Inactivation of Clostridium perfringens spores and vegetative cells by photolysis and TiO2 photocatalysis with H2O2

Due to public health concerns related to the generation of dangerous by-products from conventional systems of water disinfection, innovative technologies based on the generation of oxidant radicals are being developed. The aim of this work is to evaluate the bactericidal activity of different treatments with light (λ: 320-800 nm), TiO₂ (1 g L⁻¹) and H₂O₂ (0.04 mM) on the viability of vegetative cells and spores of Clostridium perfringens. After spiking a natural water sample (from the Ebro River, Zaragoza (Spain)), the population of vegetative cells was of 10⁸ CFU·100 mL⁻¹ and of spores about 10³ CFU·100 mL⁻¹. Treatments without radiation source (TiO₂, H₂O₂, TiO₂/H₂O₂) show a poor level of inactivation (<0.5 log) on both bacterial forms. The light treatment achieves a vegetative cell inactivation of 1.2 log after 5 min of treatment and <0.5 log on spores after 30 min. The combined light/TiO₂ system increases the level of disinfection with a vegetative cell removal in the order of 6 log after 5 min and 0.6 log of spores after 5 min. Light/H₂O₂ and light/TiO₂/H₂O₂ treatments also significantly increase the disinfection of vegetative cells of C. perfringens (>6 log). Regarding spores, light/H₂O₂ and light/TiO₂/H₂O₂ treatments achieve constant inactivation of 1 log after 5 min of treatment. The application of a light/TiO₂/H₂O₂ treatment does not increase the level of inactivation with regard to the level reached by the light/TiO₂ and light/H₂O₂ systems. This fact shows there is no a significant interaction between TiO₂ and H₂O₂ under the conditions studied.     

Response of Aspergillus flavus spores to nitric oxide fumigations in atmospheres with different oxygen concentrations

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. NO fumigation must be conducted under ultralow oxygen conditions because NO reacts with O₂ to produce nitrogen dioxide (NO₂). In this study, NO fumigations under different O₂ concentrations were conducted on Aspergillus flavus spores to determine effectiveness of NO and NO₂ in inactivating spores. Spores on gridded cellulose filter discs in Petri dishes were subjected to six fumigation treatments including a control with varying levels of NO under different O₂ conditions for 3 h at 15 °C. The discs with spores were then cultured on Aspergillus Differentiation medium plates after fumigation for four days at 25 °C to count A. flavus colonies. Untreated control discs each had over 50 A. flavus colonies. Three fumigation treatments with 0.1% NO₂ or 1.0% NO caused complete inactivation of A. flavus spores. The study demonstrated that NO fumigation with certain levels of NO₂ can effectively inactivate A. flavus spores. The results suggest that NO fumigation has potential to be an alternative treatment to control both pests and microbes on stored products.     

Technical note: Advantages of the self-organizing controller for high-pressure sterilization equipments

A study of a self-organizing controller is implemented in a way that response to controlled system follows the desired given by the model. The self-organizing controller has proven to be a valuable tool in sterilization equipment in order to verify the capacity of the response to any change in the pressure or temperature. Basically, this type of controller is based on the Self-Organizing Map (SOM) that is a neural network algorithm of unsupervised learning. The new ideas include clustering visualization, interactive training and one-dimension arrays.     

Antimicrobial activity of Satureja montana L. essential oil against Clostridium perfringens type A inoculated in mortadella-type sausages formulated with different levels of sodium nitrite

This research evaluated the antimicrobial effect of the winter savory (Satureja montana L.) essential oil (EO) against Clostridium perfringens type A (ATCC 3624) inoculated in mortadella-type sausages formulated with different levels of sodium nitrite (NaNO₂: 0 ppm, 100 ppm and 200 ppm) in addition to EO at concentrations of 0.0%, 0.78%, 1.56% and 3.125% stored at 25 °C for 30 days. The EO extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry (CG-MS) was tested in vitro using an agar well diffusion method for determination of minimum inhibitory concentration (MIC) on C. perfringens. According to compositional analysis of the winter savory EO, 26 chemical compounds were identified, and the major constituents were thymol (28.99%), p-cymene (12.00%), linalool (11.00%) and carvacrol (10.71%). The results obtained showed that EO applied at a concentration of 1.56%, which was defined as the MIC, exhibited antimicrobial activity against C. perfringens in the in vitro assays, and the transmission electron microscopy (TEM) revealed structural damage and cell lysis of C. perfringens caused by EO treatment. A synergistic effect between NaNO₂ and EO was observed. In mortadella-type sausages formulated with 100 ppm of NaNO₂ and EO at all concentrations tested, the population of target microorganisms was reduced (p ≤ 0.05) compared to control samples during all storage period. This data suggests the potential combined use of savory EO and minimal amounts of the synthetic additive, NaNO₂ to control C. perfringens in mortadella, which goes according to current market trends, where consumers are requesting natural products.     

Kinetic studies on high-pressure inactivation of Bacillus stearothermophilus spores suspended in food matrices

Bacillus stearothermophilus spores ATCC 7953 can effectively be inactivated by high-pressure treatment, but only if it is applied at elevated temperatures; however, these temperatures are much lower compared to the temperature level used in heat inactivation under atmospheric pressure. Temperature and pressure in a range between 60 and 120°C and 50–600 MPa were applied to inactivate spores suspended in mashed broccoli and in cocoa mass. Utilizing an empirical mathematical model, derived from nth order kinetics, the survival curves of the spore strain investigated could be described accurately. The model can predict the impact of combined action of pressure and temperature on spore reduction. It was demonstrated that the inactivation of B. stearothermophilus spores ATCC 7953 improved with increasing treatment intensity. Beside intrinsic microbial inactivation mechanisms, the role of the pressure-induced shift in crystallization temperature of fat on spore inactivation in cocoa mass is discussed.     

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

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

Sporeforming bacteria in beer: Occurrence,diversity, presence of hop resistance genes and fate in alcohol-free and lager beers

The aim of this study was to assess the occurrence of sporeforming bacteria in different types of beers (n = 163) and to assess the presence of hor genes in the isolates. Additionally, the study aimed to evaluate the fate of five representative sporeforming bacteria harboring horA and horC genes in alcohol-free and lager beers. Two hundred and sixty (n = 260) sporeforming bacteria belonging to eight different genera were isolated from beers, i.e., Bacillus (n = 118), Paenibacillus (n = 89) and Brevibacillus (n = 41), Lysinbacillus (n = 6), Cohnella (n = 3), Rummeliibacillus (n = 1), Alicyclobacillus (n = 1), and Anoxybacillus (n = 1), respectively. A predominance of members within the Bacillus cereus sensu lato (n = 72; 27.1%), followed by B. megaterium (n = 18; 7%), P. validus (n = 16; 6.1%), P. humicus (n = 13; 5%), P. alginolyticus (n = 13; 5%) and Br. brevis (n = 13; 5%) was observed in beer samples analyzed. Only 5% (n = 14) out of 260 sporeforming bacterial isolates recovered from beers harbored one or both horA and horC genes. Only one (0.3%) isolate, i.e., Bacillus cereus sensu lato (identified as B. thuringiensis LMQA 206) presented both horA and horC genes. None of the five bacterial sporeforming strains harboring horA or horC genes inoculated was able to grow in the beers throughout the storage period studied, and no spoilage was detected. The results of this study indicated a widespread occurrence of sporeforming bacteria in several types of beers from different brands, highlighting that measures should be taken to reduce the occurrence of sporeforming bacteria considering stability and safety concerns.     

Bacterial spore inactivation at 45-65 °C using high pressure processing: Study of Alicyclobacillus acidoterrestris in orange juice

High pressure processing (HPP) is a new non-thermal technology commercially used to pasteurize fruit juices and extend shelf-life, while preserving delicate aromas/flavours and bioactive constituents. Given the spoilage incidents and economic losses due to Alicyclobacillus acidoterrestris in the fruit juice industry, the use of high pressure (200 MPa – 600 MPa) in combination with mild temperature (45 °C–65 °C) for 1–15 min, to inactivate these spores in orange juice were investigated. As expected, the higher the temperature, pressure and time, the larger was the A. acidoterrestris inactivation. The survival curves were described by the first order Bigelow model. For 200 MPa, D_45 °C = 43.9 min, D_55 °C = 28.8 min, D_65 °C = 5.0 min and z-value = 21.3 °C. At 600 MPa, D_45 °C = 12.9 min, D_55 °C = 7.0 min, D_65 °C = 3.4 min and z-value = 34.4 °C. Spores were inactivated at 45 °C and 600 MPa, and at 65 °C only 200 MPa was needed to achieve reduction in spore numbers.     

Susceptibility of Clostridium perfringens to antimicrobials produced by lactic acid bacteria: Reuterin and nisin

The effectiveness as antimicrobials of lactic acid bacteria produced compounds reuterin and nisin was assessed against vegetative cells and spores of Clostridium perfringens isolates (from ovine milk obtained in farms with diarrheic lambs) and C. perfringens CECT 486 (type A toxin producer). We also tested the inhibitory effect of lysozyme and sodium nitrite on Clostridium. Minimal inhibitory concentrations (MIC) of antimicrobials were determined in modified RCM (mRCM) and milk by using a broth microdilution method, after 7 d at 37 °C under anaerobic conditions. The sensitivity of C. perfringens to the tested antimicrobials was strain and culture medium-dependent. In general, vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 2.03–16.25 mM) inhibited the growth of vegetative cells and the outgrowth of spores of all tested C. perfringens, both in mRCM and milk, with higher resistance in milk. Nisin (MIC values 0.78–12.5 μg/ml) was also effective against vegetative cells and spores of tested C. perfringens in both culture media. However, lysozyme (up to 400 μg/ml) did not control the growth of any of the tested Clostridium. Sodium nitrite only inhibited the outgrowth of spores of two C. perfringens isolates at the maximum concentration assayed (300 μg/ml) exclusively in mRCM medium. These results suggest that reuterin and nisin have the potential to control the growth of C. perfringens, and might help to ensure safety at different stages of the food chain. Future studies in food/feed products would be necessary to further corroborate this hypothesis.     

A wireless biosensor using microfabricated phage-interfaced magnetoelastic particles

A micro-scale, free standing, wireless biosensor has been developed using magnetoelastic particles composed of an amorphous iron–boron binary alloy. Upon the application of an external magnetic field, these particles exhibit a characteristic resonance frequency, determined by their size and mass, due to the phenomena of magnetoelasticity. The particles are produced using the microelectronic fabrication techniques of photolithography and physical vapor deposition (sputtering). The biosensor is formed by coating the magnetoelastic particle with a thin layer of gold and immobilizing a biomolecular recognition element (bacteriophage) on the surfaces. Bacteriophage genetically engineered to bind Bacillus anthracis spores was used in this set of experiments as the detection probe. Once these targeted spores come into contact with the biosensor, the phage will bind selectively with only that pathogen, thereby increasing the particle's mass and causing a shift in the resonance frequency. Due to the magnetic nature of the sensing platform, this resonance frequency shift may be detected remotely by a wireless scanning device, presenting a distinct advantage over other techniques. A good correlation between the actual number of spores bound to the sensors and the calculated attached mass, based upon resonance frequency shifts, was obtained from the experiments.