Aerobic spore-forming bacteria associated with ropy bread: Identification, characterization and spoilage potential assessment.

Aerobic spore-forming (ASF) bacteria have been reported to cause ropiness in bread. Sticky and stringy degradation, discoloration, and an odor reminiscent of rotting fruit are typical characteristics of ropy bread spoilage. In addition to economic losses, ropy bread spoilage may lead to health risks, as virulent strains of ASF bacteria are not uncommon. However, the lack of systematic approaches to quantify physicochemical spoilage characteristics makes it extremely difficult to assess rope formation in bread. To address this problem, the aim of this study was to identify, characterize and objectively assess the spoilage potential of ASF bacteria associated with ropy bread. Hence, a set of 82 ASF bacteria, including isolates from raw materials and bakery environments as well as strains from international culture collections, were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and their species identity confirmed by 16S rRNA and gyrA or panC gene sequencing. A standardized approach supported by objective colorimetric measurements was developed to assess the rope-inducing potential (RIP) of a strain by inoculating autoclaved bread slices with bacterial spores. In addition, the presence of potential virulence factors such as swarming motility or hemolysis was investigated. This study adds B. velezensis, B. inaquosorum and B. spizizenii to the species potentially implicated of causing ropy bread spoilage. Most importantly, this study introduces a standardized classification protocol for assessing the RIP of a bacterial strain. Colorimetric measurements are used to objectively quantify the degree of breadcrumb discoloration. Furthermore, our results indicate that strains capable of inducing rope spoilage in bread often exhibit swarming motility and virulence factors such as hemolysis, raising important food quality considerations.

Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC-MS) of spoilage compounds and spore's resistance to thermal shocks.

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

Pilot-scale production of Bacillus subtilis MSCL 897 spore biomass and antifungal secondary metabolites in a low-cost medium.

OBJECTIVES: Bacillus subtilis is a plant growth promoting bacterium (PGPB) that acts as a microbial fertilizer and biocontrol agent, providing benefits such as boosting crop productivity and improving nutrient content. It is able to produce secondary metabolites and endospores simultaneously, enhancing its ability to survive in unfavorable conditions and eliminate competing microorganisms. Optimizing cultivation methods to produce B. subtilis MSCL 897 spores on an industrial scale, requires a suitable medium, typically made from food industry by-products, and optimal temperature and pH levels to achieve high vegetative cell and spore densities with maximum productivity. RESULTS: This research demonstrates successful pilot-scale (100 L bioreactor) production of a biocontrol agent B. subtilis with good spore yields (1.5 × 109 spores mL-1) and a high degree of sporulation (>80%) using a low-cost cultivation medium. Culture samples showed excellent antifungal activity (1.6-2.3 cm) against several phytopathogenic fungi. An improved methodology for inoculum preparation was investigated to ensure an optimal seed culture state prior to inoculation, promoting process batch-to-batch repeatability. Increasing the molasses concentration in the medium and operating the process in fed-batch mode with additional molasses feed, did not improve the overall spore yield, hence, process operation in batch mode with 10 g molasses L-1 is preferred. Results also showed that the product quality was not significantly impacted for up to 12 months of storage at room temperature. CONCLUSION: An economically-feasible process for B. subtilis-based biocontrol agent production was successfully developed at the pilot scale.

Growth of Clostridium perfringens in cooked chicken during cooling: One-step dynamic inverse analysis, sensitivity analysis, and Markov Chain Monte Carlo simulation.

The objective of this study was to determine the kinetic parameters and apply Markov Chain Monte Carlo (MCMC) simulation to predict the growth of Clostridium perfringens from spores in cooked ground chicken meat during dynamic cooling. Inoculated samples were exposed to various cooling conditions to observe dynamic growth. A combination of 4 cooling profiles was used in one-step inverse analysis with the Baranyi model as the primary model and the cardinal parameters model as the secondary model. Six kinetic parameters of the Baranyi model and the cardinal parameters model, including Q0, Ymax, µopt, Tmin, Topt, and Tmax, were estimated. The estimated Tmin, Topt, and Tmax were 14.8, 42.9, and 50.5 °C, respectively, with a µopt of 5.25 h-1 and maximum cell density of 8.4 log CFU/g. Correlation analysis showed that both Q0 and Ymax are weakly correlated to other parameters, while the remaining parameters are mostly mildly to strongly correlated with each other. Although it may be difficult to estimate highly correlated parameters using a single temperature profile, one-step analysis with multiple different temperature profiles helped estimate them successfully. The estimated parameters were used as the prior information to construct the posterior distribution for Bayesian analysis. MCMC simulation was used to predict the bacterial growth using different dynamic temperature profiles for validation of the accuracy of the predictive models. The MCMC simulation results showed that the Bayesian analysis produced more accurate predictions of bacterial growth during cooling than the deterministic method. With Bayesian analysis, the root-mean-square-error (RMSE) of prediction was only 0.1 log CFU/g with all residual errors within ±0.25 log CFU/g. Therefore, Bayesian analysis is recommended for predicting the growth of C. perfringens in cooked meat during cooling.

Low-cost cultivation and sporulation of alkaliphilic Bacillus sp. strain AK13 for self-healing concrete.

The alkaliphilic, calcium carbonate precipitating Bacillus sp. strain AK13 can be utilized in concrete for self-repairing. A statistical experimental design was used to develop an economical medium for its mass cultivation and sporulation. Two types of screening experiment were first conducted to identify substrates that promote the growth of the AK13 strain: the first followed a one-factor-at-a-time factorial design and the second a two-level full factorial design. Based on these screening experiments, barley malt powder and mixed grain powder were identified as the substrates that most effectively promoted the growth of the AK13 strain from a range of 21 agricultural products and by-products. A quadratic statistical model was then constructed using a central composite design and the concentration of the two substrates was optimized. The estimated growth and sporulation of Bacillus sp. strain AK13 in the proposed medium were 3.08 ± 0.38 × 108 and 1.25 ± 0.12 × 108 CFU/ml, respectively, which meant that the proposed low-cost medium was approximately 45 times more effective than the commercial medium in terms of the number of cultivatable bacteria per unit price. The spores were then powdered via a spray-drying process to produce a spore powder with a spore count of 2.0 ± 0.7 × 109 CFU/g. The AK13 spore powder was mixed with cement paste, yeast extract, calcium lactate, and water. The yeast extract and calcium lactate generated the highest CFU/ml for AK13 at a 0.4:0.4 ratio compared to 0.4:0.25 (the original ratio of the B4 medium) and 0.4:0.8. Twenty-eight days after the spores were mixed into the mortar, the number of vegetative cells and spores of the AK13 strain had reached 106 CFU/g within the mortar. Cracks in the mortar under 0.29 mm were healed in 14 days. Calcium carbonate precipitation was observed on the crack surface. The mortar containing the spore powder was thus concluded to be effective in terms of healing micro-cracks.

Stochastic modeling of variability in survival behavior of Bacillus simplex spore population during isothermal inactivation at the single cell level using a Monte Carlo simulation.

The control of bacterial reduction is important to maintain food safety during thermal processing. The goal of this study was to illustrate and describe variability in bacterial population behavior during thermal processing as a probability distribution based on individual cell heterogeneity regarding heat resistance. Toward this end, we performed a Monte Carlo simulation via computer, and compared and validated the simulated estimations with observed values. Weibullian fitted parameters were estimated from the kinetic survival data of Bacillus simplex during thermal treatment at 94 °C. The variability in reductions of bacterial sporular populations was illustrated using Monte Carlo simulation based on the Weibull distribution of the parameters. In particular, variabilities in viable spore counts and survival probability of the B. simplex spore population were simulated in various replicates. We also experimentally determined the changes in survival probability and distributions of survival spore counts; notably, these were successfully predicted by the Monte Carlo simulation based on the kinetic parameters. The kinetic parameter-based Monte Carlo simulation could thus successfully illustrate bacterial population behavior variability during thermal processing as a probability distribution. The simulation approach may contribute to improving food quality through risk-based processing designs and enhance risk assessment model accuracy.

Growth of Clostridium perfringens in sous vide cooked ground beef with added grape seed extract.

The growth of Clostridium perfringens from spore inocula was studied in sous vide cooked ground beef with added 0 to 3% grape seed extract (GSE). C. perfringens did not grow at 4 °C with or without GSE present. Lag time (LT) was 95 h in control samples at 15 °C, whereas 1-3% GSE addition significantly (p < .05) extended LT to 244 h or longer. Generation time (GT) in 3% GSE added beef was similar to that of control (19 h, 3% GSE versus 18 h, control) at 15 °C. At 20 °C, GT was 1.5 h in samples without GSE; however, 1-3% GSE addition extended GT about 2-3 folds (p < .05). Lag time at 20 °C was 23 h in control samples, while LT was 40-59 h in samples containing GSE. Interestingly, GSE did not affect LT at 25 °C; however, significantly (p < .05) longer GT was observed in 3% GSE added samples than the other sample groups. Additionally, GSE from 1 to 3% in beef extended the period needed to reach 6 log cfu/g at 15 or 20 °C, while 3% GSE was required at 25 °C. The findings suggest that GSE exhibits concentration and temperature dependent inhibitory effect on growth of C. perfringens in sous vide cooked ground beef. Grape seed extract can be used to extend the shelf-life and ensure the microbiological safety of sous vide cooked meat products.

Low cost medium for spore production of Bacillus KKU02 and KKU03 and the effects of the produced spores on growth of giant freshwater prawn (Macrobrachium rosenbergii de Man).

In order to extend the shelf life of 2 high potential Bacillus probiotic isolates which were Bacillus KKU02 and Bacillus KKU03, the spore forms of these 2 Bacillus isolates were studied for using as probiotic instead. The low cost medium for spore production of these 2 Bacillus isolates was examined in order to produce probiotic spores for feeding the shrimps. It was found that cassava at 100 g L(-1) and supplemented with 20.0 g L(-1) dextrose, 0.1 g L(-1) MgSO4 and 2.0 g L(-1) (NH4)2SO4 showed the highest spore concentration at about 1 x 10(8) CFU mL(-1). The effects of feeding these 2 Bacillus spores on the growth of giant freshwater prawns were further examined. The spores of Bacillus KKU02 and Bacillus KKU03 (-10(7) spore mL(-1)) in pure and mixed culture forms were mixed with commercial prawn feed (200 mL kg(-1)) to give six feed treatments. Body length and weight of the prawns in mixed spore culture tanks after rearing for 90 days (13.5 cm and 59.8 g, respectively) were significantly higher (p = 0.05) than other treatments. The treated prawns were further challenged with Aeromonas hydrophila for 7 days. The percentages of survival after the challenge in the prawns fed with the mixed spores (46.8%) were also found significantly higher (p = 0.05) than others groups, except the mixed live cell treatment (60%). These results indicated that the spores of Bacillus KKU02 and Bacillus KKU03 had a high potential for using as commercial probiotics.

Glycerol-based sterilization bioindicator system from Bacillus atrophaeus: development, performance evaluation, and cost analysis.

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D (160 °C) value of 6.6 ± 0.1 min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3 ± 1.2 × 10(8) CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48 h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24 h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8 % (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.

A novel cost-effective medium for the production of Bacillus thuringiensis subsp. israelensis for mosquito control.

Bacillus thuringiensis subsp. israelensis (Bti) has been used for mosquito-control programmes the world-wide. Indeed, the large-scale production of Bti for mosquito control is very expensive due to the high cost of its culture. In the present study, we attempted to widen the scope in developing cost-effective culture medium for Bti production, based on the raw materials available on the biosphere, including coconut cake powder, CCP (Cocos nucifera), neem cake powder, NCP (Azadirachta indica) and groundnut cake powder, GCP (Arachis hypogea). Among these raw materials, the biomass production of Bti, sporulation and toxin synthesizing from 'CCP' in combination with mineral salt (MnCl(2)) was comfortably satisfactory. Bioassays with mosquito species (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) and field trials were also satisfactory. The present investigation suggests that coconut cake-based culture medium can be used as an alternative for industrial production of Bti in mosquito-control programme. Therefore, the study is very important from the point of effective production of Bti from cost-effective culture medium for the control of mosquito vectors.

Development of a low-cost sterilization biological indicator using Bacillus atrophaeus by solid-state fermentation.

The production of biological indicators involving bacterial sporulation and multi-step downstream processes has been described. The goal of the present work was to use fermented material as the final product in a biological indicator, thereby reducing processing steps and costs. The performance of three different inexpensive supports (vermiculite, sand, and sugarcane bagasse) was assessed by determining Bacillus atrophaeus sporulation during solid-state fermentation and by assessing the direct use of the fermentation products in the subsequent steps of the process. All three supports allowed spore production of between 10(7) and 10(9) CFU g(-1). Sand proved to be the best inert support enabling the direct use of the fermented product due to its easy homogenization, filling properties, and compatibility with recovery medium. Bacterial adhesion to the sand surface was supported by biofilm formation. The resistance to sterilization of the dried fermentation product was evaluated. For dry-heat resistance (160°C), the D value was 6.6 min, and for ethylene oxide resistance (650 mg/L), the D value was 6.5 min. The cost reduction of this process was at least 48%. No previous studies have been published on the application of sand as a support in solid-state fermentation for the production of biological indicators.

Effect of input data variability on estimations of the equivalent constant temperature time for microbial inactivation by HTST and retort thermal processing.

UNLABELLED: Consumer demand for food safety and quality improvements, combined with new regulations, requires determining the processor's confidence level that processes lowering safety risks while retaining quality will meet consumer expectations and regulatory requirements. Monte Carlo calculation procedures incorporate input data variability to obtain the statistical distribution of the output of prediction models. This advantage was used to analyze the survival risk of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and Clostridium botulinum spores in high-temperature short-time (HTST) milk and canned mushrooms, respectively. The results showed an estimated 68.4% probability that the 15 sec HTST process would not achieve at least 5 decimal reductions in M. paratuberculosis counts. Although estimates of the raw milk load of this pathogen are not available to estimate the probability of finding it in pasteurized milk, the wide range of the estimated decimal reductions, reflecting the variability of the experimental data available, should be a concern to dairy processors. Knowledge of the C. botulinum initial load and decimal thermal time variability was used to estimate an 8.5 min thermal process time at 110 °C for canned mushrooms reducing the risk to 10⁻9 spores/container with a 95% confidence. This value was substantially higher than the one estimated using average values (6.0 min) with an unacceptable 68.6% probability of missing the desired processing objective. Finally, the benefit of reducing the variability in initial load and decimal thermal time was confirmed, achieving a 26.3% reduction in processing time when standard deviation values were lowered by 90%. PRACTICAL APPLICATION: In spite of novel technologies, commercialized or under development, thermal processing continues to be the most reliable and cost-effective alternative to deliver safe foods. However, the severity of the process should be assessed to avoid under- and over-processing and determine opportunities for improvement. This should include a systematic approach to consider variability in the parameters for the models used by food process engineers when designing a thermal process. The Monte Carlo procedure here presented is a tool to facilitate this task for the determination of process time at a constant lethal temperature.

Effect of olive powder and high hydrostatic pressure on the inactivation of Bacillus cereus spores in a reference medium.

The aim of this work was to study the effect of olive powder combined with high hydrostatic pressure (HHP) on the inactivation of Bacillus cereus spores, to use it as an additional control hurdle in beverages pasteurised by this technology. With this purpose, reference medium prepared at different concentrations of olive powder was inoculated with B. cereus spores and subjected to different pressure treatments. The outgrowth capacity of the treated spores was then determined at 20°C and 32°C. The addition of olive powder was found to slightly reduce the effectiveness of HHP, although in post-treatment storage there was an increased bacteriostatic effect in the samples with 2.5% of olive powder at both temperatures in the samples pressurised at 400 and 500 MPa, and only at 20°C in the samples pressurised at 200 MPa. The addition of olive powder therefore had an additive effect with storage temperature and HHP processing and could act as an additional control hurdle during the shelf-life of products pasteurised by HHP technologies or in the case of cold-chain breakage.

Challenges in risk assessment and predictive microbiology of foodborne spore-forming bacteria.

Mathematical description of the behavior of bacterial foodborne pathogens and concepts of risk assessment were first applied to spore-forming bacteria and specially to Clostridium botulinum with numerous works dealing with spores heat destruction to ensure the safety of canned foods or with their germination and growth probability in foods. This paper discusses two aspects which appear specific to pathogenic sporeformers in comparison to vegetative microorganisms, that is, firstly, the extreme intra-species biodiversity of spore-forming bacteria and its consequences for risk assessment and, secondly, the modeling of spore germination and outgrowth processes. The intra-species biodiversity of spore-forming bacteria has a great impact on hazard identification, exposure assessment and hazard characterization leading thus to an extremely variable individual poisoning risk for consumers. The germination and outgrowth processes were shown independent at the single cell level and although numerous studies were performed to study the effect of spores treatments and growth conditions on these two events, the mathematical modeling and the prediction of these processes is still challenging today. The difficulties to accurately assess the biodiversity and the germination and outgrowth processes of spore-forming bacteria lead to a substantial uncertainty in risk estimates related to the exposure to these microorganisms. Nevertheless, significant progress have been made these last years improving the relevance of quantitative risk assessments for spore-forming bacteria and decreasing the risk uncertainty. Despite these difficulties, risk assessment still constitutes a valuable tool to justify the implementation of management options.

Quantitative risk assessment for hazards that arise from non-proteolytic Clostridium botulinum in minimally processed chilled dairy-based foods.

A modular process risk model has been constructed that describes the manufacture of dairy dessert products and hazards that arise from non-proteolytic Clostridium botulinum. The model describes batch manufacture and consumer storage of a family size generic dairy dessert but includes a realistic quantification that could apply to a specific food product. The dairy dessert sector is an expanding part of the UK market. The model includes modules that describe spore loads in raw materials, spore inactivation during thermal processing, volume partition and the population kinetics for non-proteolytic C. botulinum during sequential isothermal storage regimes. Where possible elements of uncertainty and variability are identified explicitly. The model is constructed as a belief network from published data and expert opinions. The model provides marginal probabilities, and associated sensitivities, for a range of endpoint measures centred on the toxicity of a single retail unit after an extended period of storage. The decimal reduction time for non-proteolytic C. botulinum spore populations at the highest (hold) temperature of the primary thermal process and the highest temperature experienced during poorly controlled (consumer) storage are dominant factors determining risks. Priorities for additional information to support risk assessments have been identified.

Assessment of Clostridium perfringens spore response to high hydrostatic pressure and heat with nisin.

The elimination of spores from low-acid foods presents food-processing and food-safety challenges to high-pressure processing (HPP) developers as bacterial spores are extremely resistant to pressure. Therefore, the effects of pressure (400-800 MPa), temperature (35-95 °C), and nisin (0-496 IU/mL) on the inactivation of Clostridium perfringens AS 64701 spores at various pressure-holding times (7.5-17.5 min) were explored. A second-order polynomal equation for HPP- and nisin-induced inactivation of C. perfringens spores was constructed with response surface methodology. Experiment results showed that the experimental values were shown to be significantly in agreement with the predicted values because the adjusted determination coefficient (R (Adj)²) was 0.9708 and the level of significance was P < 0.0001. The optimum process parameters (obtained by solving the quadratic polynomal equation) for a six-log cycle reduction of C. perfringens AS 64701 spores were pressure of 654 Mpa, temperature of 74 °C, pressure-holding time of 13.6 min, and nisin concentration of 328 IU/mL. The validation of the model equation for predicting the optimum response values was verified effectively by ten test points that were not used in the establishment of the model. Compared with conventional HPP techniques, the main process advantages of HPP-nisin combination sterilization in the UHT milk are, lower pressure, temperature, natural preservative (nisin), and in a shorter treatment time. The synergistic inactivation of bacteria by HPP-nisin combination is a promising and natural method to increase the efficiency and safety of high-pressure pasteurization.

Spatial simulations of myxobacterial development.

Many bacteria exhibit multicellular behaviour, with individuals within a colony coordinating their actions for communal benefit. One example of complex multicellular phenotypes is myxobacterial fruiting body formation, where thousands of cells aggregate into large three-dimensional structures, within which sporulation occurs. Here we describe a novel theoretical model, which uses Monte Carlo dynamics to simulate and explain multicellular development. The model captures multiple behaviours observed during fruiting, including the spontaneous formation of aggregation centres and the formation and dissolution of fruiting bodies. We show that a small number of physical properties in the model is sufficient to explain the most frequently documented population-level behaviours observed during development in Myxococcus xanthus.

Use of sensitivity analysis to aid interpretation of a probabilistic Bacillus cereus spore lag time model applied to heat-treated chilled foods (REPFEDs).

The microbiological safety and quality of REfrigerated Processed Foods of Extended Durability (REPFEDs) relies on a combination of mild heat treatment and refrigeration, sometimes in combination with other inhibitory agents that are not effective when used alone. In this context, the output of a probabilistic model predicting the lag time of heat-treated Bacillus cereus spores under realistic heat-treatment profile and chilled supply-chain conditions, has been investigated using a sensitivity analysis technique. Indeed, knowing that there was uncertainty in the model (e.g. due to lack of data to build the model input probability density function), the objective of the analysis was to evaluate if the variability associated with some inputs (e.g. the consumers' refrigerator temperature values reported in Europe and US markets were different) had a significant impact on the model output, i.e. on the expected lag time of heat-treated B. cereus spores in REPFEDs. To do so, the uncertainty and variability associated with the various model inputs have been identified and then separated using a second order Monte Carlo decomposition. Concerning the variability, there was a significant difference between the chilled supply-chains (Europe, US) and between the raw material groups (low, medium or high contamination levels). For example, in the European market, after a heat treatment of 90 degrees C for 10 min, with a high raw material contamination level, the predicted 5th percentile of the lag time was 17 days, while it was 35 days with a low raw material contamination level. This was confirmed with an ANOVA. The impact of the uncertainty on the lag time has been illustrated graphically by building confidence intervals around its 5th percentile. A sensitivity analysis based upon uncertainty and variability decomposition is clearly a complex and time consuming exercise; however, it provides a greater confidence (greater transparency and better understanding) in the model output when making food safety decisions (e.g. determining the safe shelf-life of REPFEDs).

Egg yolk enhances early sporulation and toxicity of Bacillus sphaericus H5a5b for small-scale production of a mosquito control agent.

Bacillus sphaericus has been widely used in mosquito control programs, but the production of this bacterium is a little tricky as it does not utilize carbohydrates and requires proteinaceous substrates, which are expensive. In this study, we developed a cost-effective medium that resulted in a lower cost and shorter fermentation time. The locally available raw material, egg yolk was used and the level of sporulation, toxicity and biomass were compared with the conventional medium. Use of the egg yolk culture medium significantly shortened fermentation time to 15 h and yielded high activity, equivalent to that of conventional medium against 3rd instar Culex quinquefasciatus. Conventional NYSM medium required 21 h to attain the maximum activity and biomass. Hence, the egg yolk-based culture medium appears to be suitable and economical for the small-scale production of B. sphaericus.

Selection and optimization of Bacillus atrophaeus inoculum medium and its effect on spore yield and thermal resistance.

Bacillus atrophaeus's spores are used as biological indicators to monitor sterilization processes and as a Bacillus anthracis surrogate in the development and validation of biosafety methods. The regular use of biological indicators to evaluate the efficiency of sterilization processes is a legal requirement for health services. However, its high cost hinders its widespread use. Aiming at developing a cost-effective inoculum medium, soybean molasses and nutrient-supplemented vinasse were evaluated for their effectiveness in solid-state fermentation (SSF). In biomass production, the results demonstrated that all tested compositions favor growth by providing the nutritional demands of the microorganism. Optimum casein peptone and soybean molasses concentration (1.0%, 2.5%, or 4.0%) was determined by a 2((2-0)) factorial experimental design. The results have showed a positive influence of peptone on biomass production. In order to define peptone final concentration (4.0% or 6.0%), a 2(2) factorial experimental design was used. An optimized medium containing 4.0% soybean molasses and 4.0% casein peptone was similar in performance to a synthetic control medium (tryptone soy broth) in dry-heat thermal-resistant spore production by SSF. An experiment performed under optimum SSF conditions resulted in 1.9 x 10(10) CFU g(-1) dry matter with D (160 degrees C) = 5.2 +/- 0.2 min.