Inactivation Of Spores Research Articles

Impact of Ultra-High-Pressure Homogenisation on the Inactivation of Bacillus pumilus and Bacillus subtilis Spores in Sheep and Cow Milk.

The efficacy of ultra-high-pressure homogenisation (UHPH) in inactivating Bacillus pumilus ATCC 27142 and Bacillus subtilis ATCC 6633 spores suspended in sheep and cow milk was investigated. The UHPH treatment was conducted at 200 and 250 MPa with an inlet temperature of 85 °C, resulting in homogenising valve temperatures of 117 °C and 127 °C, respectively. To isolate the role of temperature and pressure in the inactivation of bacterial spores, the UHPH treatment was repeated at 250 MPa with a lower inlet temperature of 70 °C that resulted in a valve temperature of 117 °C. Increasing the pressure and valve temperature resulted in increased inactivation. At 250 MPa with a valve temperature of 127 °C, greater than 5 log CFU/mL reduction was achieved in B. pumilus and B. subtilis spores in both milk types. Reductions of 0.61 ± 0.03 log CFU/mL and 0.62 ± 0.09 log CFU/mL in B. pumilus spores and 1.18 ± 0.04 log CFU/mL and 1.30 ± 0.07 log CFU/mL in B. subtilis spores were obtained at 250 MPa with a valve temperature of 117 °C in sheep and cow milk, respectively. The spore inactivation was influenced by both the pressure and temperature, suggesting a synergistic effect, with the latter playing a critical role in the lethality of the treatment. No significant differences in the inactivation of either strain was observed between sheep and cow milk.

Strategies for effective high pressure germination or inactivation of Bacillus spores involving nisin.

Extremely resistant spore-forming bacteria are widely distributed in nature. They infiltrate the food chain and processing environments, posing risks of spoilage and food safety. Traditional heat-intensive inactivation methods often negatively affect the product quality. HP germination-inactivation offers a potential solution for better preserving sensitive ingredients while inactivating spores. However, the presence of ungerminated (superdormant) spores hampers the strategy's success and safety. Knowledge of strategies to overcome resistance to HP germination is vital to progress mild spore control technologies. Our study contributes to the evaluation and development of mild preservation processes by evaluating strategies to enhance the HP germination-inactivation efficacy. Mild preservation processes can fulfill the consumers' demand for safe and minimally processed food.

The oxidative stress mechanism of Bacillus cereus spores induced by atmospheric pressure plasma jet

Emerging plasma technology has demonstrated its effectiveness in inactivating bacteria and spores, showing wide applicability in environment cleaning and in the food supply chain. Studies have revealed that the main reason of plasma-mediated bacterial inactivation were contributed to the oxidative damage on inner membranes (IMs) of cell, while few research paid attention to the oxidation stress process of lipid in spores’ IMs. This study investigated the impact of atmospheric pressure plasma jet (APPJ) treatment on spore inactivation, focusing on oxidative stress mechanisms and changes in membrane lipid composition. Results demonstrated that APPJ treatment induced significant oxidative stress within spores, characterized by elevated levels of reactive oxygen and nitrogen species (RONS). This oxidative stress led to the peroxidation of glycerophospholipids, particularly phosphatidylglycerol, phosphatidylcholine, and cardiolipin, major components of spore membranes. Subsequently, the rearrangement of membrane lipid and the formation of lipid hydroperoxide, caused by the released cations and RONS, accelerated membrane instability. These changes correlated with increased membrane permeability and structural damage observed via flow cytometry, indicating the critical role of membrane lipid peroxidation in spore inactivation. Furthermore, the study highlighted the potential of APPJ as a robust technology for enhancing food safety by targeting spore-forming pathogens throughout the food supply chain.

Inactivation kinetics of Bacillus cereus and Aspergillus niger spores in dehydrated onion shreds after pulsed light and infrared treatments

Fresh onions are dehydrated to increase their shelf-life. Primarily, open dehydration techniques like solar dehydration come with the problem of contamination through natural air convection. A solar conduction dryer that uses conduction, convection, and radiation for dehydration of food samples is exploited in this study. The food samples are often contaminated by Bacillus and Aspergillus species spores. As a remedy, pulsed light treatment as a non-thermal technology and infrared treatment as a thermal technology are studied and compared. Bacillus cereus and Aspergillus niger spores are chosen as a representative of bacterial and fungal contamination in onions. Dehydrated onion shreds with varying water activities (0.4, 0.5, 0.6) were treated. The spore inactivation was best described by Weibull model as compared with first-order model. Scanning electron microscopy images of the microbial cells showed surface distortions on the bacterial and fungal spores. The effect of the treatment technologies on the colour, flavour (thiosulphinate and pyruvic acid concentration), total phenolic and flavonoid content, and ascorbic acid concentration are compared. Overall, pulsed light treatment showed promising inactivation with a maximum log reduction of 4.5 log B. cereus spores·g−1 and 3.1 log A. niger spores·g−1 at 2.131 J·cm−2 in samples with water activity 0.6. The inactivation rate increased with an increase in water activity. The colour was better retained in pulsed light treated samples. The thiosulphinate content (9.24 μmol·g−1), total phenolics (0.268 mg GAE·g−1), and flavonoid content (0.344 mg QE·g−1) in the sample were improved upon pulsed light exposure.Graphical

Role of Dipicolinic Acid in Heat Resistance of Spores of Clostridium botulinum and Clostridium sporogenes PA3679 by Thermal and Pressure-assisted Thermal Processing

Dipicolinic acid (DPA) is a major constituent of spores and reportedly provides protection against inactivation by various thermal processes; however, the relationship between DPA and resistance towards pressure-assisted thermal processing is not well understood. Thermal and pressure-assisted thermal inactivation studies of Clostridium botulinum nonproteolytic strains QC-B and 610-F, proteolytic strain Giorgio-A, and thermal surrogate Clostridium sporogenes PA3679 spores suspended in ACES buffer (0.05 M, pH 7.0) were performed to determine if a relationship exists between DPA release and log reduction of spores. Thermal inactivation at 80, 83, and 87 °C for nonproteolytic strains and 101, 105, and 108 °C for the proteolytic strain and thermal surrogate were conducted. Pressure-assisted thermal inactivation for nonproteolytic strains at 83 °C/600 MPa and for the proteolytic strain and thermal surrogate at 105 °C/600 MPa were performed. Surviving spores were enumerated by 5-tube MPN method for log reductions and analyzed for released DPA by liquid chromatography-tandem mass spectrometry. The correlation between MPN log reductions, released DPA, and D-values were calculated. A positive correlation between released DPA and log reduction of spores was observed for QC-B and 610-F at 80 and 83 °C (r = 0.6073 − 0.7755; P < 0.01). At 87 °C, a positive correlation was detected for 610-F (r = 0.4242, P < 0.05) and no correlation was observed for QC-B (r = 0.1641; P > 0.05). A strong, positive correlation (r = 0.8359 − 0.9284; P < 0.05) between released DPA and log reduction of spores was observed for Giorgio-A at 101, 105, and 108 °C, and a strong, positive correlation (r = 0.8402; P < 0.05) was observed for PA3679 at 101 °C. A positive correlation (r = 0.5646 − 0.6724; P < 0.01) was observed for QC-B, 610-F, and Giorgio-A after pressure-assisted thermal treatment. No correlation (r = 02494; P > 0.05) was found for PA3679 after pressure-assisted thermal treatment. These results suggest a correlation exists between DPA release and heat resistance; however, the level of correlation varied between strains and temperatures. The findings from this research may aid in the development of spore inactivation strategies targeting the thermal resistance profiles of various strains of C. botulinum spores.

Impact of metal ions on PMS/Cl− disinfection efficacy: Enhancing or impeding microbial inactivation?

Peroxymonosulfate (PMS) is an eco-friendly disinfectant gaining attention. This study examined the influence of metal ions (Co(II), Cu(II), Fe(II)) on PMS disinfection with chloride ions (Cl−) against waterborne microorganisms, encompassing both bacteria and fungal spores. The findings elucidated that metal ions augment the inactivation of bacteria in the PMS/Cl− system while concurrently impeding the inactivation of fungal spores. Specifically, the PMS/Co(II)/Cl− process increased E. coli inactivation rates by 2.25 and 2.75 times compared to PMS/Co(II) and PMS/Cl−, respectively. Conversely, PMS/Me(II)/Cl− generally exhibited a diminished inactivation capacity against the three fungal spores compared to PMS/Cl−, albeit surpassing the efficacy of PMS/Me(II). For instance, the inactivation levels of A. niger by PMS/Cl−, PMS/Cu(II)/Cl−, and PMS/Cu(II) are 4.47-log, 1.92-log, and 0.11-log, respectively. Notably, fungal spores demonstrated a substantially higher resistance to disinfectants compared to bacteria. Differences in microbial susceptibility were linked to cell wall structure, composition, antioxidant defenses, and reactive species generation, such as hydroxyl radicals (•OH), sulfate radicals (SO4•−), and reactive chlorine species (RCS). This study demonstrated the novel and unique phenomenon of metal ions' dual role in modulating the PMS/Cl− disinfection process, which has not been reported before and has important implications for the field of water treatment.

Characterization of high-pressure-treated Bacillus subtilis spore populations using flow cytometry — Shedding light on spore superdormancy at 550 MPa

Mild spore inactivation can be challenging in industry because of the remarkable resistance of bacterial spores. High pressure (HP) can trigger spore germination, which reduces the spore's resistance, and thereby allows mild spore inactivation. However, spore germination is heterogenous. Some slowly germinating or non-germinating spores called superdormant spores remain resistant and can survive. Therefore, superdormant spores need to be characterized to understand the causes of their germination deficiency.Bacillus subtilis spores were pressurized for 50 s – 6 min at a very high pressure (vHP) level of 550 MPa and 60 °C in buffer to trigger germination. For a rapid quantification of the remaining ungerminated superdormant spores, flow cytometry (FCM) analysis was validated using single cell sorting and growth analysis. FCM based on propidium iodide (PI) and SYTO16 can be used for 550 MPa-superdormant spores after short vHP treatments of ≤1 min and post-HP incubation at 37 °C or 60 °C. The need for a post-HP incubation is particular for vHP treatments. The incubation was successful to separate FCM signals from superdormant and germinated spores, thus allowing superdormant spore quantification. The SYTO16 and PI fluorescence levels did not necessarily indicate superdormancy or apparent viability. This highlights the general need for FCM validation for different HP treatment conditions.The ∼7 % of ungerminated, i.e., superdormant, spores were isolated after a vHP treatment (550 MPa, 60 °C, 43–52 s). This allowed the characterization of vHP superdormant spores for the first time. The superdormant spores had a similar dipicolinic acid content as spores of the initial dormant population. Descendants of superdormant spores had a normal vHP germination capacity. The causes of vHP superdormancy were thus unlikely linked to the dipicolinic acid content or a permanent genetic change. Isolated superdormant spores germinated better in a second vHP treatment compared to the initial spore population. This has not been observed for other germination stimuli so far. In addition, the germination capacity of the initial spore population was time-dependent. A vHP germination deficiency can therefore be lost over time and seems to be caused by transient factors. Permanent cellular properties played a minor role as causes of superdormancy under chosen HP treatment conditions.The study gained new fundamental insights in vHP superdormancy which are of applied interest. Understanding superdormancy helps to efficiently develop a strategy to avoid superdormant spores and hence to inactivate all spores. The development of a mild HP spore germination-inactivation process aims at better preserving the food quality.

Programmed temperature control for inactivation of Clostridium perfringens spores in cooked chicken with different germinants

This study designed a new programmed temperature control (PTC) process for cooked chicken, aiming to increase the efficacy of Clostridium perfringens spore inactivation through inducing spore germination at different levels of dormancy. The results showed that the use of PTC treatment in the presence of germinant AGFK (a mixture of l-asparagine, D-glucose, D-fructose, and KCl) lead to a reduction of exceeding 6 lg CFU/mL spores in a model system after being subjected to 90 °C for 20 min. A significant up-regulation of gerKA, gerKC and gerAA relative expression suggested that the PTC treatment may induce more spore germination. Meanwhile, there was an observed rise in the release of pyridine-2,6-dicarboxylic acid, accompanied by a steady decline in refractivity. In the cooked chicken meat undergone a cooling process, the number of C. perfringens spores were reduced by approximately 5 lg CFU/g after the PTC treatment and heated at 90 °C for 20 min. In conclusion, the PTC treatment can effectively enhance the inactivation of C. perfringens spores by inducing more spore germination at different levels of dormancy during cooked meat preparation.

Mechanistic insight into roles of α/β-type small acid-soluble proteins, RecA, and inner membrane proteins during bacterial spore inactivation by ohmic heating.

Ohmic heating (OH) (i.e. heating by electric field) more effectively kills bacterial spores than traditional wet heating, yet its mechanism remains poorly understood. This study investigates the accelerated spore inactivation mechanism using genetically modified spores. We investigated the effects of OH and conventional heating (CH) on various genetically modified strains of Bacillus subtilis: isogenic PS533 (wild type_1), PS578 [lacking spores' α/β-type small acid-soluble proteins (SASP)], PS2318 (lacking recA, encoding a DNA repair protein), isogenic PS4461 (wild type_2), and PS4462 (having the 2Duf protein in spores, which increases spore wet heat resistance and decreases spore inner membrane fluidity). Removal of SASP brought the inactivation profiles of OH and CH closer, suggesting the interaction of these proteins with the field. However, the reemergence of a difference between CH and OH killing for SASP-deficient spores at the highest tested field strength suggested there is also interaction of the field with another spore core component. Additionally, RecA-deficient spores yielded results like those with the wild-type spores for CH, while the OH resistance of this mutant increased at the lower tested temperatures, implying that RecA or DNA are a possible additional target for the electric field. Addition of the 2Duf protein markedly increased spore resistance both to CH and OH, although some acceleration of killing was observed with OH at 50V/cm. In summary, both membrane fluidity and interaction of the spore core proteins with electric field are key factors in enhanced spore killing with electric field-heat combinations.

Impact of pH and High-Pressure Pasteurization on the Germination and Development of Clostridium perfringens Spores under Hyperbaric Storage versus Refrigeration.

This study aimed to evaluate hyperbaric storage at room temperature (75-200 MPa, 30 days, 18-23 °C, HS/RT) on Clostridium perfringens spores in brain-heart infusion broth (BHI-broth) at pH 4.50, 6.00, and 7.50 and coconut water (pH 5.40). Both matrices were also pasteurized by high pressure processing (600 MPa, 3 min, 17 °C, HPP) to simulate commercial pasteurization followed by HS, in comparison with refrigeration (5 °C, RF). The results showed that, at AP/RT, spores' development occurred, except at pH 4.50 in BHI-broth, while for RF, no changes occurred along storage. Under HS, at pH 4.50, neither spore development nor inactivation occurred, while at pH 6.00/7.50, inactivation occurred (≈2.0 and 1.0 logs at 200 MPa, respectively). Coconut water at AP/RT faced an increase of 1.6 logs of C. perfringens spores after 15 days, while for RF, no spore development occurred, while the inactivation of spores under HS happened (≈3 logs at 200 MPa). HPP prior to HS seems to promote a subsequent inactivation of C. perfringens spores in BHI-broth at pH 4.50, which is less evident for other pHs. For HPP coconut water, the inactivation levels under HS were lower (≈2.0 logs at 200 MPa). The Weibull model well described the inactivation pattern observed. These results suggest that HS/RT can be simultaneously used as a tool to avoid C. perfringens spores' development, as well as for its inactivation, without the application of high temperatures that are required to inactivate these spores.

Inactivation of Bacillus cereus spores by ohmic heating: Efficiency and changes of spore biological properties

Bacillus cereus spores pose a significant concern during food processing due to their high resistance to environmental stress. Ohmic heating (OH) is an emerging and alternative heating technology with potential for inactivating such spores. This study evaluated the inactivation effects and the biological property changes of Bacillus cereus spores during OH treatments. OH effectively inactivated spores in milk, orange juice, broth, rice soup, and buffer solution in less time than oil bath heating (OB). A decrease in NaCl content improved spore inactivation at the same temperature. Spores were more sensitive to acid at 80–85 °C with OH treatment. Furthermore, OH at 10 V/cm and 50 Hz could reduce the spore resistance and inhibit an increase in spore hydrophobicity and spore aggregation. Both heating methods resulted in significant dipicolinic acid (DPA) leakage and damage to the cortex and inner membranes of the spores. However, OH at 10 V/cm and 50 Hz had the lowest DPA leakage and inflicted the least damage to the inner membrane. The damage to the spore's inner membrane was considered the primary reason for inactivation by OB and OH treatments. Still, OH at 10 V/cm and 50 Hz might also block the germination or outgrowth of treated spores or cause damage to the spore core.

Evaluation of germicidal ultraviolet-C disinfection in a real-world outpatient health care environment

The coronavirus disease 2019 pandemic has highlighted the need for effective infection control in outpatient healthcare settings. Germicidal ultraviolet-C (GUV) light, known for inactivating microorganisms by damaging their deoxyribonucleic acid or ribonucleic acid, offers a potential solution. This study examines the efficacy of GUV air disinfection systems in real-world outpatient environments. We deployed upper-room and far-UV GUV fixtures in 3 outpatient facilities, assessing their impact on bacterial loads through air and surface sampling and bioindicator tests. Occupancy was also monitored. While manual air and surface sampling did not show a significant difference in bacterial loads between control and Ultraviolet C-treated groups, bioindicator tests demonstrated a high level of spore inactivation (up to 99.7% for upper-room GUV and 96.26% for far-UV). Occupancy levels did not significantly influence these outcomes. The discrepancy between bioindicator efficacy and environmental sampling results suggests limitations in the latter's ability to accurately capture environmental bioburden. Bioindicators proved to be reliable for in-situ validation of Ultraviolet C surface disinfection. Bioindicators are effective for validating GUV surface disinfection efficacy in healthcare settings, though further research is needed to optimize environmental sampling methods for assessing GUV's impact on real-world bacterial loads.

Effects of field strength and frequency on inactivation of Clostridium sporogenes PA3679 spores during ohmic heating

The study explored the effects of electric field strength, temperature, and frequency on the inactivation of Clostridium sporogenes PA3679 spores (a surrogate of Clostridium botulinum) within buffer. The experiments were conducted at three different field strengths (30, 40, and 50 V/cm), temperatures (110, 120, and 130 °C), and frequencies (60, 1000, and 5000 Hz). Ohmic heating resulted in significantly greater reduction of C. sporogenes compared to conventional heating. Electric field strength significantly influenced spore inactivation. It was found that lower frequencies led to quicker rates of inactivation. Given that high field strengths have dual benefits of increasing both heating and spore kill rate, the possibility exists to process foods to sterility without a hold. This is expected to result in improved product quality, reduced process times and increased efficiency.

Fungicidal efficiency of DBD cold plasma against Aspergillus niger on dried jujube

This study investigated the fungicidal efficiency and mechanism of action of dielectric barrier discharge cold atmosphere plasma (DBD-CAP) in inactivating Aspergillus niger (A. niger) spores. The disinfection efficacy and quality of dried jujube used as the processing application object were also studied. The results indicated that the Weibull + Tail model performed better for spore inactivation curves at different voltages among various treatment times, and the spore cells were reduced by 4.05 log (cfu/mL) in spores suspension at 70 kV after 15 min of treatment. This disinfection impact was further supported by scanning electron microscope (SEM) and transmission electron microscopy (TEM) images, which showed that the integrity of the cell membrane was damaged, and the intracellular content leaked out after DBD-CAP treatment. Elevated levels of reactive oxygen species (ROS) during the treatment increased the relative conductivity of cells, and leakage of nucleic acids and proteins further supported the disinfection impact. Additionally, the growth and toxicity of surviving A. niger spores after treatment were also greatly reduced. When DBD-CAP was applied to disinfecting dried jujube, the spore number exhibited a 2.67 log cfu/g reduction after treatment without significant damage observed onto the quality (P > 0.05).

Synergic effect of multi-frequency thermosonication and germinant on Alicyclobacillus acidoterrestris spore inactivation and underlying mechanisms

Alicyclobacillus acidoterrestris spores have strong stress resistance, and their spores cannot be eliminated by pasteurization, which is considered a major threat to the beverage industry. In this study, based on the spore control strategy of “germinate to eradicate”, the spores were germinated by multi-frequency thermosonication (MFTS) combined with germinant. The process parameters were optimized, and the underlying mechanism was explored. The results showed that when A. acidoterrestris spores were treated with MFTS (60 °C, 20/40/50 kHz) and 300 mM AGFK for 30 min, incubated at 45 °C for 2 h, and heated to 80 °C for 20 min, a maximum 1.4 ± 0.04 log reduction can be achieved. The spores showed the darkest phase and the best germination effect after MFTS treatment, but leakage of proteins and electrolytes inside the spores were also caused. The surface of the vegetative cells formed by spore after MFTS treatment shrank extensively, and the shape was irregular. Spore resistance was reduced, making them more likely to die after pasteurization. This study could provide theoretical evidence for the technological innovation and practical application of A. acidoterrestris spores hazard control in the beverage industry.

Inactivation of fungal spores using household BDD device as a POU purification device: Application evaluation, influencing factors, and mechanisms

Fungal contamination is commonly found in water supply systems, with filamentous fungi posing a particular threat through secondary pollution and potential risks to drinking water safety. In this study, an electrolytic device, with boron-doped diamond (BDD) as the anode material, was proposed for use as a point-of-use (POU) water treatment device to address the fungal contamination in tap water. The BDD device was found to effectively inactivate Aspergillus terrestris spores under different processing current intensities, water hardness, and alkalinity conditions. Moreover, in practical tap water applications, it also exhibited effective inactivation of Aspergillus terrestris spores and good efficiency in removing antibiotic-resistant genes (ARGs). During the electrolysis, various active species are produced, including ozone, reactive chlorine, and extracellular reactive oxygen species (eROS) such as •OH, •O2− and •1O2. Addtionally, spores produce excessive intracellular reactive oxygen species (iROS) under stimulus. These species could damage the cell membrane, resulting in DNA and TN leakage, a decrease in cellular ATP content, and destruction of surface morphology. This study provides a novel solution for eliminating fungal contamination in water endpoints.

Inactivation of Nosema spp. with zinc phthalocyanine

Most honey bee pathogens, such as Vairimorpha (Nosema), cannot be rapidly and definitively diagnosed in a natural setting, consequently there is typically the spread of these diseases through shared and re-use of beekeeping equipment. Furthermore, there are no viable treatment options available for Nosema spores to aid in managing the spread of this bee disease. We therefore aimed to develop a new method using novel Zinc Phthalocyanine (ZnPc) as a photosensitizer for the photodynamic inactivation of Nosema spores that could be used for the decontamination of beekeeping equipment. Nosema spores were propagated for in vitro testing using four caged Apis mellifera honey bees. The ZnPc treatment was characterized, encapsulated with a liposome, and then used as either a 10 or 100 µM treatment for the freshly harvested Nosema spores, for either a 30 and or 60-minute time period, under either light or dark conditions, in-vitro, in 96-well plates. In the dark treatment, after 30-min, the ZnPc 100 µM treatment, caused a 30 % Nosema mortality, while this increased to 80 % at the same concentration after the light treatment. The high rate of anti-spore effects, in a short period of time, supports the notion that this could be an effective treatment for managing honey bee Nosema infections in the future. Our results also suggest that the photo activation of the treatment could be applied in the field setting and this would increase the sterilization of beekeeping equipment against Nosema.

UV-Induced Spectral and Morphological Changes in Bacterial Spores for Inactivation Assessment.

The ability to detect and inactivate spore-forming bacteria is of significance within, for example, industrial, healthcare, and defense sectors. Not only are stringent protocols necessary for the inactivation of spores but robust procedures are also required to detect viable spores after an inactivation assay to evaluate the procedure's success. UV radiation is a standard procedure to inactivate spores. However, there is limited understanding regarding its impact on spores' spectral and morphological characteristics. A further insight into these UV-induced changes can significantly improve the design of spore decontamination procedures and verification assays. This work investigates the spectral and morphological changes to Bacillus thuringiensis spores after UV exposure. Using absorbance and fluorescence spectroscopy, we observe an exponential decay in the spectral intensity of amino acids and protein structures, as well as a logistic increase in dimerized DPA with increased UV exposure on bulk spore suspensions. Additionally, using micro-Raman spectroscopy, we observe DPA release and protein degradation with increased UV exposure. More specifically, the protein backbone's 1600-1700 cm-1 amide I band decays slower than other amino acid-based structures. Last, using electron microscopy and light scattering measurements, we observe shriveling of the spore bodies with increased UV radiation, alongside the leaking of core content and disruption of proteinaceous coat and exosporium layers. Overall, this work utilized spectroscopy and electron microscopy techniques to gain new understanding of UV-induced spore inactivation relating to spore degradation and CaDPA release. The study also identified spectroscopic indicators that can be used to determine spore viability after inactivation. These findings have practical applications in the development of new spore decontamination and inactivation validation methods.

Atmospheric non-thermal plasma inactivation of Ascosphaera apis, the causative agent of chalkbrood disease in honeybee

Ascosphaera apis is a worldwide pathogenic fungi of honeybees that can cause a decline in bee populations. In this study, we investigated the antifungal activity of non-thermal plasma on fungal growth. Spore inactivation after exposure to gas plasma by liquid phase and plasma activated water (PAW) and pathogenicity of A. apis in vivo were also examined. The results demonstrated that the mycelial growth of fungi was completely inhibited after argon plasma treatment. Both gas plasma and PAW exposures resulted in a significant decrease of A. apis spore numbers, maximum reduction of 1.71 and 3.18-fold, respectively. Germinated fungal spores on potato dextrose agar were also reduced after plasma treatment. SEM analysis revealed a disruption in the morphological structure of the fungal spores. The pathogenicity of A. apis on honeybee larvae was decreased after spores treated by gas plasma and PAW with a disease inhibition of 63.61 ± 7.28% and 58.27 ± 5.87%, respectively after 7 days of cultivation. Chalkbrood in honey bees have limited control options and our findings are encouraging. Here, we demonstrate a possible alternative control method using non-thermal plasma for chalkbrood disease in honeybees.

Influence of Temperature, Light, and H2O2 Concentration on Microbial Spore Inactivation: In‐Situ Raman Spectroscopy Combined with Optical Trapping

To gain insight on chemical sterilization processes, the influence of temperature (up to 70 °C), intense green light, and hydrogen peroxide (H2O2) concentration (up to 30% in aqueous solution) on microbial spore inactivation is evaluated by in‐situ Raman spectroscopy with an optical trap. Bacillus atrophaeus is utilized as a model organism. Individual spores are isolated and their chemical makeup is monitored under dynamically changing conditions (temperature, light, and H2O2 concentration) to mimic industrially relevant process parameters for sterilization in the field of aseptic food processing. While isolated spores in water are highly stable, even at elevated temperatures of 70 °C, exposure to H2O2 leads to a loss of spore integrity characterized by the release of the key spore biomarker dipicolinic acid (DPA) in a concentration‐dependent manner, which indicates damage to the inner membrane of the spore. Intensive light or heat, both of which accelerate the decomposition of H2O2 into reactive oxygen species (ROS), drastically shorten the spore lifetime, suggesting the formation of ROS as a rate‐limiting step during sterilization. It is concluded that Raman spectroscopy can deliver mechanistic insight into the mode of action of H2O2‐based sterilization and reveal the individual contributions of different sterilization methods acting in tandem.