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Thermal processing remains the major technology in food industry while it inevitably destructs sensory, nutrition and functional ingredients of food. In recent years, there has been a growing interest in non-thermal process. Spores are the dormant state of vegetative cells and have unique multilayers structure. A lot of research focused on the inactivation of spores due to its extremely resistance. This review focused on the inactivation effects and mechanisms of spores by non-thermal processing. High-pressure processing inactivated spores by two step pathways. First moderate high pressure induced the germination of spores, and the resistance decreased rapidly. Then the germinated spores inactivated as followed. Some non-thermal techniques such as ultrasound and ultraviolet radiation had synergistic effects and were usually applied as hurdle technologies.

The ever-increasing applications of enzymes are limited by the relatively poor performance in harsh processing conditions. As a result, there are constant innovations in immobilization protocols for improving biocatalyst activity and stability. Bacterial spores are cheap to generate and highly resistant to environmental stress. The spore core is sheathed by an inner membrane, the germ cell wall, the cortex, outer membrane, spore coat and in some species the exosporium. The spore surface is anion-rich, hydrophobic and contains several reactive groups capable of interacting and stabilizing enzyme molecules through electrostatic forces, hydrophobic interactions and covalent bonding. The probiotic nature of spores obtained from non-toxic bacterial species makes them suitable carriers for the enzyme immobilization, especially food-grade enzymes or those intended for therapeutic use. Immobilization on spores is by direct adsorption, covalent attachment or surface display during the sporulation phase. Hindrances to the immobilization on spore matrix include the production rates, operational instability, and reduced catalytic properties due to conformational changes in enzyme. This paper reviews bacterial spore as a heterofunctional support matrix gives reasons why probiotic bacillus spores are better options and the diverse technologies adopted for spore-enzyme immobilization. It further suggests directions for future use and discusses the commercialization prospects.

International audience; Bacterial spores can cause significant problems such as food poisoning (like neurotoxin or emetic toxin) or serious illnesses (like anthrax or botulism). This dormant form of bacteria, made of several layers of barriers which provide extreme resistance to many abiotic stresses (radiation, temperature, pressure, etc.), are difficult to investigate in situ. To better understand the biological and chemical mechanisms involved and specific to spores resistance, the acquisition of environmental parameters is necessary. For that purpose, our research has been focused on the detection and analysis of a unique spore component, dipicolinic acid (DPA), used as the main in situ metabolite for sporulating bacteria detection. In its native form, DPA is only weakly fluorescent but after Ultraviolet irradiation at the wavelength of 254 nm (UVc), DPA photoproducts (DPAp) exhibit a remarkable fluorescence signal. These photoproducts are rarely identified and part of this study gives new insights offered by mass spectrometry (MS) in the determination of DPA photoproducts. Thanks to DPA assay techniques and fluorescence spectrometry, we highlighted the instability of photoproducts and introduced new assumptions on the effects of UVc on DPA. Studies in spectroscopy and microscopy allowed us to better understand these native probes in bacterial spores and will allow the implementation of a new method for studying the physico-chemical parameters of spore resistance.

Removing arsenic from aquatic environments has become an urgent problem worldwide. In this study, Ti 4+ - loaded bacterial spore were adopted as a novel adsorbent (spore@Ti 4+ microspheres) for the adsorption efficient removal of arsenite (As(III)) and arsenate (As(V)). The developed adsorbents were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR) and X-ray photoelectron spectroscopy (XPS). Results indicated that the adsorption kinetics was well described by a pseudo-second-order kinetic model, and the adsorption process rapidly achieved equilibrium within 15 min at pH 7.0. The adsorption mechanism was also investigated. The maximum adsorption capacities for As(III) and As(V) were 97.26 mg g −1 and 137.01 mg g −1 , respectively, based on the isothermal studies. These properties suggest that spore@Ti 4+ microspheres can be potentially applied in water treatment.

Since the 2001 anthrax attacks, researchers have focused on the development of a bacterial spore biomarker detector with rapid response time and high sensitivity and selectivity. Hence, a ratiometric fluorescent nanosensor based on lanthanide-functionalized attapulgite (Atta) was designed and prepared using dye-doped Atta as an internal reference. Atta, which is famous because of Maya Blue, is a hydrated magnesium aluminum silicate present in nature, with pronounced adsorption and a large specific surface area rich in silicon hydroxyl. The adsorption of dye molecules benefits the formation of an internal reference with orange fluorescence. Meanwhile, silicon hydroxyl would be available for terbium (Tb 3+ ) complex grafting, which favors the sensitization of the bacterial spore biomarker. With increasing dipicolinic acid (DPA) concentrations, energy transfer from DPA to Tb 3+ gradually enhanced, thereby resulting in strong and predominant green fluorescence. The ratiometric fluorescence intensity is highly sensitive. It has a limit of detection of 9.8 nM, which is much lower than the infectious dosage of Bacillus anthracis spores (60 μM) for humans. The rapid response time (10s), high selectivity, and reliable and practical performances in actual applications make this ratiometric fluorescent nanosensor useful in the detection of biomarkers for various bacterial spores. Moreover, the reported construction of visual ratiometric detection system follows the sustainable development idea of ‘‘from nature, for nature, into the nature’’ and promotes the use of naturally renewable resources to produce promising and eco-friendly functional materials for various applications.

The advantage of using high pressure (HP) cycling treatment compared with continuous HP treatment was investigated for the inactivation of bacterial spores. The effects of parameters such as pulse number, pressure level, treatment temperature, compression and decompression rates, and time between pulses were evaluated. For this purpose, Bacillus subtilis and B. cereus spores (108 and 106 CFU/mL respectively) were suspended in 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution, tryptone salt (TS) buffer solution, or infant milk and treated by HP cycling at 300–400 MPa, at 38–60 °C, for 1–5 pulses. Pressure cycling reduced the number of viable spores by 1.8 and 5.9 log respectively for B. subtilis and B. cereus species. Continuous HP treatments were performed at the same pressure and temperature for similar treatment durations. Our results showed that the spore inactivation ratio was correlated with the cumulative exposure time to pressure rather than to effects of the cycling process. Greater spore inactivation caused by HP cycling was observed only when faster compression and decompression rates were applied, probably due to adiabatic heating. A three-step kinetic model was developed, which seemed to support our hypothesis regarding the mechanisms of inactivation by pressure cycling and continuous HP treatments. Industrial relevance The resistance of bacterial spores to HP limits the industrial applications to refrigerated food products. In this study, we investigated the use of pressure cycling as a means to improve spore baroinactivation at moderate temperatures (T

We report an extremely sensitive and ultra-low-cost PVDF-based SERS substrate using an easy fabrication process that can be conducted in a laboratory and scaled up for industrial applications. Hydrophilic PVDF membranes were sequentially dipped in ethanol and aqueous sodium chloride solution, following which multi-shaped Ag nanoparticles were deposited on the surface of the membranes by suction filtration. The simple solvating pretreatment led to an approximately 188-fold enhancement in the SERS signal intensity of R6G. Using the hydrophilic PVDF-based SERS substrate, subnanomolar sensitivity for R6G was achieved using 3 μL of sample volume on a 3×3 mm2 SERS substrate within 1 min detection time and without the need for concentration or drying processes. To demonstrate the feasibility of the developed SERS substrate as a sensor for bacterial spore detection, it was applied for the detection of the representative biomarker dipicolinic acid (DPA). The limits of detection of DPA and Bacillus spores were measured as 1 ppm and 5×103 spores/mL, respectively. The optimized configuration of low substrate area and wet micro-analyte volume makes the PVDF-based SERS substrate a simple and low-cost tool for the trace-level detection of small molecules in a rapid, sensitive, and high-throughput manner.

This study compares the crack healing performances of biological self-healing concretes using cell/nutrient dropping and immobilization methods to produce microbially induced calcium carbonate precipitation (MICP). Vegetative cells and microencapsulated bacterial spores of Bacillus sphaericus (LMG 22257) were used to heal pre-cracked mortars. Mortar specimens were cured for 7 and 28 days before pre-cracking, and then repaired under wet-dry cycles for 7 days. Comparisons were made in terms of crack-width and crack-area healing and load recovery after repairing. Results showed that the vegetative cell dropping method was more effective in closing cracks and recovering ultimate load after repair than the method of using immobilized microencapsulated bacterial spores for mortar repair. In fact, specimens mixed with microencapsulated bacterial spores exhibited lower ultimate loads than comparable control specimens.

cited By 3; International audience; In this work, our goal was to study the capability of a single metallic oxide to neutralize a chemical agent and to exhibit an antibacterial effect. We tested two types of magnesium oxides, MgO. The first MgO sample tested, which commercial data size characteristic was −325 mesh (MgO-1) destroyed in 3 h, 89.7% of paraoxon and 93.2% of 4-nitrophenol, the first degradation product. The second MgO sample, which commercial data size was

Methods for microbial inactivation are important in the food industry; however, conventional external heating (CH) reduces food quality. Accordingly, the nonthermal effects of ohmic heating (OH) on Bacillus subtilis spores in a sodium chloride aqueous solution at 101°C (i.e., the boiling point), as well as the effects of electric field intensity and frequency during OH, were investigated. Survival kinetics were compared between OH and external CH. The inactivation effect on B. subtilis was greater for all electric field conditions (5, 10, and 20 V/cm) than for CH. In particular, 20 V/cm showed a significantly higher inactivation effect (P0.05) on B. subtilis than those of CH at 8, 10, 12, 14, and 16 min. The survival data were fitted to various primary kinetic models. In the Weibull model and the log-linear model, there were significant differences (P0.05) in the rate parameters δ and k

High pressure (HP) processing, typically at 600 MPa in combination with mild temperatures, makes it possible to produce safe and high quality foods while inactivating pathogenic microorganisms such as Enterohaemorrhagic Escherichia coli (EHEC). In the last decade much research was conducted to understand the inactivation of spores at high pressures and elevated temperatures. Bacterial spore inactivation by HPP in complex food matrices is assessed with an emphasis on mechanisms of inactivation and treatment of products that have low or non-uniform water activity (a w ) profiles. Low a w can be the result of a high concentration in solutes and the presence of oils/fats or other ingredients in the food. The synergistic effect of pressure and temperature leads to lower thermal loads applied to the product and consequently less unwanted compounds can be formed during the process. Future applications of high pressure and elevated temperature processes are assed. These processes could lead to healthier products, safer products and a smarter usage of emerging technologies. Industrial relevance The next step for high pressure processing in addition to pasteurization could be for food sterilization in combination with elevated temperatures. This would have a big impact on many sectors of the industry. Elevated temperatures could be used in existing “cold” HP systems using insulated carriers. The extra cost and construction problems could be minimized. Product quality and health attributes could be increased in comparison to conventional technologies.

The germination of ingested spores is often a necessary first step required for enabling bacterial outgrowth and host colonization, as in the case of Clostridioides difficile (C. difficile) infection. Spore germination rate in the colon depends on microbiota composition and its level of disruption by antibiotic treatment since secretions by commensal bacteria modulate primary to secondary bile salt levels to control germination. Assessment of C. difficile spore germination typically requires measurement of colony-forming units, which is labor intensive and takes at least 24 hours to perform but is regularly required due to the high recurrence rates of nosocomial antibiotic-associated diarrhea. We present a rapid method to assess spore germination by using high throughput single-cell impedance cytometry (>300 events/s) to quantify live bacterial cells, by gating for their characteristic electrophysiology versus spores, so that germination can be assessed after just 4 hours of culture at a detection limit of ~100 live cells per 50 μL sample. To detect the phenotype of germinated C. difficile bacteria, we utilize its characteristically higher net conductivity versus that of spore aggregates and non-viable C. difficile forms, which causes a distinctive high-frequency (10 MHz) impedance phase dispersion within moderately conductive media (0.8 S/m). In this manner, we can detect significant differences in spore germination rates within just 4 hours, with increasing primary bile salt levels in vitro and using ex vivo microbiota samples from an antibiotic-treated mouse model to assess susceptibility to C. difficile infection. We envision a rapid diagnostic tool for assessing host microbiota susceptibility to bacterial colonization after key antibiotic treatments.

Co-cultured bacterial spores from alkaliphilic and alkali-tolerant strains exhibit superior performance compared with those from single strains because of their higher resistance to pH and harsh environments, which promotes biofilm formation and CaCO3 precipitation. In this study, spray-dried co-cultured bacteria spores have been incorporated into cement mortar to explore their effects on its self-healing, mechanical properties, and durability. They showed high survival, decreased mortar sorptivity, and well-crystalline CaCO3 formation; on the other hand, the 28-day compressive strength of the mortar was severely reduced, and the water porosity raised.

Bacterial endospores through their strong resistance to both chemical and physical hurdles constitute a risk for food industry. Inactivation strategies are based on thermal and/or chemical treatments but rely on incomplete knowledge of the mechanisms of inactivation. Alternative strategies were suggested to achieve food safety while improving product quality. One of them relies on the successive germination and inactivation by pasteurization of bacterial spores. However, to date, a gap of knowledge on bacterial spore germination remains and hinders such an application for food sterilization. Geobacillus stearothermophilus ATCC 7953 spore germination mechanisms were investigated by in situ fluorometry combined with plate counts. G. stearothermophilus spores' inner membrane was stained with Laurdan fluorescent dye. While nutrient pathways showed no strong germination with the combinations tested, successful germination up to 3 log10 could be achieved using 60 mmol l−1 calcium-dipicolinic acid (CaDPA) at 55 °C for 2 h. A model for the CaDPA germination mechanism in two phases could be derived which suggested a potential key role of cortex fragments in the germination path, before completion of the cortex degradation. Additionally, it was confirmed that the germination potential of CaDPA, which does not rely on nutrient receptors, is a widespread germination trigger across spore formers. Understanding germination mechanisms and the limitations of different germination paths is important for the development of multi-hurdle approaches to achieve commercial sterility with reduced thermal load.

Recent incidents of foodborne illness associated with spices as the vehicle of transmission prompted this examination of U.S. retail spices with regard to Bacillus cereus. This study focused on the levels of aerobic-mesophilic spore-forming bacteria and B cereus spores associated with 247 retail spices purchased from five states in the United States. Samples contained a wide range of aerobic-mesophilic bacterial spore counts ( 107 CFU/g). Using a novel chromogenic agar, B. cereus and B. thuringiensis spores were isolated from 77 (31%) and 11 (4%) samples, respectively. Levels of B. cereus were

The benefits of high pressure processing (HPP) for microbial inactivation in food production include reduced thermal treatment and minimized effects on sensory and nutritional profiles. These benefits have resulted in increasing commercial production of high pressure pasteurized foods. In this review, the current state of the art in terms of vegetative cell and bacterial spore inactivation by HPP in complex food matrices is assessed with an emphasis on mechanisms of inactivation and treatment of products that have low or non-uniform water activity (aw) profiles. Low aw can be the result of a high concentration in solutes, the presence of oils/fats, or the physical removal of water through dehydration. Microbial inactivation in low aw environments remains a particular challenge for HPP and studies on microbial inactivation observed in the different types of low aw food matrices are reviewed in detail. Industrial relevance HPP-treated food products with low aw have been on the market since the nineties, but the mechanisms of microbial inactivation at low aw are still not well understood, which hinders the development of new applications in low or inhomogeneous aw food. This review summarizes the state of the art in terms of HPP microbial inactivation mechanisms in model systems and various low aw food environments. Thereby, it identifies existing and potential new applications as well as the current gaps and future research needs.

Summary The bacterial spore can rapidly convert from a dormant to a fully active cell. Here we study this remarkable cellular transition in Bacillus subtilis and reveal the identity of the newly synthesized proteins throughout spore revival. Our analysis uncovers a highly ordered developmental program that correlates with the spore morphological changes and reveals the spatial and temporal molecular events fundamental to reconstruct a cell. As opposed to current knowledge, we found that translation takes place during the earliest revival event, termed germination, a process hitherto considered to occur without the need for any macromolecule synthesis. Furthermore, we demonstrate that translation is required for execution of germination and relies on the bona fide translational factors RpmE and Tig. Our study sheds light on the spore revival process and on the vital building blocks underlying cellular awakening, thereby paving the way for designing new antimicrobial agents to eradicate spore-forming pathogens., Graphical Abstract, Highlights • We reveal the identity of the newly synthesized proteins throughout spore revival • We define the timeline of molecular events occurring during spore revival • Protein synthesis occurs during germination and is essential for its execution • RpmE and Tig are required for protein synthesis during germination, Sinai et al. describe the spatial and temporal molecular events occurring throughout the remarkable awakening process of a dormant bacterial spore and show that, in contrast to current knowledge, protein synthesis takes place during the earliest revival event, termed germination, and is required for its execution.

The major objectives of this study were to determine the incidence of psychrotolerant spore formers from REPFEDS marketed in Belgium, and their diversity and characteristics. Spore formers in general were found as spores on 38.3% of the food samples and in 85% food products types evaluated. 76% of the food samples containing spore formers had spores before enrichment. A total of 86 spore formers were isolated from the samples. 28 of 86 bacterial spore formers (32.6%) were capable of vegetative growth at 7 °C. 96% (27/28) of these psychrotolerant spore formers were determined to belong to Bacillus or related genera. According to a (GTG)5-PCR analysis, 24 of these 28 isolates were genetically distinct from each other. 10.7% (3/28) of the bacilli were determined to belong to the Bacillus cereus group, namely B. cereus (chicken curry and Edam cheese) and Bacillus mycoides (Emmental cheese). Almost half of the bacilli (12/27) were putatively identified as Bacillus pumilus, which occurs ubiquitously in nature and has been associated with outbreaks of foodborne disease. Only one psychrotolerant clostridium, Clostridium tyrobutyricum, was isolated in the study. The results of this study show the highly diverse ecology and spoilage potential of psychrotolerant spore formers in REPFEDs marketed in Belgium.

Bacillus endospores have a wide variety of important medical and industrial applications. This is an overview of the fundamental aspects of the life cycle, spore structure and factors that influence the spore resistance of spore-forming Bacillus. Bacillus atrophaeus was used as reference microorganism for this review because their spores are widely used to study spore resistance and morphology. Understanding the mechanisms involved in the cell cycle and spore survival is important for developing strategies for spore killing; producing highly resistant spores for biodefense, food and pharmaceutical applications; and developing new bioactive molecules and methods for spore surface display.

A new generation of high pressure homogenizers reaching up to 400 MPa offers opportunities for spore inactivation and high pressure sterilization of foods. A Stansted Fluid Power ultra-high pressure homogenization (UHPH) unit was tested to inactivate bacterial spores in a model buffer system. Bacillus subtilis PS832 and Geobacillus stearothermophilus ATCC7953 spores’ thermal resistances were assessed (D, z-value and Ea). The pressure and valve temperature were monitored during UHPH. Residence times under pressure and high temperature were estimated and enabled comparison with thermal inactivation, indicating the estimated thermal contribution to inactivation. Spore germination was also assessed but no germination was observed. Up to five log10 B. subtilis and two log10 G. stearothermophilus spores were inactivated by the harshest treatments (> 300 MPa –Tvalve > 145 °C for ~ 0.24 s). The inactivation profiles were similar to the predicted thermal inactivation suggesting that the valve temperature might be a dominant parameter leading to bacterial spore inactivation. Industrial relevance This work showed the UHPH potential for spore inactivation and the need of highly thermoresistant surrogates for process validation, for instance, G. stearothermophilus ATCC7953 spores. Both these findings are highly relevant for industrial application as, to date, no surrogate for this process was suggested while the first patents for the technology transfer to industry are being issued.

Summary Background Non-thermal plasma has received much attention for elimination of microbial contamination from a range of surfaces. Aim This study aimed to determine the effect of a range of dielectric barrier discharge high voltage atmospheric cold plasma (HVACP) parameters for inactivation of Bacillus atrophaeus spores inside a sealed package. Methods A sterile polystyrene Petri dish containing B. atrophaeus spore strip (spore population 2.3 × 10 6 /strip i.e. 6.36 log 10 /strip) was placed in a sealed polypropylene container and was subjected to HVACP treatment. The HVACP discharge was generated between two aluminium plate electrodes using a high voltage of 70kV RMS . The effects of process parameters, including treatment time, mode of exposure (direct/indirect), and working gas types, were evaluated. The influence of relative humidity on HVACP inactivation efficacy was also assessed. The inactivation efficacy was evaluated using colony counts. Optical absorption spectroscopy (OAS) was used to assess gas composition following HVACP exposure. Findings A strong effect of process parameters on inactivation was observed. Direct plasma exposure for 60s resulted in ≥6 log 10 cycle reduction of spores in all gas types tested. However, indirect exposure for 60s resulted in either 2.1 or 6.3 log 10 cycle reduction of spores depending on gas types used for HVACP generation. The relative humidity (RH) was a critical factor in bacterial spore inactivation by HVACP, where a major role of plasma-generated species other than ozone was noted. Direct and indirect HVACP exposure for 60s at 70% RH recorded 6.3 and 5.7 log 10 cycle reduction of spores, respectively. Conclusion In summary, a strong influence of process parameters on spore inactivation was noted. Rapid in-package HVACP inactivation of bacterial spores within 30–60s demonstrates the promising potential application for reduction of spores on medical devices and heat-sensitive materials.

This report summarizes the current state of knowledge on the persistence of biological agents on drinking water infrastructure (such as pipes) along with information on decontamination should persistence occur. Decontamination options for drinking water infrastructure have been explored for some biological agents, but data gaps remain. Data on bacterial spore persistence on common water infrastructure materials such as iron and cement-mortar lined iron show that spores can be persistent for weeks after contamination. Decontamination data show that common disinfectants such as free chlorine have limited effectiveness. Decontamination results with germinant and alternate disinfectants such as chlorine dioxide are more promising. Persistence and decontamination data were collected on vegetative bacteria, such as coliforms, Legionella and Salmonella. Vegetative bacteria are less persistent than spores and more susceptible to disinfection, but the surfaces and water quality conditions in many studies were only marginally related to drinking water systems. However, results of real-world case studies on accidental contamination of water systems with E. coli and Salmonella contamination show that flushing and chlorination can help return a water system to service. Some viral persistence data were found, but decontamination data were lacking. Future research suggestions focus on expanding the available biological persistence data to other common infrastructure materials. Further exploration of non-traditional drinking water disinfectants is recommended for future studies.

Outgrowth heterogeneity of bacterial spore populations complicates both prediction and efficient control of spore outgrowth. In this study, the impact of mild preservation stresses on outgrowth of Bacillus cereus ATCC 14579 spores was quantified during the first stages of outgrowth. Heterogeneity in outgrowth of heat-treated (90 °C for 10 min) and non-heat-treated germinated single spores to the maximum micro-colony stage of 256 cells was assessed by direct imaging on Anopore strips, placed on BHI plates at pH 7 and pH 5.5, without and with added NaCl or sorbic acid (HSA). At pH 7 non-heated and heat-treated germinated spores required 6 h to reach the maximum microcolony stage with limited heterogeneity, and these parameters were only slightly affected with both types of spores when incubated at pH 7 with added NaCl. Notably, the most pronounced effects were observed during outgrowth of spores at pH 5.5 without and with added NaCl or HSA. Non-heat-treated germinated spores showed again efficient outgrowth with limited heterogeneity reaching the maximum microcolony stage after 6 h at pH 5.5, which increased to 12 h and 16 h with added NaCl and HSA, respectively. In contrast, heat-treated spores displayed a strong delay between initial germination and swelling and further outgrowth at pH 5.5, resulting in large heterogeneity and low numbers of fastest growers reaching the maximum microcolony stage after 10, 12 and 24 h, without and with added NaCl or HSA, respectively. This work shows that Anopore technology provides quantitative information on the impact of combined preservation stresses on outgrowth of single spores, showing that outgrowth of germinated heat-treated spores is significantly affected at pH 5.5 with a large fraction of spores arrested in the early outgrowth stage, and with outgrowing cells showing large heterogeneity with only a small fraction committed to relatively fast outgrowth. Keywords Bacillus cereus; Spores; Outgrowth; Heterogeneity; Anopore; Stress

Bacterial spore interactions with planar surfaces in aquatic environments, including adhesive forces and force–distance profiles, are influenced by the geometry and physicochemical properties of the system. The characteristics of spores of Bacillus thuringiensis (Bt) are determined using electron microscopy and electrokinetic measurements. The average size of the spores is 1.57 μm long and 0.86 μm wide, and the zeta potential values are negative for the solutions used in this work. The zeta potentials of the spores and mica surfaces used in the experiments are measured as a function of pH and ionic strength. The Derjaguin, Landau, Verwey and Overbeek (DLVO) theory is employed to predict the interaction force between the spores and planar surfaces as a function of the separation distance, and a force balance is used to explain the adhesive force. Theoretical estimations are compared to experimental measurements obtained from atomic force microscopy (AFM). The DLVO-based calculations are consistent with AFM force measurements, while the calculated adhesive force shows some deviations from the measurements. The deviations can be minimized by considering the roughness of the Bt spore and substrate surfaces. Results are important in the understanding of spore interactions with environmental surfaces in aquatic systems.

The sporicidal effect of hydrogen peroxide (H2O2) in the sterilization of low density polyethylene film (LDPE) was evaluated using a Central Composite Design (CCD). The effects of contact time (5-19s), bath temperature (23–70 °C) and concentration of H2O2 (0–35%) in an immersion bath were investigated. A 16 cm2 film surface was evenly inoculated with 100 μL of the test microorganism Bacillus subtilis var. globigii ATCC9372 spores. The effective H2O2 sporicidal activity was demonstrated at concentrations from 18 to 35% and in a temperature range from 46 to 70 °C, resulting in 2–7 decimal reductions of B. subtilis spores. A quadratic mathematical model representative of the action of H2O2 on the B. subtilis spores was developed as a function of concentration, time and temperature. Test specimens sanitized with 28% H2O2 at 60 °C for 8 s showed 4 decimal reductions. In the same sterilization procedure, but extending the time to 16 s, this value increased to 7 decimal reductions, demonstrating the efficiency of H2O2 as a function of contact time. The sterilization system tested showed satisfactory performance in the sterilization of LDPE films, being capable of reaching up to 7 decimal reductions of the bacterial spore population.

Studies about interaction of plasmas with microorganisms usually focus on bacteria, bacteria spores or fungal spores; little attention has been paid to plasma treatment of fungal species in their vegetative state. The aim of this work is to evaluate the effect of air plasma treatment at atmospheric pressure on mycelia of the agricultural relevant fungal species Ascochyta pinodella and Fusarium culmorum concerning growth inhibition and morphologic alterations. Radial growth measurements on plasma-treated and heat-treated mycelia plugs and light microscopy imaging of isolated and plasma-treated hyphae were carried out. Growth inhibition of the vegetative state of fungi due to plasma treatment was found as well as deformation and disruption of hyphae.

Spores of Geobacillus stearothermophilus and Bacillus subtilis adhering to the surface of stainless steel (SS) and polypropylene (PP) were subjected to rinsing in water or NaOH solution to study their removability. The spores were made adherent through deposition from suspension under humid and dry conditions. The humid condition kept the surface wet throughout the deposition, whereas the dry condition made them dry up finally. Experimental results demonstrated that drying significantly enhanced both quantity and strength of the spore adhesion for both types of surface. No removal of the spores adhering to surfaces exposed to dry environment was observed through water rinsing with shear force application. Even with 0.2% NaOH, they were hardly removed irrespective of temperature. Although 1% NaOH removed or inactivated most of spores on SS surface, it left considerable viable spores on PP surface. Thus enhanced alkali tolerance was indicated for spores adhering to surfaces, especially made of PP, exposed to dry environment.

Effective decontamination of environments contaminated by Bacillus spores remains a significant challenge since Bacillus spores are highly resistant to killing and could plausibly adhere to many non-biological as well as biological surfaces. Decontamination of Bacillus spores can be significantly improved if the chemical basis of spore adherence is understood. In this research, we investigated the surface adhesive properties of Bacillus subtilis and Bacillus anthracis spores. The spore thermodynamic properties obtained from contact angle measurements indicated that both species were monopolar with a preponderance of electron-donating potential. This was also the case for spores of both species missing their outer layers, due to mutation. Transport of wild type and mutant spores of these two species was further analyzed in silica sand under unsaturated water conditions. A two-region solute transport model was used to simulate the spore transport with the assumption that the spore retention occurred within the immobile region only. Bacillus spore adhesion to the porous media was related to the interactions between the spores and the porous media. Our data indicated that spore surface structures played important roles in spore surface properties, since mutant spores missing outer layers had different surface thermodynamic and transport properties as compared to wild type spores. The changes in surface thermodynamic properties were further evidenced by infrared spectroscopy analysis.

The effectiveness of ohmic and conventional heating for reducing spores of Alicyclobacillus acidoterrestris was investigated in commercial pasteurized orange juice. The kinetic parameters (D- and z-values) were determined during ohmic and conventional heating. The effects of temperature (70. 80, and 90 °C) and heating time (0, 10, 15, 20, and 30 min) on inactivation of A. acidoterrestris spores during ohmic heating in orange juice were significant (P < 0.05). For 70°C, the voltage gradient also had an effect on inactivation kinetics. At 30 V/cm, D-values at 70, 80, and 90°C were 58.48, 12.24, and 5.97 min, respectively. D-values at corresponding temperatures for conventionally heated spores were 83.33, 15.11, and 7.84 min, respectively. Results showed significantly higher lethality for spores treated with ohmic heating than for spores treated with conventional heating. Conventional heating was ineffective for pasteurizing orange juice, whereas the maximum ohmic heating treatment applied at 30 V/cm was sufficient to inactivate 5 log units of A. acidoterrestris spores. Copyright ©, International Association for Food Protection., Scientific and Technical Research Council of Turkey (project no. 106 O 240)

Both atmospheric pressure plasma and photo-catalyst metal oxide titanium dioxide (TiO2) are well known for their microorganism inactivation and chemical material decomposition abilities. In this work, radio-frequency atmospheric pressure plasma and TiO2 are used together to inactivate Bacillus subtilis spores that have a very high degree of environmental resistance to ultra-violet (UV) photons and heat. The combinational use of the plasma and TiO2 demonstrates an enhanced performance of B. subtilis spore inactivation by showing a decrease in the decimal reduction time of as large as 40% compared with the use of plasma alone. A significant increase of hydroxyl (OH) and excited oxygen atomic emission line (O I) intensities in the presence of TiO2 suggests that the atmospheric pressure plasma assisted by TiO2 is very effective at generating reactive oxygen radicals, which is known to be a dominant factor in bacterial spore inactivation. Possible TiO2 activation mechanism by the plasma is investigated.

Bacterial spores are the ultimate (stress) ‘survival capsules’. They allow strains from the Bacillus and Clostridium species to survive harsh environmental conditions. In addition to the decision to enter sporulation the decision to do the reverse (germinate) is also a decisive event after which there is no return. Generally it is observed that the behaviour of spores towards the environment is not homogeneous. In fact in many cases it is even quite heterogeneous, certainly upon subjecting the spores to a thermal stress treatment. Genome information coupled to high resolution single-cell analysis techniques allow us currently to analyse signalling events of individual cells. In the area of food preservation the next challenge is to couple the newly acquired mechanistic data to the physiologically observed heterogeneity in spore behaviour.The current paper will introduce the background of physiological heterogeneity while discussing the molecular processes that likely contribute to the observed heterogeneity in outgrowth. The discussion is set in the framework of contemporary and future needs for single-cell data integration in order to enhance the mechanistic basis of food preservation and spoilage models targeting bacterial spores.

A semi-automated bacterial spore detection system (BSDS) was developed to detect biological threat agents (e.g., Bacillus anthracis) on-site. The system comprised an aerosol sampler, micro-fluidic chip-A (for spore germination and cell lysis), micro-fluidic chip-B (for extraction and detection of genomic DNA) and an analyzer. An aerosol with bacterial spores was first collected in the collection chamber of chip-A with a velocity of 300 l/min, and the chip-A was taken off from the aerosol sampler and loaded into the analyzer. Reagents packaged in the chip-A were sequentially applied into the chamber. The genomic DNA extract from spore lyzate was manually transferred from chip-A to chip-B and loaded into the analyzer. Genomic DNA in chip-B was first trapped on a glass bead column, washed with various reagents, and eluted to the detection chamber by sequential auto-dispensing. Isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) with fluorescent measurement was adopted to amplify and detect target DNA. Bacillus subtilis was the stimulant of biological warfare agent in this experiment. Pretreatment conditions were optimized by examining bacterial target DNA recovery in the respective steps (aerosol collection, spore germination, cell lysis, and DNA extraction), by an off-chip experiment using a real-time polymerase chain reaction quantification method. Without the germination step, B. subtilis spores did not demonstrate amplification of target DNA. The detection of 104 spores was achieved within 2 h throughout the micro-fluidic process.

This report provides a statistical analysis of a Bacillus atrophaeus spore aerosol generated by opening a spore containing letter in an office, simulating the effects of an “anthrax letter”. Spore aerosols were collected by five sets of high-resolution slit to agar samplers in a vacated office building (338.4 m2, 1015.2 m3) during 34 replicates of a single control scenario. Results showed that 0.1 g of B. atrophaeus spores loaded in a letter were easily detected throughout the building following the opening of the letter. Areas under the curve (AUCs) were used to summarize each replicate scenario, and the variability of the AUCs was examined to assess the sample size requirements for future scenario analyses. Based on this analysis a minimum of five replicates of each scenario will be required for future statistical comparison of new scenarios intended to determine the effectiveness of existing or proposed anthrax letter response protocols.

Protein A chromatography media require sanitization between batches as well as prior to long-term storage. While sodium hydroxide (NaOH) is probably one of the most widely used sanitants within the bioprocess industry, it cannot be used with silica- or controlled pore glass (CPG)-based adsorbents due to the instability of the base matrix at high pH. Benzyl alcohol is commonly used for sanitizing such adsorbents, though extended contact times may be required to meet desired microbial log reduction values, especially for fungal and bacterial spore formers. With the rising market need for monoclonal antibody therapeutics, higher manufacturing throughput may be required. In such cases, a shorter sanitization cycle would be extremely beneficial to maximize manufacturing throughput and productivity. This paper describes the development of a new synergistic sanitant solution, designated PAB (120 mM phosphoric acid, 167 mM acetic acid, 2.2% benzyl alcohol) that delivers improved microbial kill kinetics, enabling sanitization times of 2-3h at room temperature, while maintaining acceptable adsorbent stability. Both the approaches taken to establish the effectiveness of the improved solution as well as confirmation of its process compatibility are covered here.

The technology for over-expressing NADPH-cytochrome P450 reductase (CPR), a diflavin-containing enzyme, offers the opportunity to develop enzymatic systems for environmental detoxication and bioconversions of drugs, pesticides and fine chemicals. In this study, Bacillus subtilis was chosen to express rat CPR (rCPR) because of its capacities for high protein production and spore formation. rCPR was expressed in B. subtilis DB104 under the transcriptional control of an IPTG-inducible fusion promoter of P(groE) and P(tac). The expressed rCPR was released into the culture medium after sporulation by autolysis of the host cell. It was associated with and displayed on the spore surfaces; this was confirmed by measuring rCPR activity in purified spores and analyzing its accessibility to anti-rCPR antibodies using flow cytometry. The spore-displayed rCPR was able to reduce cytochrome c and ferricyanide, and also assisted in the O-deethylation of 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin (EFC) by human cytochrome P450 1A2, indicating that it was functionally active. Spore surface display of rCPR in B. subtilis appears to be useful for preparing cytochrome P450-related enzymes, and spore biocatalysts of rCPR are likely to have wide biotechnological applications.

The shift of the dissociation equilibrium under pressure and temperature plays a decisive role in the inactivation of bacterial spores. To obtain the dissociation equilibrium shift in water and buffer systems, the thermodynamic dissociation constants in a wide range of pressure and temperature were modelled. Heat and pressure inactivation of Geobacillus stearothermophilus spores at different initial pH-values in ACES and phosphate buffer confirmed the modelled data. Thermal inactivation in both buffers at 114, 122 and 127 °C resulted in higher logarithmic reductions with ACES. Contrary, after pressure treatment at 500, 600 and 900 MPa with 80 °C phosphate buffer showed higher inactivation. These results indicated the different dissociation equilibrium shifts in buffer systems by heat and pressure.

Holographic sensors for the detection of Bacillus species spore germination and vegetative growth are described. Reflection holograms were fabricated using a diffusion method for the distribution of ultra-fine silver bromide grains into pre-formed polymer films, followed by holographic recording using a frequency doubled Nd:YAG (532 nm) laser. Changes in holographic replay wavelength or diffraction intensity were used to characterise the swelling behaviour or structural integrity of a range of holographic matrices in response to various extracellular products of bacterial spore germination and vegetative metabolism. Divalent metal ion-sensitive holograms containing a methacrylated analogue of nitrilotriacetic acid (NTA) as the chelating monomer were successfully used to monitor Ca2+ ions released during B. subtilis spore germination in real-time, which was within minutes of sample addition; the holographic response manifested as a 16 nm blue-shift in diffraction wavelength over the progress of germination. Similarly, pH-sensitive holograms comprising methacrylic acid (MAA) as the ionisable monomer were responsive to changes in pH associated with early vegetative metabolism following germination of B. megaterium spores; a visually perceptible blue-shift in holographic replay wavelength of 75 nm was observed. Casein and starch-based holographic matrices, prepared by co-polymerisation of the appropriate substrate with acrylamide, were used to detect exo-enzymes released during later stages of B. megaterium and B. subtilis vegetative cell growth; holographic responses of both matrices were visible as a reduction in diffraction intensity due to progressive fringe disruption caused by enzymatic cleavage. The combined monitoring of various germination and growth events using the range of aforementioned holographic sensors provides a novel, comprehensive means for the detection of viable bacterial spores.

The effectiveness of a formulated bio-nematicide product containing lyophilized bacteria spores of Bacillus firmus was evaluated against root-knot nematodes (RKN) in greenhouse and field experiments. A decrease of second stage juveniles hatching from eggs was recorded by using the bio-nematicide at a dose of 0.9 g kg(-1) of soil while further a decrease was recorded by doubling the dose. However, the mortality rate decreased as the inoculurn level increased. Exposure of either second stage juveniles or egg masses to temperatures of 35-40 degrees C for 1-4 weeks had a marked effect on their survival. In a field experiment, the bio-nematicide was evaluated for its potential to control RKN either as a stand-alone method or in combination with soil solarization. The latter was tested for 15-30 days and the bionematicide was applied just before soil coverage with the plastic sheet or just after its removal. Soil solarization either for 15-30 days provided satisfactory control of RKN. The combination of soil solarization with the bio-nematicide improved nematode control and gave results similar to the chemical treatment. (c) 2007 Elsevier Ltd. All rights reserved.

Spore photoproduct lyase (SPL) is a “Radical-SAM” repair enzyme which catalyzes the cleavage of spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine), a specific lesion found in bacterial spore DNA, to thymine monomers by a free-radical mechanism. The enzyme requires S-adenosyl- l -methionine (SAM) and a [4Fe–4S] cluster for activity. SPL from Bacillus subtilis has been difficult to isolate and characterize due to problems with the solubility and stability of the overexpressed protein in Escherichia coli and the lability of the [Fe–S] cluster, even if the protein was purified under strictly anaerobic conditions. In order to overcome these problems we searched for another SPL enzyme and we found that the recombinant SPL enzyme from Clostridium acetobutylicum, isolated either aerobically or anaerobically from overexpressing E. coli, behaves more stably than the B. subtilis one. We report here a complete spectroscopic and biochemical characterization of this enzyme. In particular we show for the first time that, using HYSCORE spectroscopy, SAM binds to the cluster as observed in the case of other members of the “Radical-SAM” enzyme family such as the activases of pyruvate formate lyase and ribonucleotide reductase.

Dormant spores of Bacillus , Clostridium and related species can survive for years, largely because spore DNA is well protected against damage by many different agents. This DNA protection is partly a result of the high level of Ca 2+ -dipicolinic acid in spores and DNA repair during spore outgrowth, but is primarily caused by the saturation of spore DNA with a group of small, acid-soluble spore proteins (SASP), which are synthesized in the developing spore and then degraded after completion of spore germination. The structure of both DNA and SASP alters upon their association and this causes major changes in the chemical and photochemical reactivity of DNA.

Deprotection of the fully blocked disacharide allyl O-(2-amino-4, 6-O-benzylidene-3-O-[(R)-1-carboxyethyl]-2-deoxy-β -D-glucopyranosyl-1’ , 2-lactam)-(1→ 4)-2-acetamido-3, 6-di-O-benzyl-2-deoxy-β -D-glucopyranoside by selective de-O-allylation and parallel removal of the benzylidene and O-benzyl groups is described. The resulting β -muramyl lactam-(1→ 4)-GlcNAc disaccharide is characterised as the per-O-acetylated derivative by 1H and 13C NMR spectroscopy and X-ray structure analysis. Conformational analysis about glycosidic bond of repeating units of bacterial spore cortex is based on experimental data and molecular modelling.

Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat. The 65-kDa CotA protein is an abundant component of the outer coat layer. CotA is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light. Here, we report the structure of CotA at 1.7-A resolution, as determined by x-ray crystallography. This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase. The overall fold of CotA comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center. This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli. However, the three cupredoxin domains in CotA are further linked by external interdomain loops, which increase the packing level of the structure. We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role. Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that CotA may accept the largest substrates of any known laccase. Moreover, and unlike other laccases, CotA appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility. The implications of these findings for the properties of CotA, its assembly and the properties of the bacterial spore coat structure are discussed.

The effects of bacteria associated with arbuscular mycorrhizal fungal (AMF) spores on spore germination, growth in vitro and on the pea-AMF symbiosis were evaluated. Bacterial colonies were recovered from untreated Glomus clarum NT4 spores and NT4 spores decontaminated with 5% chloramine-T for 30, 45 and 60 min on five different media. Both G+ and G− bacteria were recovered from untreated NT4 spores, whereas only G+ bacteria were isolated from decontaminated spores. An in vitro assessment of the effect of spore-associated bacteria on clean, decontaminated NT4 spores revealed that (i) most of the bacteria isolated from untreated spores generally did not significantly alter spore function, (ii) some bacteria isolated from clean, decontaminated spores inhibited or stimulated NT4 spore germination, (iii) stimulation of spore germination occurred only when bacteria were in contact with spores, and (iv) inhibition of spore germination was the result of volatile bacterial metabolites. A stimulatory bacterial isolate, Bacillus pabuli LA3, significantly ( P Bacillus chitinosporus LA6a and NT4.

We describe a method of detecting bacterial endospores via quantitation of calcium uniquely associated with spore suspensions. The sensor is created by combining a fluorescent calcium-sensitive indicator, calcein, in a viscous membrane on planar, silica glass coupons. The sensor coupon is affixed to the distal end of an optical fiber, which provides excitation light and collects the fluorescence emission. Aqueous micro-droplets of bacterial spores are added to the sensor and the resulting increase in fluorescence intensity is monitored. This assay effectively exploits the high concentration of calcium ion found in the spore core and expressed on the surface, and fluorescence spectroscopy, as an indirect method to detect and quantify spore concentration. This sensor technology is applicable to near real-time detection of calcium in bacterial spore suspensions without interference from light scattering or other ambient biological materials such as bacteria, pollen, viruses, and fungal spores.

A field-portable, pyrolysis membrane-inlet quadrupole ion trap mass spectrometer has been used to characterize four pathogenic bacteria ( Bacillus anthracis, Brucella melitensis, Yersinia pestis , and Francisella tularensis ). Moreover, a variety of strains were included, prepared under various growth conditions and a range of growth stages. In these analyses, an in situ thermal hydrolysis-methylation procedure was used during pyrolysis with the reagent tetramethylammonium hydroxide. Mass spectra generated from the analysis of the four pathogens contained information related to the biochemical composition of the sample (i.e. biomarkers) including mass spectral peaks derived from methyl esters of fatty acids, DNA/RNA, and peptide/protein fragments. Using multivariate statistics, bacterial mass spectral fingerprints were analyzed to determine the variance in the data and the contribution of biomarker origin (i.e. lipid, protein, nucleic acid, etc.) for bacterial differentiation. An optimum 98.3% correct classification rate was obtained using cross validation with linear discriminant analysis (on four replicates each of 54 bacterial samples) using only biomarkers of lipid origin and the bacterial spore biomarker dipicolinic acid.

In order to obtain microorganisms or enzymes responsible for lysis of bacterial spores (endospores), microbial cells capable of utilizing damaged spores as a sole carbon source were screened among 200 samples in nature. Such microbial cells were enriched in a cluster containing at least five kinds of bacterial strains. One of the strains was isolated from the cluster and designated strain S-2. During the growth of cells of strain S-2 or in the cluster, the turbidity (emulsified white in color) of the medium containing spores was eliminated with a concomitant loss of spore viability. The finding of spore-degrading bacteria suggests that spores are not always in a long-lived form for microorganisms to survive in living cell system.

This study examined the potential for injury of Bacillus globigii (Bacillus subtilis var. niger, strain ATCC9372) spores during extrusion cooking of a corn/soybean (70/30%, w/v) mixture. The barrel temperature in zone 1 was kept constant at 80 degrees C while it was varied from 100 to 120 and 140 degrees C in zone 2. Recovery and enumeration of spores surviving the extrusion cooking process were conducted with five culture media ranging from minimal to rich in nutrient composition. Numbers of spores counted with a minimal culture medium after extrusion at the low temperature were lower compared to recovery with more complete media. All culture media, including minimal and richer, were equally effective in recovering untreated spores. At the higher extrusion temperatures all media were equally effective in recovering the small numbers of viable spores. The results indicated that the spores of the organism may be injured at lower extrusion temperatures.

Aqueous chlorhexidine gluconate (5 and 10% w/v) in combination with direct ultrasonic energy from an ultrasonic disintegrator linked to a cooling system (20 °C) decreased the viability of Bacillus subtilis spore suspensions (1 × 106 cfu/ml) to levels of 47.7 and 4.8%, respectively, after 3 h contact. No viable spores were detected after 3 h with 10% chlorhexidine in 50% isopropyl alcohol. These solutions exhibited no Sporicidal activity at 20 ° C in the absence of ultrasonic energy. Spore viability was not greatly reduced (72% viable) when sonication was performed at 20 °C over 3 h. When temperature was allowed to rise naturally in an ultrasonic bath (65 °C attained at 3 h) no viable spores were detected following 3 h indirect sonication and spore contact with alcoholic 10% chlorhexidine in glass bottles. Marked improvements in Sporicidal activity were observed with each formulation when spores were indirectly sonicated in a thin rubber membrane container. Aqueous and alcoholic 10% chlorhexidine achieved total spore kill at 3 h whilst a 99.97% reduction in viability was apparent by 2 h (43 °C) with the alcoholic solution.

To develop a novel and effective method for sterilization of bacterial spores, we examined the lethal effect of high-pressure CO 2 on spore cells of Bacillus megaterium at various pressures and temperatures and for various treatment times. The bactericidal effect was found to be enhanced with increasing temperature and treatment time. Interestingly, there was an optimum pressure in the subcritical region of CO 2 which gave the maximum inactivation effects. Under the appropriate conditions of 58 atm, 60°C and 30 h, the survival ratio of the bacterial spore could be reduced to about 10 −7 .

Laboratory test data showed a 4.8-fold increase in the sporicidal activity of glutaraldehyde with the incorporation of ultrasound. Projected times required for 100 percent killed of a 20,000 hydrated spore inoculant were reduced from 211 minutes to 44 minutes. Clinical exposure time intervals necessitated a 31/2-hour glutaraldehyde immersion versus a 30-minute cavitated glutaraldehyde chemosterilization to ensure a 100 percent kill. The feasibility of reducing the ADA-required 10-hour soak in glutaraldehyde to ensure sterility with the incorporation of ultrasonic baths is supported by these data. Tests performed also showed that the airborne contamination from an open tank method is minimal. No spores were retrieved from vapor collected by passive vacuum during cavitation of inoculated glutaraldehyde.