts below 10² CFU/g in all samples. A prevalence of uncommon serotypes 4a and 4c was observed.     

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.