Growth and inhibition by spices of growth from spores of enterotoxigenic Bacillus cereus in cooked rice

Bacillus cereus is a pathogenic spore-forming bacterium implicated in cases of diarrheal and emetic type of foodborne illness. We previously found that enterotoxigenic B. cereus is widely present in retail spices. Here we analyzed the spore heat resistance of nine diarrheal strains isolated from spices. Seven had D_95°C values ranging from 0.64 to 3.53 min while two emetic strains had D_95°C values of 7.04 min and 6.64 min. The ability of selected strains to grow in cooked rice at temperatures 20 °C, 17 °C and 12 °C was determined as well as their toxin expression capability. After 48 h, B. cereus levels of 1.26 × 10⁷ and 3.8 × 10⁷ CFU/g were obtained in cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 10⁴ CFU/g even after 48 h of incubation. The increase in the aerobic, mesophilic bacterial population (APC) and B. cereus population naturally present in 0.1% crushed pepper added to cooked rice was determined over a period of 48 h at 20 °C and 17 °C. Levels of B. cereus in pepper/rice samples reached a maximum of 1600 MPN/g at 20 °C while the APC was 2.4 × 10⁸/g at this temperature. When thyme, containing the same initial natural level of B. cereus, was added to rice in place of pepper, more than a five-fold greater level of B. cereus was detected at 20 °C. Since thyme contained initial APC of 2.5 log₁₀ less than pepper we conclude that the high APC functions in a competitive-exclusion manner, minimizing the growth of B. cereus and potentially other agents of foodborne illness. This is particularly relevant in instances of temperature abuse of foods and may explain the low incidence of foodborne illness due to B. cereus despite its widespread presence shown in previous surveys of market spices.     

Survival and growth of Bacillus cereus during Gouda cheese manufacturing

Survival and growth of Bacillus cereus was investigated during manufacturing of Gouda type cheese. The cheese was prepared in the pilot plant from pasteurised milk artificially contaminated with spores to give a final concentration of approximately 10² B. cereus spores per millilitre of cheese milk. B. cereus was enumerated by surface plating on B. cereus selective media and lactic acid bacteria were enumerated on lactic agar and MRS agar (de Man-Rogosa-Sharpe). Samples were taken for microbiological analysis of the milk before renneting, curd at cutting, at half whey removal, at final whey removal, at hooping of the curd, the cheese after pressing, after brining, after 1 week, after 2 weeks, after 4 weeks and after 6 weeks. The spores germinated into vegetative cells, which grew and reached a maximum of approximately 10⁴ CFU per gram of cheese at hooping (about 4 h after renneting). After pressing (approximately 16 h after renneting ) the viable cells were reduced to less than 10² CFU per gram. After brining (about 40 h after renneting) B. cereus was not detected in the cheese curd. At this stage the conditions of the cheese, particularly lower moisture content and a_w, lower Eh, high salt content, depleted lactose content combined with high acidity may have inhibited the growth of B. cereus. B. cereus did not affect the growth of lactic acid bacteria during cheese manufacturing. Lactic acid bacteria grew from 10⁷ to 10⁹ CFU per gram of curd during cheese manufacturing and stayed fairly constant at about 10⁹ for 6 weeks.     

The control of Clostridium botulinum during extended storage of pressure-treated,cooked chicken

High pressure processing (HPP) is used as a post-process decontamination treatment to ensure that cooked chicken is free from Listeria monocytogenes and other food poisoning bacteria. However HPP does not inactivate bacterial endospores and the conditions in cooked chicken could support the growth of Clostridium botulinum. Therefore some method for controlling the germination of spores and their subsequent growth would be necessary to ensure a safe product over an extended storage period.A five strain cocktail of non-proteolytic C. botulinum was inoculated into raw chicken mince, which was cooked and then pressure treated at 600 MPa for 2 min at 20 °C. C. botulinum survived cooking and HPP and subsequently grew in the pressure-treated, cooked chicken during storage. Germination and growth was controlled when 2% w/w sodium lactate was added and when oxygen-permeable packaging was used. Weissella viridescens was not suitable for use as a protective culture to control the growth of C. botulinum.     

Bacillus cereus in fresh ricotta: Comparison of growth and Haemolysin BL production after artificial contamination during production or post processing

Bacillus cereus is of particular concern for the production of fresh ricotta, due to the ability of spores to survive to the thermal treatment, leading to a potential germination, growth and toxin production in the product. This study aimed to evaluate the effect of a B. cereus contamination occurring in the whey used for the production of ricotta, or in the final product as post-production event.Four B. cereus strains (ATCC 14579 and three clinical isolates, GGu1, GPe2 and RCe1) were first evaluated for their ability to grow at different temperatures (from 5 to 55 °C) and spore survival rate to different thermal treatments (65, 70, 80 and 90 °C for 30, 15. 10 and 3 min, respectively). None of the strains showed to be psychrotrophic, as no growth below 10 °C was observed. Strains ATCC 14579 and GPe2 were the most resistant to thermal stresses and were selected for the inoculation tests.In the first trial, two aliquots of whey were inoculated with ATCC 14579 or GPe2 strain and used for the production of fresh ricotta samples, that were stored at 10 °C for 7 days (only GPe2) or 15 °C for 5 days (both the strains). In the second trial, the inoculation was made on fresh ricotta just after production. Samples were stored in the same conditions and analysed daily for the quantification of B. cereus vegetative cells and spores; the L2 component of Haemolysin BL was also quantified in the product.At 15 °C, a very fast germination of spores, followed by an active growth, was constantly observed in the two trials for both B. cereus strains. An earlier growth was detected in the whey-inoculated samples, suggesting the potential activation of spore germination caused by high temperatures reached during ricotta production. A slightly faster growth was observed for ATCC 14579 strain. At 10 °C, GPe2 strain showed a slow growth, with similar rates between whey- or product-inoculated ricotta samples. The production of HBL toxin was significant only in samples kept at 15 °C, starting from the 4th day of storage.In order to ensure the consumers’ protection, these results suggest the suitability of fresh ricotta as a substrate for the growth and metabolic activity of B. cereus, highlighting the need to prevent the contamination of the product and, above all, to apply a correct refrigeration during its storage.     

The antimicrobial effect of wine on Bacillus cereus in simulated gastro-intestinal conditions

This study aimed to evaluate the antimicrobial activity of wine against Bacillus cereus vegetative cells and spores. The results clearly show that wine exerts a strong inactivation effect against vegetative cells of B. cereus. The red wine tested inactivated stationary phase cultures to undetectable numbers in less than 10 s. Thus, further inactivation assays were carried out with wine diluted with water (1:4 and 1:8). Diluted wine 1:4 caused a reduction of approximately 5 log cycles on viable cell counts in 20 s. On the other hand, B. cereus spores were found to be highly resistant to wine exposure. The influence of wine components (organic acids, ethanol and phenolic compounds) was investigated on vegetative cells. The wine organic acids tested exhibited a strong inactivation effect, and, when combined with ethanol, a slight synergistic effect was observed. The wine phenolic compounds assayed displayed no activity against the vegetative cells at the concentrations tested. At the simulated gastric conditions studied (in the presence of food), wine diminished considerably the number of B. cereus viable cells in addition to the effect of the synthetic gastric fluid. The behaviour of B. cereus spores under gastro-intestinal conditions was also evaluated. In a consumption-like scenario, the addition of wine led to lower total counts (vegetative cells + spores) of B. cereus in the simulated intestine conditions, showing that wine inhibits the proliferation of the vegetative cells obtained from the germination of spores. This work provides evidence that consumption of wine during a meal may diminish the number of viable cells of B. cereus and reduces the impact of the germination of spores that may occur in the small intestine, thus lowering the risk of toxi-infection that may be caused by this pathogen.     

Degradation of acephate and its metabolite methamidophos in rice during processing and storage

The degradation of acephate and its metabolite methamidophos during different stages of commercial processing, homing processing, and storage was assessed. Residues were determined by a simple gas-chromatographic method using a flame photometry detector. Acephate and methamidophos mostly remained in rice hull fractions, and hulling significantly reduced acephate and methamidophos in rice. Commercial processing caused the loss of 86% of acephate and 35.9% of methamidophos from rough brown rice to polished rice, whereas home processing caused the loss of 83.9% of acephate and 70% of methamidophos from polished rice to cooked rice. Washing for 5, 15, and 30 min (with tap water, 0.9% NaCl, and 0.1% Na₂CO₃) caused an average loss in the range of 9.8%-35.3% of acephate and 9.7%-45.2% of methamidophos. Extending washing time and adding a small amount of soda into the washing solution can efficiently eliminate acephate and methamidophos. The stability of acephate and methamidophos in polished rice was studied at different storage intervals, from 7 to 42 days at ambient temperatures (25 °C). Methamidophos was found to be more persistent than acephate.     

Modelling the interaction of storage temperature,pH, and water activity on the growth behaviour of Listeria monocytogenes in raw and pasteurised semi-soft rind washed milk cheese during storage following ripening

The objective of this study was to investigate the growth of Listeria monocytogenes in semi-soft rind washed cheese made from raw and pasteurised milk at different storage temperatures (4, 10 and 15 °C) over a 28 day period simulating storage following ripening. Changes in water activity (a_w) and pH in cheeses were also monitored during storage. Response surface models were used to model the interaction of storage temperature and time on a_w, pH and L. monocytogenes population. Growth curves were fitted using Baranyi, modified Gompertz and Logistic models at all storage temperatures for both cheeses, and model parameters were statistically analysed. In raw and pasteurised milk cheeses, all models showed a significant (P < 0.05) increase in the specific growth rate (SGR, Day⁻¹) of L. monocytogenes with an increase in storage temperature. A higher SGR was observed for L. monocytogenes in pasteurised milk cheese (0.18–0.85 Day⁻¹) compared to raw milk cheese (0.05–0.37 Day⁻¹) at all storage temperatures studied. Response surface models indicated an increase in the L. monocytogenes population and pH with an increase in storage temperature. However, a decreasing trend in a_w for both cheese types was observed. The predicted regression model parameters for both the raw and pasteurised milk cheese showed a high correlation coefficient R² > 0.87. Overall, the L. monocytogenes population increased up to 3 log₁₀ cfu⁻g⁻¹ for both cheeses during storage following ripening. The fitted models confirmed different L. monocytogenes growth behaviour between raw and pasteurised milk cheeses, which could support the Food Business Operator in predicting growth during storage following ripening.     

Occurrence and diversity of Bacillus cereus and moulds in spices and herbs

Spices and herbs can contain toxin-producing bacteria and moulds, which can cause health problems for consumers and contribute to food spoilage and shelf-life reduction. The aims of the present work were (i) to determine the occurence and levels of B. cereus and moulds; (ii) to charactize the incidence and diversity of B. cereus emetic toxin (ces, CER), and diarrhoeal toxin-encoding genes (entFM, nheA, hblC, cytK) and toxigenic potential of Hbl toxin-producing B. cereus strains. Black ground pepper samples showed the most contamination with the highest concentration of B. cereus 2.49 log₁₀ CFU/g. Moreover, cumin contained the most prominent mould concentration level of 3.6 log₁₀ CFU/g. The most common moulds were Aspergillus and Penicillium spp. Compared to packaging type, all products acquired from the local market, except curry for B. cereus, exchibited high concentrations of B. cereus and moulds. Four genes were identified – 96% of B. cereus strains contained entFM, 94% nheA, 56% hblC, 42% cytK. None of the samples contained emetic toxin-encoding genes (ces, CER). Toxigenic potential of Hbl toxin was found in 72% of B. cereus strains. Different temperature, moisture levels and hygiene practices were observed at places of sale in local markets thus facilitating contamination and development of moulds. Moreover, the presence of B. cereus and its ability to produce toxins in spices and herbs, may suggest the need to establish microbiological criteria for mould and spore-forming bacteria in spices and herbs.     

Molecular characterization of biosurfactant producing Bacillus cereus strain DRDU1 for its potential application in bioremediation and further EOR studies

Abstract

Environmental pollution by crude oil during petroleum exploration is a global issue of concern. It largely alters the physicochemical property of soil and water which makes them toxic for crops and aquatic organisms to survive. Bacillus cereus strain DRDU1 (Genbank accession no. KF273330.1) is an endospore forming potential hydrocarbon degrader which was isolated from an automobile engine. The isolate could achieve up to 96 and 84% kerosene and crude oil degradation with respective cfu of (6.3?±?2.31)?×?10⁹ and (13.67?±?2.52)?×?10⁸ cells/mL. On the other hand it also showed its potential to degrade 74 and 67% of kerosene and crude oil in nitrate (N) and phosphate (P) deficient media with corresponding cfu (10?±?1.73)?×?10⁸ and (6?±?2.65)?×?10⁶ cells/mL. This indicates its potential to survive under nutrient stress condition. The strain was reported to be capable of producing biosurfactant which may play role in petroleum biodegradation and may be a potential tool for Microbial Enhanced Oil Recovery (MEOR) studies in future. Herein the presence of sfp gene in the isolate was confirmed which may be responsible for its ability to synthesize biosurfactant for hydrocarbon degradation and also for its stress tolerant potential. It was further characterized and the hypothetical protein sequence encoded by this gene is also proposed.     

Tenacity of Bacillus cereus and Staphylococcus aureus in dried spices and herbs

Numerous studies examined the antimicrobial effects of spice and herb extracts, whereas little is known about the effects of dry condiments on the survival of microorganisms. This study investigated the impact of dried condiments on the survival of Bacillus cereus and B. thuringiensis spores as well as Staphylococcus aureus cells. In addition, the survival variability between different strains was evaluated. Condiments (allspice, basil, cinnamon, nutmeg, oregano, paprika, parsley and pepper) were artificially contaminated by a dry spiking method using sand as carrier matrix and as control. The results show that counts of B. cereus and B. thuringiensis spores (initial spore count 5.6 ± 0.2 log₁₀ cfu/g and 6.7 ± 0.1 log₁₀ cfu/g, respectively) did not decline significantly in all condiments over a period of 50 weeks. In contrast, in some of the spiked test materials, cell counts of S. aureus (initial cell count 8.1 ± 0.5 log₁₀ cfu/g) were reduced below the detection limit of 10 cfu/g within 10 weeks of storage. D values for S. aureus ranged between 5 and 31 days depending on the strain, condiment and initial contamination level. In conclusion, dried condiments may not affect the survival of spores but can significantly affect the survival of non-spore forming bacteria. As strain variability can occur, tenacity studies should be conducted including a variety of strains.     

Short communication: Fate of major foodborne pathogens and Bacillus cereus spores in sterilized and non-sterilized Korean turbid rice wine (Makgeolli)

The objective of this study was to examine the fate of foodborne pathogens (Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus) and B. cereus spores in Korean turbid rice wine (Makgeolli). Samples of sterilized and non-sterilized turbid rice wine were inoculated with each of the vegetative bacteria or B. cereus spores at 3–4 log CFU/ml. The samples were stored at 5 °C or 22 °C, and bacterial survival was monitored over 28 days. Despite the harsh environment (alcohol content: 6–7% and pH: 3.43–3.98), long-term survival of pathogens was observed. Survival time was different depending on the type of beverage (pathogens survived longer in sterilized wine than in non-sterilized wine), cellular state (spores survived longer than vegetative cells), species (B. cereus survived longer than other species), and storage temperature (pathogens survived longer at 5 °C then at 22 °C). The number of B. cereus spores remained constant at both temperatures. The vegetative B. cereus population declined rapidly within 1 day, but then remained steady for up to 28 days (1.20–1.55 log CFU/ml in sterile wine). These results indicate that B. cereus formed spores that survived for a long time; therefore, it is possible that B. cereus may exist as spores in turbid rice wine. E. coli O157:H7, L. monocytogenes, S. Typhimurium, and S. aureus survived for up to 28, 14, 14, and 14 days, respectively, in sterilized wine at 5 °C. Thus, the health implications of the long-term survival of pathogens in alcoholic beverages should be carefully considered. The results provide new information that may be useful in predicting the potential microbiological hazards associated with turbid rice wine.     

短短芽孢杆菌和蜡状芽孢杆菌采油机理及其在大庆特低渗透油藏的应用

针对大庆外围朝阳沟油田储集层和油水特性，筛选出短短芽孢杆菌（Brevibacillus brevis）和蜡状芽孢杆菌（Bacillus cereus）作为采油菌种。实验结果表明，实验菌株作用原油烃时只降解高碳链（C20以上）饱和烷烃，降解途径以氧化降解为主，代谢产物以饱和烷基酸为主，没有低碳饱和烷烃的生成。实验菌种性能评价结果表明：微生物作用后界面张力下降50％左右，产生多种有机酸；微生物可选择性降解原油中某些中—高碳数烷烃，使原油中的长链烷烃含量相对减少，短链烃含量相对增加，原油黏度下降40％左右，含蜡量、含胶量下降，流变性得到改善。物理模拟驱油实验表明微生物提高采收率幅度可达到6．7％。应用短短芽孢杆菌和蜡状芽孢杆菌等菌种在大庆朝阳沟特低渗透油田开展的微生物单井吞吐试验和微生物驱油矿场试验取得了理想效果。图7表4参22     

Behaviour of Belgian consumers,related to the consumption,storage and preparation of cooked chilled foods

Cooked chilled foods or Refrigerated and Processed Foods of Extended Durability (REPFEDs) are a heterogeneous group of food products designed for consumer convenience. The consumer exposure to pathogenic microorganisms due to the consumption of REPFEDs is influenced by the industrial production process and by consumer behaviour. A consumer survey was organised and conducted to assess the consumption frequency, storage time, reheating practices and perception of and respect for the product's ‘use by’ date. The survey was conducted at a local food fair in Belgium with 874 respondents correctly completing the questionnaire. Over three quarters (77.5%, n = 677) had consumed at least one REPFED over the last year. Consumption frequency was the highest between the ages of 18 and 30. Nine out of ten consumers were able to give an acceptable estimate of the shelf life of REPFEDs (e.g. less than four weeks). By contrast, only half of the consumers (53.3%) fully respected the ‘use by’ date as indicated on the packaging. The majority of the remaining consumers (39.6%) would consume the product until three days past the ‘use by’ date; 2.5% of the consumers would still consume the product after more than three days past the ‘use by’ date and 4.6% did not consider the ‘use by’ date. In addition, only half of the consumers fully complied with the reheating instructions on the label, 36.5% only partially followed these instructions and 13.4% did not follow them at all. Data analysis showed that consumers, who did not respect the ‘use by’ date, were also less likely to follow the reheating instructions. To determine the distribution of the time a REPFED spends in a consumer fridge, the consumers were asked how frequently they bought REPFEDs and how they stored them. Using this information an attempt was made to construct a time-to-consumption (TTC) distribution. This TTC demonstrated that approx. 20% of REPFEDs was consumed on the day of purchase; about half (52.9%) were consumed within two days of purchase, 75.5% within four days and over 90% during the first week. These short storage times are likely to reduce the exposure to psychrotrophic microorganisms present in REPFEDs.     

Extracellular enzyme production and enterotoxigenic gene profiles of Bacillus cereus and Bacillus thuringiensis strains isolated from cheese in Turkey

The aim of the present study was to investigate the biochemical characteristics, extracellular enzyme production and enterotoxigenic genes contents of 6 Bacillus cereus and 22 Bacillus thuringiensis strains, isolated from 100 cheese samples in Turkey. Crystal morphologies of B. thuringiensis strains were found either spherical (n = 12) or spherical and irregular-shaped (n = 10) by phase contrast microscopy. B. cereus and B. thuringiensis strains were found to produce extracellular enzymes, respectively: gelatinase (83% and 91%), DNase (83% and 77%), lecithinase (83% and 95%), protease on skim milk agar (100% and 100%), protease on milk agar (100% and 91%), protease on casein agar (83% and 77%), xylanase (100% and 45%), and cellulase (0% and 41%), and amylase (83% and 27%). All of the strains, except for Bt-D1, hydrolyzed Tween 20 (96%), but not Tween 80 or tributyrin. Pectinolytic activity was obtained to be the least frequent (4%). PCR analysis showed that all strains contained nheA, nheB, nheC and hblD genes. The hblA and hblC genes were present in 2 and 4 of B. thuringiensis strains, respectively. The bceT gene was detected in 1 B. cereus and 9 B. thuringiensis strains. The entFM gene was detected more frequently in B. thuringiensis (82%) than in B. cereus strains (50%). To our knowledge, this is the first report about the isolation and identification of enterotoxigenic B. cereus and B. thuringiensis strains from cheese samples in Turkey.     

Sporulation dynamics and spore heat resistance in wet and dry biofilms of Bacillus cereus

Environmental conditions and growth history can affect the sporulation process as well as subsequent properties of formed spores. The sporulation dynamics was studied in wet and air-dried biofilms formed on stainless steel (SS) and polystyrene (PS) for Bacillus cereus ATCC 10987 and the undomesticated food isolate B. cereus NIZO 4080. After harvesting and maturation, the wet heat resistance of spores obtained from these biofilms was tested and compared to planktonic and agar plate-derived spores. Drying/air exposure of the preformed 24 h old biofilms accelerated spore formation for both strains and resulted in higher final spore percentages. Prolonged dry incubation of more than three days triggered germination of spores in the biofilms of ATCC 10987. Spores harvested from wet biofilms on SS displayed the highest heat resistance compared to liquid, agar plate and PS biofilm derived spores. The D_95 °C values for these spores were 17 and 22 min for NIZO 4080 and ATCC 10987, respectively, which was 2 and 1.2 fold higher compared to planktonic spores of these strains. Spores obtained from dried biofilms of ATCC 10987 displayed reduced heat resistance compared to wet biofilm spores. The results indicate that environmental conditions encountered by biofilms affect sporulation dynamics and spore heat resistance, thus affecting subsequent quality issues and safety risks related to these biofilms.     

Distribution and expression of the enterotoxin genes of Bacillus cereus in food products from Jiangxi Province,China

Bacillus cereus is a major foodborne pathogen that can cause a type of diarrhea. Diarrheal syndrome results from the ingestion of food products contaminated with enterotoxin-producing B. cereus. This study investigated the presence of four enterotoxins genes in 130 B. cereus isolated from various food products from Jiangxi, China. The expression levels of the enterotoxin genes in three B. cereus strains of different origins were subsequently analyzed. All of the B. cereus strains harbor at least 1 enterotoxin gene, whereas 34 strains harbor 2 enterotoxin genes, 44 strains harbor 3 enterotoxin genes, and 47 strains carry all of the four enterotoxin genes. The detection rates of the cytK, hblD, nheA, and entFM enterotoxin genes in all B. cereus isolates were 71%, 43%, 92%, and 99%, respectively. Moreover, the food matrix significantly affected the expression of these enterotoxin genes. The majority of the enterotoxin genes were also downregulated in four food matrices, indicating that the relative expression of certain enterotoxin genes, especially the entFM gene, was lower in a real food matrix than in laboratory broths. Hence, these data revealed that B. cereus is a serious health hazard and that the food matrix may influence the virulence expression of B. cereus. Our results provide better insights into the physiology of the microorganism grown in food products.     

Mathematical modeling and growth kinetics of Clostridium sporogenes in cooked beef

Clostridium sporogenes PA 3679 is a common surrogate for proteolytic Clostridium botulinum for thermal process development and validation. However, little information is available concerning the growth kinetics of C. sporogenes in food. Therefore, the objective of this study was to investigate the growth kinetics of C. sporogenes in cooked beef under different temperature conditions.Ground beef samples, inoculated with C. sporogenes spores, were incubated at temperatures between 8 and 47 °C to examine the growth of this microorganism. Two primary models (Huang and Baranyi models) were used to analyze the growth data. The Ratkowsky square-root model was used as the secondary model to evaluate the effect of temperature on bacterial growth rate and lag time. The USDA IPMP 2013, a free data analysis tool for predictive microbiology, was used in data analysis.No growth of C. sporogenes was observed at temperatures below 15 °C for up to 25 days. At temperatures between 20 and 47 °C, C. sporogenes grew in cooked beef. The growth curves could be analyzed by both Huang and Baranyi models. The root mean squared error (RMSE) was 0.375 for the Huang model, and 0.441 for the Baranyi model with a global h₀ of 10.46. The nominal minimum growth temperature (T₀) estimated from the Huang model was 15.5 °C, which was 12.7 °C for the Baranyi model. The maximum growth temperature was 48.0 and 48.3 °C for the Baranyi and Huang models, respectively. The lag times and specific growth rates of C. sporogenes observed in this study were reasonably close to the data reported in the literature for C. botulinum under comparable conditions, suggesting that C. sporogenes may be used as a potential surrogate for evaluating the growth of C. botulinum in cooked meats during cooling. However, a direct comparison of growth kinetics between the two microorganisms is needed to confirm the suitability of C. sporogenes as a surrogate of C. botulinum.     

Probabilistic evaluation of Clostridium perfringens potential growth in order to validate a cooling process of cooked dishes in catering

The cook-chill operation, a widely used process within central cooking facilities, implies that cooked dishes have to be quickly cooled down immediately after cooking in order to prevent any food-borne diseases due to Clostridium perfringens. The food service operators are obliged to validate the efficiency of the cooling process in the context of their HACCP plan. To perform this cooling process, they can either ensure compliance with some reference criterion (maximal cooling duration) or demonstrate that the cooling operation is safe by carrying out prior tests.This document presents an experimental method to validate the sanitary efficiency of the cooling process. In the studied kitchen the food was cooked and, successively, distributed into plastic containers, cooled down in blast-chiller and then stored in cold room.The tests consisted in monitoring food temperature at the center of the containers from the end of cooking until the beginning of cold room storage. The measurements were performed during 6 cycles of food preparation.From the monitored time–temperature profiles, thermal cooling equations were established for modeling.Each parameter of these thermal equations was linked with a distribution fitted with the experimental data. Then these thermal equations were coupled with predictive microbiology equations. A probabilistic calculation of the C. perfringens potential growth was carried out by using @Risk software. The proposed method required only simple monitoring equipments and could be easily implemented in central kitchens under the usual working conditions.The cooling conditions in the studied kitchen could be considered as satisfactory because the calculations give only a 0.4% probability that the C. perfringens potential growth was equal or greater than 1 log10 cfu/g.     

Quantification and differentiation of Bacillus cereus group species in spices and herbs by real-time PCR

Spores of Bacillus (B.) cereus group species are frequent contaminants in foodstuffs including spices and herbs. However, the distribution of individual B. cereus group species is unknown as standard cultural methods are insufficient for differentiation. Real-time PCR is an alternative method to detect, differentiate and quantify B. cereus group species in food.In our study we applied a combination of previously described real-time PCR assays to detect and quantify the B. cereus group (excluding B. cytotoxicus) with simultaneous discrimination of B. pseudomycoides and cry1-positive B. thuringiensis as well as differentiation of B. weihenstephanensis from B. cereus group species with non-rhizoid colony morphology. For testing food matrices, which can also include PCR inhibiting substances, an internal amplification control was included. In total, five DNA extraction kits were tested on pure spore suspensions to select the one with the best recovery rate when coupled to real-time PCR. The Qiagen DNeasy Blood & Tissue Kit performed best with a limit of detection (LOD) of approximately 100 cfu/ml for spores of each B. cereus, B. weihenstephanensis, B. thuringiensis and B. pseudomycoides strain. However, applied to allspice, paprika, pepper and oregano artificially contaminated with B. cereus spores the LOD was ≥10⁵ cfu/g. In contrast, using the open-formula cetyltrimethylammonium bromide (CTAB) method LODs of 10² to 10³ cfu/g were achieved for paprika, pepper and oregano. For allspice, the LOD was 10⁶ cfu/g.Our quantitative multiplex real-time PCR coupled to DNA extraction by the CTAB method provides a sensitive culture independent technique with the potential to quantitatively detect and differentiate B. cereus group species in several spices and herbs.