Geobacillus Stearothermophilus Spores Research Articles

Microwave Oven Application for the Preparation and Sterilization of Microbiological Culture Media: A Feasible Method with An Adapted Water Bath and Perforable Cap

There is currently increased interest in the use of alternatives to autoclaved culture media, in order to maintain the properties of the media, while saving energy and time. In this study, we assess a new system for culture media preparation, using a conventional microwave with a water bath and a glass bottle with a rubber cap that allows depressurization. Sterilization, using the proposed system (1000 W, 3 to 20 min), was compared with autoclaving for the preparation of tryptone soy agar (TSA), tryptone soy broth (TSB), Sabouraud 4% dextrose agar (SDA), and violet red bile glucose agar (VRBG). Microwave exposure for 7 min yielded sterile TSA plates. The productivity of both sterilization methods was assessed using the pour plate method, and significant increases in the growth of certain micro-organisms after using a microwave were observed for every culture medium, especially those that were sterilized by boiling (VRBG). The kinetics of microbial destruction showed that Escherichia coli and Bacillus subtilis spores were destroyed after 3 and 7 min in a microwave, respectively, while three decimal reductions were obtained for Geobacillus stearothermophilus spores after 15 min in an autoclave. This new sterilization method could be a feasible, rapid, and economical method to prepare microbiological media, with a quality similar to that obtained through autoclaving.

Performance evaluation of the sterilization process with Bowie & Dick test and biological indicator in the quality control of a blood bank in Peru.

Managing contaminated waste in blood banks is a crucial process that must end with the safe disposal of blood products. In this sense, practical methods and indicators must be used to guarantee the operability of autoclave equipment and the sterility of treated waste to mitigate the risk of biological exposure and hospital biosafety. We designed a cross-sectional study to evaluate the performance the autoclaving process with Bowie & Dick test and biological indicator at the Hemotherapy and Blood Bank Unit of the Cayetano Heredia Hospital in Peru. Fifty autoclaving processes were carried out independently for the Bowie & Dick tests and biological indicators based on Geobacillus stearothermophilus spores. Autoclaving was programmed at 134°C for 3.5 minutes for the Bowie & Dick test, while, for the biological indicator, it was programmed at 121°C for 5 minutes. Both in the presence of contaminated waste. The autoclaving process evaluated by the Bowie & Dick test had a compliance rate of 80% (CI95: 66.3%-90.0%), while, by the biological indicator, 90% (CI95: 78.2%-96.7%). We did not find significant differences between the results of both tests (P = .689; Fisher exact test). The compliance rate in the autoclaving process within the blood bank of a Peruvian Hospital is acceptable; however, there are actions for continuous improvement, especially at the vacuum level in the autoclaving process.

Sterilization of yak milk by direct steam injection (DSI) and effects on milk quality

To facilitate development of new yak milk products with high added value, sterilization effects of direct steam injection (DSI) were evaluated. The Z value (temperature increase necessary to reduce inactivation time by 90%) of Geobacillus stearothermophilus spores (ATCC 7953; 106 CFU mL−1) in yak milk was 5.88 °C. Heating of the milk above 153.86 °C for 0.116 s achieved complete inactivation of ATCC 7953 spores; i.e., commercially-acceptable sterility. The native lactoferrin and β-lactoglobulin (β-Lg) contents of the milk decreased by 40 and 30% after DSI treatment, respectively, whereas the α-lactalbumin (α-La) content did not differ significantly; the overall extent of denaturation of heat sensitive proteins was lower than in UHT milk. The furosine and lactulose contents in DSI treated yak milk were ≤1.11 mg/10 g of protein and 0.368 mg/10 mL, respectively, similar to those in pasteurized milk. The pH, Hunter color values and aroma were significantly influenced by DSI treatment, but the overall sensory acceptability of DSI treated yak milk was assessed as good. These findings indicate that DSI has great potential for producing yak milk and processed yak milk products with considerable added value.

The novel sterilization device: the prototype testing

Currently, there are numerous methods that can be used to neutralize pathogens (i.e., devices, tools, or protective clothing), but the sterilizing agent must be selected so that it does not damage or change the properties of the material to which it is applied. Dry sterilization with hydrogen peroxide gas (VHP) in combination with UV-C radiation is well described and effective method of sterilization. This paper presents the design, construction, and analysis of a novel model of sterilization device. Verification of the sterilization process was performed, using classical microbiological methods and flow cytometry, on samples containing Geobacillus stearothermophilus spores, Bacillus subtilis spores, Escherichia coli, and Candida albicans. Flow cytometry results were in line with the standardized microbiological tests and confirmed the effectiveness of the sterilization process. It was also determined that mobile sterilization stations represent a valuable solution when dedicated to public institutions and businesses in the tourism sector, sports & fitness industry, or other types of services, e.g., cosmetic services. A key feature of this solution is the ability to adapt the device within specific constraints to the user’s needs.

Suitability of Sodium Pyruvate as Decomposer of Carried‐over Liquid Hydrogen Peroxide

AbstractThe aim of the present study was to evaluate the concentration of sodium pyruvate in growth media by using the most‐probable number method to decompose carried‐over liquid hydrogen peroxide (H2O2) without negative influences on correct enumeration of Geobacillus stearothermophilus spores. An equivalent molar ratio of sodium pyruvate and H2O2 was verified to be sufficient for a complete decomposition. The results showed that by exposition of G. stearothermophilus spores in different liquid hydrogen peroxide and by carry‐over of concentrations ranging from 3.56 · 10−6 up to 1.69 · 10−4 mol, a sodium pyruvate concentration of 0.05 mol L−1 in growth media was most efficient for the recovery of spores.

Photocatalytic oxidation conveyor "PCOC" system for large scale surface disinfection.

In this paper, we present a surface decontamination system that substitutes traditional chemicals and scrubbing agents, which will be useful for the general public during a pandemic. The technique is based on a hybrid process in which UV-C light and its photons interact with metal oxide nano-catalysts to generate hydroxyl radicals, which can enhance the deactivation process, and the system can work even in the shadow regions via a dry process. The optimum number of UV light sources in combination with TiO2 nanoparticles catalysts on aluminum plates have been used synergistically in the system. The UV dose in the disinfection chamber has been optimized, which is between 60 and 500 mJ/cm2 throughout the disinfection chamber. The concentration of hydroxyl radicals is reported more than 25 000 ions/cm3 within the disinfection chamber. These ions are circulated throughout the disinfection volume. The disinfection efficiency has been tested on bacteria and spores, and the obtained results are correlated. Around 8 log reductions in the counts of the test bacteria of Escherichia coli and Klebsiella pneumoniae have been achieved in just 2minof exposure in the continuous operation of the system. Tests have also been performed on Geobacillus stearothermophilus spores, and the method described here is the result of multiple tests, a review of the scientific literature, and the incorporation of current laboratory practice. The deactivation tested in the system is larger than that of known bacteria and viruses in terms of UV-doses, signifying its utility during the pandemic.

Evaluation of a Rapid Biological Spore Test for Dental Instrument Sterilization

This study evaluated the reliability of a new rapid biological spore test (BST) for determining the sterilization efficacy of dental steam autoclaves within 20 minutes, as compared to a conventional BST requiring 2 days of incubation after autoclave exposure. A total of 177 pairs of BST, each composed of a rapid test (Celerity™ 20 Steam Biologic Indicator, Steris) and a conventional BST (Attest™ 1262 Biological Indicator, 3M), both containing Geobacillus stearothermophilus spores, were placed into steam autoclaves loaded with instruments, and subjected to either sterilizing (157 pairs) or non-sterilizing conditions (20 pairs). Celerity™ BST was then incubated for 20 minutes at 57°C, with the growth medium evaluated spectrophotometrically for fluorescent α-glucosidase signal changes (no change with successful sterilization; increased fluorescence after failed sterilization). Attest™ BST was incubated for 48 hours at 57°C, after which a pH-based color change in the culture broth was visually assessed (no change in purple color with successful sterilization; change to yellow color with failed sterilization). Celerity™ and Attest™ BST both accurately identified successful sterilization, with no G. stearothermophilus spore growth from either BST after exposure to sterilizing steam autoclave conditions (100% agreement between 157 pairs of each BST). Both BST also accurately detected unsuccessful sterilization, with all tested ampoules positive for G. stearothermophilus spore germination after non-sterilizing steam autoclave time periods. Both BST exhibited 100% sensitivity, specificity, and accuracy for detection of sterilizing steam autoclave conditions. Celerity™ BST, after only 20 minutes incubation, performed equally as well as a BST requiring 48 hours incubation in determining the sterilization efficacy of dental steam autoclaves. Rapid BST offer earlier detection of sterilization failure before potentially contaminated dental instruments are used in clinical patient care.

Influence of Superheated Steam Temperature and Moisture Exchange on the Inactivation of Geobacillus stearothermophilus Spores in Wheat Flour-Coated Surfaces

Influence of Superheated Steam Temperature and Moisture Exchange on the Inactivation of Geobacillus stearothermophilus Spores in Wheat Flour-Coated Surfaces

Evaluation of hydrogen peroxide efficacy against AZD1222 chimpanzee adenovirus strain in the recombinant COVID-19 vaccine for application in cleaning validation in a pharmaceutical manufacturing industry.

This study aimed to evaluate the performance of hydrogen peroxide vapour (HPV) to inactivate the chimpanzee adenovirus AZD1222 vaccine strain used in the production of recombinant COVID-19 vaccine for application in cleaning validation in pharmaceutical industries production areas. Two matrixes were tested: formulated recombinant COVID-19 vaccine (FCV) and active pharmaceutical ingredient (API). The samples were dried on stainless steel and exposed to HPV in an isolator. One biological indicator with population >106 Geobacillus stearothermophilus spores was used to validate the HPV decontamination cycle as standard. HPV exposure resulted in complete virus inactivation in FVC (≥5·03 log10 ) and API (≥6·40 log10 ), showing HPV efficacy for reducing chimpanzee adenovirus AZD1222 vaccine strain. However, the optimum concentration and contact time will vary depending on the type of application. Future decontamination studies scaling up the process to the recombinant COVID-19 vaccine manufacturing areas are necessary to evaluate if the HPV will have the same or better virucidal effectivity in each specific production area. In conclusion, HPV showed efficacy for reducing AZD1222 chimpanzee adenovirus strain and can be a good choice for pharmaceutical industries facilities disinfection during recombinant COVID-19 vaccine production.

Large-scale decontamination of disposable FFP2 and FFP3 respirators by hydrogen peroxide vapour, Finland, April to June 2020.

BackgroundThe shortage of FFP2 and FFP3 respirators posed a serious threat to the operation of the healthcare system at the onset of the COVID-19 pandemic.AimOur aim was to develop and validate a large-scale facility that uses hydrogen peroxide vapour for the decontamination of used respirators.MethodsA multidisciplinary and multisectoral ad hoc group of experts representing various organisations was assembled to implement the collection and transport of used FFP2 and FFP3 respirators from hospitals covering 86% of the Finnish population. A large-scale decontamination facility using hydrogen peroxide vapour was designed and constructed. Microbiological tests were used to confirm efficacy of hydrogen peroxide vapour decontamination together with a test to assess the effect of decontamination on the filtering efficacy and fit of respirators. Bacterial and fungal growth in stored respirators was determined by standard methods.ResultsLarge-scale hydrogen peroxide vapour decontamination of a range of FFP2 and FFP3 respirator models effectively reduced the recovery of biological indicators: Geobacillus stearothermophilus and Bacillus atrophaeus spores, as well as model virus bacteriophage MS2. The filtering efficacy and facial fit after hydrogen peroxide vapour decontamination were not affected by the process. Microbial growth in the hydrogen peroxide vapour-treated respirators indicated appropriate microbial cleanliness.ConclusionsLarge-scale hydrogen peroxide vapour decontamination was validated. After effective decontamination, no significant changes in the key properties of the respirators were detected. European Union regulations should incorporate a facilitated pathway to allow reuse of appropriately decontaminated respirators in a severe pandemic when unused respirators are not available.

Effective ultraviolet C light disinfection of respirators demonstrated in challenges with Geobacillus stearothermophilus spores and SARS-CoV-2 virus

BackgroundThe global COVID-19 pandemic, accompanied by spikes in the number of patients in hospitals, required substantial amounts of respiratory protective devices (respirators), thereby causing shortages. Disinfection of used respirators by applying ultraviolet C (UVC) light may enable safe reuse, reducing shortages.AimTo determine whether UVC disinfection is applicable to enable repeated safe reuse of respirators.MethodsThe UVC chamber, equipped with low-pressure mercury discharge lamps emitting at 254 nm, was used to determine the sporicidal and virucidal effects. Respirators challenged with spores and viruses were exposed to various UVC energy levels. Deactivation of the biological agents was studied as well as UVC effects on particle filtration properties and respirator fit.FindingsA 5 log10 reduction of G. thermophilus spore viability by a UVC dose of 1.1 J/cm2 was observed. By simulating spores present in the middle of the respirators, a 5 log10 reduction was achieved at a UVC dose of 10 J/cm2. SARS-CoV-2 viruses were inactivated by 4 log10 upon exposure to 19.5 mJ/cm2 UVC. In case UVC must be transmitted through all layers of the respirators to reach the spores and virus, a reduction of >5 log10 was achieved using a UVC dose of 10 J/cm2. Exposure to a six-times higher UVC dose did not significantly affect the integrity of the fit nor aerosol filtering capacity of the respirator.ConclusionUVC was shown to be a mild and effective way of respirator disinfection allowing for reuse of the UVC-treated respirators.

Mathematically modelling the inactivation kinetics of Geobacillus stearothermophilus spores: Effects of sterilization environments and temperature profiles

In this study, inactivation kinetics of Geobacillus stearothermophilus spores were evaluated in different sterilization environments. The kinetics were analysed and mathematically modelled based on experimental data collected. The inactivation kinetics were measured precisely in moist heat environments using different sterilization temperatures and holding times. All measured inactivation times were shorter than the inactivation time indicated by the Biological Indicator (BI) manufacturer. Increasing sterilization efficiency was found in the following environments: air, saturated steam, wet steam, liquid water, dialysis solutions. Applying first- and second-order reaction kinetics approaches, formulas were derived from measured data that enabled bacterial inactivation to be modelled. A mathematical first-order reaction kinetic modelling approach could be taken to effectively predict inactivation kinetics for G. stearothermophilus spores based on the experimentally measured data collected in wet steam and air environments. A second-order reaction kinetics approach could be taken, however, to model measured data more accurately in liquid water and dialysis-solution environments. The mathematical models presented here can be applied to describe inactivation kinetics for G. stearothermophilus spores in different sterilization test environments or for any given sterilization temperature profile. These findings can be used to improve the quality of sterilization processes.

Antibacterial characterization of erythorbyl laurate against Geobacillus stearothermophilus spores

We evaluated the antibacterial activity of erythorbyl laurate (EL) against Geobacillus stearothermophilus and its spore in aqueous medium and in oil-in-water (O/W) emulsion. The effective concentrations of EL required to kill vegetative cells and inhibit spore germination were determined. EL exerted bactericidal effects on the vegetative cells at concentrations of 200 and 1500 ppm in the aqueous and O/W emulsion systems, respectively. In addition, EL inhibited spore germination at 200 and 2000 ppm in the aqueous and O/W emulsion systems, respectively, and treatment with concentrations exceeding 7500 ppm killed spores in the emulsion. The germination inhibition activity of EL was verified by determining the dipicolinic acid release from the spores. The antibacterial activity of EL in canned milk coffee indicated that the viable number of spores decreased with increasing EL concentration. Furthermore, the sporicidal activity of EL was greatly enhanced when the spores were exposed to heat treatment at 121 °C in the commercial milk coffee environment, which implies that EL can be used to control contamination of emulsion-based foods.

Inactivation kinetics of Geobacillus stearothermophilus spores during the sterilization in steam-NCGs (steam-air) mixtures

Little is known about the steam content in mixtures of steam and NCGs (Non-Condensable Gases), also referred to as steam-air mixtures, required for successful sterilization and about the contribution of additional liquid water in the sterilization chamber to this process. In this study, sterilization efficiency was assessed with Geobacillus stearothermophilus spores in different steam-NCGs mixtures and under different temperatures and holding times. For each experiment, water, air and bioindicators (BIs) were placed in a gas-tight metal box which then underwent one pre-defined sterilization cycle. The initial water amount, sterilization temperatures and holding times were varied. The volume fractions of steam were calculated by applying thermodynamic laws, and the steam distribution was simulated using Computational Fluid Dynamics (CFD). The inactivation of the BIs was represented as a function of different steam-NCG mixtures. The main finding is that the steam volume in the gas phase is of little importance, but the amount of liquid water available to wet BIs during sterilization is crucial. This study represents the first investigation on bacterial inactivation during the sterilization process in different steam-NCG mixtures using different amounts of liquid water. The previously unknown inactivation kinetics results may prove useful for developers and researchers and enhance medical safety.

A Computational Fluid Dynamics (CFD) model to simulate the inactivation of Geobacillus stearothermophilus spores in different moist heat sterilization environments

The aim of this study was to develop a Computational Fluid Dynamics (CFD) model to simulate the inactivation of bacterial spores of Geobacillus stearothermophilus inside a Peritoneal Dialysis Bag System (PDBS). The presented CFD model has three significant modifications in comparison to current state-of-the-art simulations of sterilization processes. (i) The CFD simulation can be used to consider the multiphase flow (water, steam, different dialysis solutions, non-condensable gases (NCGS)) inside the PDBS, the natural convection as well as the steam penetration. (ii) Experimentally obtained inactivation kinetics were added to the CFD code to enable simulation of the inactivation of G. stearothermophilus spores. (iii) The inactivation process of G. stearothermophilus spores was simulated in different sterilization environments which are present inside a PDBS. The CFD model was verified with measurements using Biological Indicators (BIs). Results showed that on the pre-CFD-simulated “worst case locations” CFD simulations and the BI-based verification were in well accordance.By using the presented CFD model, the simulation of a moist heat sterilization process can be performed for any given sterilization cycle. In addition, the model is a powerful tool that can be used to optimize steam sterilization processes and guarantee a high level of sterilization efficiency and product safety.

Effect of sporulation conditions following solid-state cultivation on the resistance of Geobacillus stearothermophilus spores for use as bioindicators testing inactivation by H2O2

Geobacillus stearothermophilus spores play an essential role in the pharmaceutical and food industries as bioindicators (BI) to evaluate inactivation effects of hydrogen peroxide in processing and packaging lines' sterilization. Unstandardized, inconsistent BI production, as is the case for currently available commercial BI, can lead to variable resistances and incorrect test results. The solution to this problem could be the standardization of BI spore production. This study assessed the effects of sporulation temperature and pH on spore resistance in the production of G. stearothermophilus ATCC 7953 spores on solid-state agar. For this purpose, spores were produced under different sporulation conditions (51–63 °C, pH 6–8), and their resistance was determined by the D-value in 35% liquid hydrogen peroxide at 25 °C. Based on these data, a mathematical equation describing the resistance as a function of the sporulation conditions was derived. The analysis showed a quadratic effect of the sporulation temperature and a linear effect of the examined domain's pH on the resistance, without interactions between these factors. We observed the highest D25C,35%H2O2-value of 309 s at sporulation conditions of 57 °C and pH 6.0.

Decontamination of respirators amid shortages due to SARS-CoV-2.

The pandemic created by SARS-CoV-2 has caused a shortage in the supplies of N95 filtering facepiece respirators (FFRs), disposable respirators with at least 95% efficiency to remove non-oily airborne particles, due to increasing cases all over the world. The current article reviewed various possible decontamination methods for FFR reuse including ultraviolet germicidal irradiation (UVGI), hydrogen peroxide vapor (HPV), microwave-generated steam (MGS), hydrogen peroxide gas plasma (HPGP), and 70% or higher ethanol solution. HPV decontamination was effective against bacterial spores (6 log10 reduction of Geobacillus stearothermophilus spores) on FFRs and viruses (> 4 log10 reduction of various types of viruses) on inanimate surfaces, and no degradation of respirator materials and fit has been reported. 70% or higher ethanol decontamination showed high efficacy in inactivation of coronaviruses on inanimate surfaces (> 3.9 log10 reduction) but it was lower on FFRs which filtration efficiency was also decreased. UVGI method had good biocidal efficacy on FFRs (> 3 log10 reduction of H1N1 virus) combined with inexpensive, readily available equipment; however, it was more time-consuming to ensure sufficient reduction in SARS-CoV-2. MGS treatment also provided good viral decontamination on FFRs (> 4 log10 reduction of H1N1 virus) along with less time-intensive process and readily available equipment while inconsistent disinfection on the treated surfaces and deterioration of nose cushion of FFRs were observed. HPGP was a good virucidal system (> 6 log10 reduction of Vesicular stomatitis virus) but filtration efficiency after decontamination was inconsistent. Overall, HPV appeared to be one of the most promising methods based on the high biocidal efficacy on FFRs, preservation of respirator performance after multiple cycles, and no residual chemical toxicity. Nonetheless, equipment cost and time of the HPV process and a suitable operating room need to be considered.

Levels and Characteristics of mRNAs in Spores of Firmicute Species.

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate. IMPORTANCE Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other Bacillales spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.

Ultrasonic-assisted supercritical CO2 inactivation of bacterial spores and effect on the physicochemical properties of oil-in-water emulsions

A combined supercritical carbon dioxide and high power ultrasound treatment (SC-CO2 + HPU) to inactivate spores while minimizing the physicochemical changes in emulsions was investigated. The inactivation kinetics were obtained for Bacillus subtilis, Bacillus pumilus and Geobacillus stearothermophilus spores and the effect of the treatment conditions on the quality of the emulsions was explored using response surface methodology. The treatment for 20 min at 95 °C and 350 bar was effective at inactivating B. subtilis and B. pumilus. Treated emulsions showed, in general, minimal changes in density and good stability, although the pH decreased and the droplet size increased. The treatment at 600 bar, 95 °C and 12.5 min permitted satisfactory B. subtilis and B. pumilus inactivation with minimal physicochemical changes in the emulsions. Therefore, the use of SC-CO2 + HPU could be a viable alternative for the preservation of emulsions using temperatures lower than those of thermal treatments.

Electron Beam Susceptibility of Enteric Viruses and Surrogate Organisms on Fruit, Seed and Spice Matrices

The objective of this study was to use high-energy electron beam (HEEB) treatments to find surrogate microorganisms for enteric viruses and to use the selected surrogates as proof of concept to investigate low-energy electron beam (LEEB) treatments for enteric virus inactivation at industrial scale on frozen blueberries. Six food matrices inoculated with HAV (hepatitis A virus), MNV S99 (murine norovirus), bacteriophages MS2 and Qβ, and Geobacillus stearothermophilus spores were treated with HEEB at 10 MeV using 4, 8 and 16 kGy doses. G. stearothermophilus spores showed the highest inactivation on all matrices except on raisins, with a dose-dependent effect. HAV reached the maximum measurable log10 reduction (> 3.2 log10) when treated at 16 kGy on raisins. MNV showed the highest resistance of all tested microorganisms, independent of the dose, except on frozen blueberries. On frozen blueberries, freeze-dried raspberries, sesame seeds and black peppercorns, HAV showed a mean inactivation level in between those of MS2 and G. stearothermophilus. Based on this, we selected both surrogate organisms as first approximation to estimate HAV inactivation on frozen blueberries during LEEB treatment at 250 keV using 16 kGy. Reductions of 3.1 and 1.3 log10 were measured for G. stearothermophilus spores and MS2, respectively, suggesting that a minimum reduction of 1.4 log10 can be expected for HAV under the same conditions.