Spore Crops Research Articles

Germination and outgrowth of Bacillus mycoides KBAB4 spores are impacted by environmental pH, quantitatively analyzed at single cell level with sporetracker

Quantifying spore germination and outgrowth heterogeneity is challenging. Single cell level analysis should provide supplementary knowledge regarding the impact of unfavorable conditions on germination and outgrowth dynamics. This work aimed to quantify the impact of pH on spore germination and outgrowth, investigating the behavior of individual spore crops, produced under optimal and suboptimal conditions. Bacillus mycoides (formerly B. weihenstephanensis) KBAB4 spores, produced at pH 7.4 and at pH 5.5 were incubated at different pH values, from pH 5.2 to 7.4. The spores were monitored by microscopy live imaging, in controlled conditions, at 30 °C. The images were analyzed using SporeTracker, to determine the state of single cells. The impact of pH on germination and outgrowth times and rates was estimated and the correlation between these parameters was quantified. The correlation between germination and outgrowth times was significantly higher at low pH. These results suggest that an environmental pressure highlights the heterogeneity of spore germination and outgrowth within a spore population. Results were consistent with previous observations at population level, now confirmed and extended to single cell level. Therefore, single cell level analyses can be used to quantify the heterogeneity of spore populations, which is of interest in order to control the development of spore-forming bacteria, responsible for food safety issues.

Comparison of Sodium Nitrite and ‘Natural’ Nitrite on the Inhibition of Spore Germination and Outgrowth of Clostridium sporogenes in Low- and High-Fat Frankfurters

In the US, sodium and potassium nitrite are regulated food preservatives that prevent the germination of Clostridium spores in cured and processed meats. In recent years, the use of vegetable-derived nitrite (i.e., vegetable nitrate fermented to nitrite) has been designated as ‘natural nitrite’ to accommodate natural meats that cannot use artificial ingredients, and such meat products can be labelled as having ‘no added preservatives’. This new status and labelling allowance for microbially-modified nitrite provides for a ‘clean label’ application of nitrite against the stigma of chemical ingredients and has found increased use within the processed meat industry. The objectives of this study were to examine Clostridium sporogenes as a pathogen-surrogate challenge organism and the use of vegetable (celery) nitrite to prevent spore germination in cooked meat products. A three-strain spore crop of C. sporogenes ATCC 3584, ATCC 19404 and ATCC BAA-2695 was applied during ingredient formulation of low and high-fat hotdogs that were divided into three sub-batches (control without nitrite, hotdogs with sodium nitrite, hotdogs with celery nitrite). In both low and high-fat processes, sodium nitrite was compared to hotdogs made with comparable levels of celery nitrite (156 ppm). All treatments were performed with duplicate trial replication and triplicate sample testing within each trial. Comparisons were analyzed by repeated measures analysis of variance to determine significant difference (p < 0.05) of time course treatments. In shelf-life assays, growth was inhibited at both 5 °C and 15 °C, even if nitrite was absent; however, spore germination and growth readily occurred at 35 °C. Comparison of nitrite effects was best evaluated at 35 °C as a permissive condition to examine the effects of nitrite treatments. Celery nitrite showed no significant difference from sodium nitrite when used in both low and high-fat hotdogs, and spore outgrowth was only observed after 2–3 days at 35 °C compared to hotdogs without nitrite. Application of bacteriocin preparations in the formulation that were effective against Listeria monocytogenes, and moderately inhibitory towards the 3-strain spore mixture of C. sporogenes, were not effective in spore control in manufactured hotdogs. The nitrite validation hotdog trials described herein demonstrates that (celery or sodium) nitrite may prevent Clostridium spore germination for 24–48 h even under permissive conditions to help keep processed meat safe.

Two complementary approaches to quantify variability in heat resistance of spores of Bacillus subtilis

Realistic prediction of microbial inactivation in food requires quantitative information on variability introduced by the microorganisms. Bacillus subtilis forms heat resistant spores and in this study the impact of strain variability on spore heat resistance was quantified using 20 strains. In addition, experimental variability was quantified by using technical replicates per heat treatment experiment, and reproduction variability was quantified by using two biologically independent spore crops for each strain that were heat treated on different days. The fourth-decimal reduction times and z-values were estimated by a one-step and two-step model fitting procedure. Grouping of the 20 B. subtilis strains into two statistically distinguishable groups could be confirmed based on their spore heat resistance. The reproduction variability was higher than experimental variability, but both variabilities were much lower than strain variability. The model fitting approach did not significantly affect the quantification of variability. Remarkably, when strain variability in spore heat resistance was quantified using only the strains producing low-level heat resistant spores, then this strain variability was comparable with the previously reported strain variability in heat resistance of vegetative cells of Listeria monocytogenes, although in a totally other temperature range. Strains that produced spores with high-level heat resistance showed similar temperature range for growth as strains that produced low-level heat resistance. Strain variability affected heat resistance of spores most, and therefore integration of this variability factor in modelling of spore heat resistance will make predictions more realistic.

Lethality enhancement of pressure-assisted thermal processing against Bacillus amyloliquefaciens spores in low-acid media using antimicrobial compounds

Pressure-assisted thermal processing (PATP; 500–700 MPa, 90–121 °C) offers new opportunities to sterilize low-acid foods while preserving quality attributes to an extent greater than is possible with traditional thermal processing (TP). This study was conducted to evaluate the possibility of enhancing PATP lethality against the spores of Bacillus amyloliquefaciens, by sensitizing the spores with selected antimicrobial compounds (including emphasis on the use of natural antimicrobials) prior to treatment. A spore crop of B. amyloliquefaciens TMW 2.479 Fad 82, that had previously shown high resistance to combined pressure-heat treatment, was prepared on Nutrient Agar medium supplemented with 10 mg L−1 MnSO4·H2O and incubated at 32 °C for 3 d. Spores were inoculated (at ∼107–108 CFU mL−1 inoculum level) in HEPES buffer (pH ≤ 7.0) or selected low-acid foods (pH 5.2–5.6) with or without added antimicrobial compounds. The samples were then treated at 600 MPa and 105 °C (PATP) or 0.1 MPa and 105 °C (TP) for various holding times. Among different compounds tested, low-molecular-weight chitosan, and combination of chitosan with some surfactants were most effective (P < 0.05) in enhancing the PATP and TP lethality. This study suggests that certain antimicrobials can be added to the low-acid media prior to PATP or TP treatment to enhance the efficacy of the process. The treatment allows sterilization of low-acid foods at lower process temperatures thus ensuring better preservation of quality attributes.

Effect of Sporulation Temperature on the Resistance of Clostridium botulinum Type A Spores to Thermal and High Pressure Processing

The purpose of this study was to determine the effect of sporulation temperature on the resistance of Clostridium botulinum type A spores of strains 62A and GiorgioA to thermal and high pressure processing (HPP). Spore crops produced in Trypticase-peptone-glucose-yeast extract broth at four incubation temperatures (20, 27, 37, and 41°C) were harvested, and heat resistance studies were conducted at 105°C (strain 62A) and 100°C (strain GiorgioA). Resistance to HPP was evaluated by subjecting the spores to a high pressure (700 MPa) and temperature combination (105°C, strain 62A; 100°C strain GiorgioA) in a laboratory-scale pressure test system. The decimal reduction time (D-value) was calculated using the log-linear model. Although the time to sporulation for GiorgioA was shorter and resulted in higher spore concentrations than for 62A at 20, 27, and 37°C, GiorgioA did not produce a sufficient spore crop at 41 °C to be evaluated. The heat resistance of 62A spores was greatest when produced at 27°C and decreased for spore crops produced above or below 27°C (D105°C-values: 20°C, 1.9 min; 27°C, 4.03 min; 37°C, 3.66 min; and 41°C, 3.5 min; P < 0.05). Unlike 62A, the heat resistance behavior of GiorgioA spores increased with rising sporulation temperature, and spores formed at the organism's optimum growth temperature of 37°C were the most resistant (D100°C-values: 20°C, 3.4 min; 27°C, 5.08 min; and 37°C, 5.65 min; P < 0.05). Overall, all spore crops were less resistant to pressure-assisted thermal processing than thermal treatment alone. Sporulation temperature has an effect on the resistance of C. botulinum spores to heat and HPP, and is characteristic to a particular strain. Knowledge of the effect of sporulation temperature on the resistance of C. botulinum spores is vital for the production of spores utilized in thermal and high pressure inactivation studies.

Poster #M113 THREATENING AND INTRUSIVE LIFE EVENTS AS PRECURSORS TO PSYCHOTIC DISORDERS

The microbiological criteria of commercial cocoa powder are defined in guidelines instituted by the cocoa industry. Twenty-five commercial samples were collected with the aim of assessing the compliance with the microbiological quality guidelines and investigating the occurrence and properties of aerobic Thermoresistant Spores (ThrS). Seventeen samples complied with the guidelines, but one was positive for Salmonella, five for Enterobacteriaceae and two had mould levels just exceeding the maximum admissible level. The treatment of the cocoa powder suspensions from 100 °C to 170 °C for 10 min, revealed the presence of ThrS in 36% of the samples. In total 61 ThrS strains were isolated, of which the majority belonged to the Bacillus subtilis complex (65.6%).Strains resporulation and spore crops inactivation at 110 °C for 5 min showed a wide diversity of heat-resistance capacities. Amplified fragment length polymorphism analysis revealed not only a large intraspecies diversity, but also different clusters of heat-resistant spore-forming strains. The heat-resistance of spores of six B. subtilis complex strains was further examined by determination of their D and z-values.We concluded that B. subtilis complex spores, in particular those from strain M112, were the most heat-resistant and these may survive subsequent preservation treatments, being potentially problematic in food products, such as chocolate milk.

Study of the influence of sporulation conditions on heat resistance of Geobacillus stearothermophilus used in the development of biological indicators for steam sterilization

Biological indicators are important tools in infection control via sterilization process monitoring. The use of a standardized spore crop with a well-defined heat resistance will guarantee the quality of a biological indicator. Ambient factors during sporulation can affect spore characteristics and properties, including heat resistance. The aim of this study is to evaluate the main sporulation factors responsible for heat resistance in Geobacillus stearothermophilus, a useful biological indicator for steam sterilization. A sequence of a three-step optimization of variables (initial pH, nutrient concentration, tryptone, peptone, beef extract, yeast extract, manganese sulfate, magnesium sulfate, calcium chloride and potassium phosphate) was carried out to screen those that have a significant influence on heat resistance of produced spores. The variable exerting greatest influence on G. stearothermophilus heat resistance during sporulation was found to be the initial pH. Lower nutrient concentration and alkaline pH around 8.5 tended to enhance decimal reduction time at 121°C (D(121°C)). A central composite design enabled a fourfold enhancement in heat resistance, and the model obtained accurately describes positive pH and negative manganese sulfate concentration influence on spore heat resistance.

Kinetics of Bacillus coagulans spore inactivation in tomato juice by combined pressure–heat treatment

The efficacy of combined pressure–heat treatment was evaluated for the inactivation of Bacillus coagulans spores in tomato juice. A spore crop of B. coagulans 185A was prepared on nutrient agar plus 500mgL−1 dextrose and 3mgL−1 MnSO4·H2O, incubated at 50°C for 7d. Spores were suspended (1.7×108CFUmL−1) in tomato juice (pH 4.23) and treated at 600MPa and process temperatures of 75, 85, 95, 100 and 105°C, or 0.1MPa and process temperatures of 100 and 105°C for ≤15min. The inactivation data was fitted to estimate kinetic inactivation parameters using selected linear and non-linear models. The models were evaluated using the mean square error, regression coefficient and accuracy factor. Depending upon the process temperature, a combined pressure–heat treatment reduced the spore population by 1.5–3.4 logs during the 30s pressure come-up time. Pressure holding times of 10, 7, 4 and 3min and 40s were sufficient to inactivate the spores to below the detection limit (<10CFUmL−1) at 600MPa and 75, 85, 95, 100 and 105°C, respectively with no microbial growth during subsequent storage of samples at 37°C. In contrast, the application of heat alone needed holding times of 13 and 4min, at 100 and 105°C, respectively to inactivate the spores to undetectable level with no growth during storage. The D-values of spores inactivated by combined pressure–heat treatment ranged from 0.19 (600MPa, 105°C) to 2.10min (600MPa, 75°C), while they were 1.66 and 0.59min at 100 and 105°C, respectively under atmospheric conditions. Kinetic model analysis indicated relatively good fits of linear and Weibull models to the pressure–heat inactivation data at process temperatures of 75–95°C. The log-logistic model provided best fitting at process temperatures of 95–105°C. This study suggests that combined pressure–heat treatment may be used as a viable alternative to inactivate B. coagulans spores in acidic food products, like tomato juice.

Influence of fertiliser application on the occurrence and colonisation of arbuscular mycorrhizal fungi (AMF) under maize/Centrosema and sole maize systems

Soil fertility management practices can influence colonisation of crops by arbuscular mycorrhizal fungi (AMF) and their abundance. The effects of different rates of nitrogen-phosphorus-potassium (NPK) fertiliser on AMF occurrence and colonisation were studied in maize/Centrosema pascuorum and sole maize systems. The NPK treatments were at rates (kg/ha): 0-10-30, 45-10-30, and 0-0-0 (control). The AMF spore populations were enumerated by direct counting under a microscope. Nutrient uptake was calculated as the product of nutrient concentration and shoot dry weight, and maize yield was estimated per ha. In the maize/Centrosema system, spore count, AMF colonisation, and nutrient uptake (except N) decreased with NPK 45-10-30 compared with 0-10-30, although maize yields were comparable at the two fertiliser levels. In the sole maize system, fertiliser application did not influence AMF spore abundance, but colonisation, nutrient uptake, and crop yield increased significantly (P &lt; 0.05) with NPK 45-10-30. Maize yield increased by 1200% under the maize/Centrosema system compared with sole maize at NPK 0-10-30. The lowest values for all parameters were obtained under the control treatments. Colonisation of AMF, nutrient uptake, and maize yield were positively correlated. The maize/Centrosema system can maximise AMF benefits to increase yield and also reduce fertiliser input into agricultural soils, while application of N fertiliser is important to increase yield in the sole maize system.

USE OF ATTENUATED TOTAL REFLECTANCE INFRARED MICROSPECTROSCOPY TO DISCRIMINATE BACILLUS SPORES

In the present study, the potential of Fourier-transform infrared microspectroscopy to differentiate and discriminate Bacillus spp. spores that sporulated in two nonselective media were evaluated. Spore crops of Bacillus subtilis (ATCC 11779), Bacillus thuringiensis (ATCC 13368, ATCC 35866 and ATCC 55177), Bacillus amyloliquefacies (ATCC 496763) and Bacillus stearothermophilus (ATCC 7953) were obtained with Nutrient and Trypticase Soy Agar supplemented with 10 ppm of MnSO4.H2O. Spores were sonicated, washed with deionized water, and pellets (108 cfu/mL) were deposited onto the grids of hydrophobic membrane filters and dried to produce a uniform and thin film. Spectra were collected in the attenuated total reflectance mode in the mid-infrared region (4,000–700 cm−1). Our classification models, soft independent modeling of class analogy obtained from derivatized infrared spectra (1,800–900 cm−1), showed that the discrimination among spore crops was mainly attributed to infrared frequencies of spore coat proteins. PRACTICAL APPLICATIONS Despite regulatory efforts and emergence of new processing technologies, food-related illnesses remain a major concern for consumers and producers. Detection of bacterial spores using conventional methods is time consuming and challenging. Therefore, there is a need to develop rapid, simple and cost-effective methods to detect Bacillus spores. Fourier-transform infrared microspectroscopy is a viable technology to meet these demands.

Microbiota of cocoa powder with particular reference to aerobic thermoresistant spore-formers

The microbiological criteria of commercial cocoa powder are defined in guidelines instituted by the cocoa industry. Twenty-five commercial samples were collected with the aim of assessing the compliance with the microbiological quality guidelines and investigating the occurrence and properties of aerobic Thermoresistant Spores (ThrS). Seventeen samples complied with the guidelines, but one was positive for Salmonella, five for Enterobacteriaceae and two had mould levels just exceeding the maximum admissible level. The treatment of the cocoa powder suspensions from 100 °C to 170 °C for 10 min, revealed the presence of ThrS in 36% of the samples. In total 61 ThrS strains were isolated, of which the majority belonged to the Bacillus subtilis complex (65.6%). Strains resporulation and spore crops inactivation at 110 °C for 5 min showed a wide diversity of heat-resistance capacities. Amplified fragment length polymorphism analysis revealed not only a large intraspecies diversity, but also different clusters of heat-resistant spore-forming strains. The heat-resistance of spores of six B. subtilis complex strains was further examined by determination of their D and z-values. We concluded that B. subtilis complex spores, in particular those from strain M112, were the most heat-resistant and these may survive subsequent preservation treatments, being potentially problematic in food products, such as chocolate milk.

Characterization of Bacillus sporothermodurans IC4 spores; putative indicator microorganism for optimisation of thermal processes in food sterilisation

Spore-forming bacteria with high heat resistance increasingly challenge industrial sterilisation processes in foods. To ensure stability of manufactured foods, generally worst case scenarios are applied often leading to unwanted over processing of foods. This means bigger requirements of energy and larger emissions of CO2 into the atmosphere. A heat-resistant spore-forming bacterium, Bacillus sporothermodurans, was tested for its ability to produce consistently highly heat-resistant spore crops to be used to more precisely determine the minimal thermal process that is compatible with microbial stability, thus avoiding the indicated overprocessing. Isothermal survival curves of B. sporothermodurans IC4 spores were determined in distilled water at temperatures up to 125 °C using the capillary tube technique and a thermoresistometer Mastia. B. sporothermodurans consistently produced crops of heat-resistant spores with kmax values at 121 °C up to 0.46 min−1. After consecutive sporulation cycles, the measured heat resistance of B. sporothermodurans was not reduced. Survival curves showed shoulders that were characterized by means of existing models (Geeraerd, Weibull) that were compared with the classical, log-linear one. Shoulders are a common phenomenon in heat-resistant spore-forming bacteria and may be related to the need of more stringent processing conditions. B. sporothermodurans spores were considered adequate to optimize sterilisation processes of low acid foods under a wide variety of conditions. Establishing precise heat treatments can be a way to guarantee food safety and stability while reducing the use of energy and contaminating emissions.

Effects of minerals on sporulation and heat resistance of Clostridium sporogenes

In this study, various mineral supplements, such as chloride salts (CaCl 2, MgCl 2, MnCl 2, FeCl 2 and KCl) supplying cations and calcium salts (CaCl 2, CaCO 3, CaSO 4, Ca(OH) 2 and CaHPO 4) supplying anions, were tested if they could stimulate the sporulation of Clostridium sporogenes, a surrogate microorganism for C. botulinum. Of the cations tested, the addition of CaCl 2 showed a slightly, but not significantly, greater increase in spore levels within 3 weeks of incubation, compared to that of the other cations. The optimum concentration of CaCl 2 was 0.5%, which yielded nearly 10 4 CFU/ml of spores. Of the anions tested, CaCO 3 promoted sporulation within one week, which was the most effective compound for promoting rapid sporulation among the minerals tested. CaSO 4 produced a pattern of sporulation similar to that of CaCl 2. While CaHPO 4 resulted in the maximum production of spores after 4 weeks, Ca(OH) 2 failed to induce sporulation. With an optimized concentration of 0.5% CaCO 3, the spore yield was approximately 10 5 CFU/ml. The spores prepared in sporulation medium with CaCO 3 (pH 5.0) had slightly, but not significantly, higher D values than those produced with CaCl 2 (pH 5.0) at temperatures ranging from 113 to 121 °C. However, no significant differences were observed in Z values (both 10.76 °C). In a large scale spore production, D 121 °C values of the spore crops prepared with CaCl 2 and CaCO 3 and resuspended in phosphate buffer (pH 7.0) were found to be both 0.92 min. In conclusion, our data suggest that CaCO 3 is highly effective in reducing sporulation time as well as enhancing heat resistance.

THERMAL RESISTANCE OF BACILLUS STEAROTHERMOPHILUS SPORES IN DRIED PASTA AT DIFFERENT STAGES OF REHYDRATION

The thermal resistance of Bacillus stearothermophilus spores at different stages of rehydration was examined. Inoculation was achieved by rehydrating pasta samples in water containing 1-mL spore crop (10 6 spores/mL); the product was heat sealed in sterile pouches and processed in a laboratory retort. Thermal inactivation of spores was carried out using 10 time intervals at 121C for moisture contents, 70, 90, 105, 115, 125 and 145% dry basis. The death curves obtained consisted of two phases, an initial rapid decline (1.5 to 2.6 log reductions) followed by a slower, linear decrease of survivors from which the decimal reduction time (D value) was calculated. D 121 values were found to decrease with increasing moisture content and ranged from 4.6 to 6.5 min. The thermal resistance constant (z value) was examined to assess the effect of temperature (110-125C) on lethality, z values decreased with increasing moisture content and ranged from 10.7 to 15.6C. The results of this study demonstrate that temperature and moisture content influence the effectiveness of the intended heat process. For products that are rehydrated during heat treatment, the moisture content of the product is a critical factor. Therefore, if complete rehydration is not achieved, this may result in the heat process being less effective for the destruction of microorganisms, which may have implications in regard to food safety.

Assessment of Heat Resistance of Bacterial Spores from Food Product Isolates by Fluorescence Monitoring of Dipicolinic Acid Release

This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105 degrees C, 120 degrees C, and 131 degrees C, respectively. The estimated Z values were 6.3 degrees C, 6.1 degrees C, and 9.7 degrees C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (Tc), the temperature at which half the DPA content has been released within a fixed incubation time. We found Tc values for spores from Bacillus strains 168, A163, and IC4 of 108 degrees C, 121 degrees C, and 131 degrees C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.

Microbiological Validation of Microwave‐Circulated Water Combination Heating Technology by Inoculated Pack Studies

ABSTRACTA 915‐MHz Microwave‐Circulated Water Combination (MCWC) heating technology was validated for a macaroni and cheese product using inoculated pack studies. Before the tests, heat resistances of a Clostridium sporogenes (PA 3679) spore crop were determined in neutral phosphate buffer and macaroni and cheese product. Trays of macaroni and cheese products were subjected to 3 processing levels: target process (F0= 2.4), under target process (F0= 1.2), and over target process (F0= 4.8). The inoculated packs were evaluated by count‐reduction method and end‐point method. The microbial results showed that microbial destruction resulting from MCWC heating technology matched the calculated degree of sterilization (F0 value). This study suggests that the MCWC heating technology has potential in sterilizing packaged foods.

Quantifying the Rate of Release and Escape of Phytophthora infestans Sporangia from a Potato Canopy

ABSTRACT A means for determining the rate of release, Q (spores per square meter per second), of spores from a source of inoculum is paramount for quantifying their further dispersal and the potential spread of disease. Values of Q were obtained for Phytophthora infestans sporangia released from an area source of diseased plants in a potato canopy by comparing the concentrations of airborne sporangia measured at several heights above the source, with the concentrations predicted by a Lagrangian Stochastic simulation model. An independent estimate of Q was obtained by quantifying the number of sporangia per unit area of source at the beginning of each sampling day by harvesting diseased plant tissue and enumerating sporangia from these samples. This standing spore crop was the potential number of sporangia released per area of source during the day. The standing spore crop was apportioned into time segments corresponding to sporangia concentration measurement periods using the time trace of sporangia sampled above the source by a Burkard continuous suction spore sampler. This apportionment of the standing spore crop yielded potential release rates that were compared with modeled release rates. The two independent estimates of Q were highly correlated (P = 0.003), indicating that the model has utility for predicting release rates for P. infestans sporangia and the spread of disease between fields.

Enhanced sporulation in Bacillus subtilis grown on medium containing glucose:ribose

Spore crops of Bacillus subtilis PS 346 were increased by the addition of a combination of glucose and ribose to the sporulating medium. The increase in spore yield was over 100% higher in glucose:ribose-containing cultures, compared with cells grown on glucose as a sole carbon and energy source. Spore crops obtained from cultures grown on glucose:ribose had similar thermal resistance and hydrodynamic mean radius to those obtained when cultivated solely on glucose- or ribose-containing media. When other combinations of dual carbohydrates were tested it was apparent that those substrates primarily channelled through the pentose phosphate pathway gave enhanced sporulation. This effect was also observed by supplementing glucose-containing media with pyruvate but not citrate or malate. Those substrates that are channelled through the glycolytic pathway were also less effective in this respect. From the results obtained it appears that the enhancement of sporulation could be attributed to alteration in carbon and electron flow through metabolic pathways leading, directly or indirectly, to metabolites that play a role in sporogenesis regulation.

Rapid particle size distribution analysis of Bacillus spore suspensions

Knowledge of the homogeneity of a spore crop is an essential criteria for the subsequent investigation of their properties. Two methods of analysis have been compared; particle size distributions of Bacillus spores suspensions were analysed by laser light scattering with a Malvern Mastersizer; and scanning transmission electron microscopy (STEM) was used to record images of 50–100 individual spores. With an image particle sizing analysis software the dimensions (length, breadth, perimeter) of single spores were determined. Good agreement between the average individual spore dimensions determined by electron microscopy and those obtained by light scattering was evident. The Mastersizer was convenient and rapid to use but the image analysis provided the opportunity to distinguish between spores, vegetative cells and other particles. Spore suspensions were further separated from vegetative cells by centrifugation in continuous and discontinuous Percoll (modified colloidal silica) density gradients (1.09–1.13 g/ml). Electron microscopy revealed an adhesion of colloidal silica particles to the spore surface which could only be partially removed by repetitive washing. High silica contents were therefore detected by X-ray microanalysis. Percoll did not effect differential scanning calorimetric analysis of spores.

Influence of sporulation awon heat resistance and germination ofPenicillium roquefortispores

Spores ofPenicillium roquefortiwere produced using potato dextrose agar which was solute agjusted using either NaCl, sucrose, or glycerol to water activity (aw) levels of: 0·99 (control), 0·97, 0.·90 and 0·88. All spore crops, regardless of the sporulation awon which they were produced, exhibited near total germination within 24 h at 22°C in sterile water. However, when spores were initially heated either in water (42°C, 2 h or 55°C, 10 min) or in air (60°C, 20 min) and then germinated, levels were observed to decrease particularly for spores produced on 0·88 awadjusted media. Germination levels were also observed to decrease with time when harvested spores were initially suspended in sterile distilled water (0 to 24 h, 2°C) and then heated. Control spores (0·99 aw) showed no effects. Heat studies of spores suspended in water were used to calculate thermal decimal reduction times (D). Control spores and spores produced at 0·88 awwith glycerol, exhibitedD55°Cvalues of 10·3and 2·3 min, respectively. Specific effects attributable to the nature of the solute appeared to influenceDvalues; however, no clear trend was observed. The results of this study indicated that spores that were produced at reduced awbecame heat-sensitized perhaps owing to injury and/or damage incurred during sporogenesis.