Effect of novel and conventional food processing technologies on Bacillus cereus spores.

This chapter provides a summary of the effect of thermal and non-thermal processing technologies on Bacillus cereus spores, a well-known pathogenic bacterium associated with foodborne illnesses. B. cereus has been frequently detected in rice, milk products, infant food, liquid eggs products and meat products all over the world. This Gram positive, rod-shaped, facultative anaerobe can produce endospores that can withstand pasteurization, UV radiation, and chemical reagents commonly used for sanitization. B. cereus spores can germinate into vegetative cells that can produce toxins. The conventional regime for eliminating spores from food is retorting which uses the application of high temperature (121 °C). However, at this temperature, there could be a significant amount of loss in the organoleptic and functional qualities of the food components, especially proteins. This leads to the research on the preventive measures against germination and if possible, to reduce the resistance before using a non-thermal technology (temperatures less than retorting-121 °C) for inactivation. This chapter reviews the development and success of several food processing technologies in their ability to inactivate B. cereus spores in food.

Light tolerance of extended spectrum ß-lactamase producing Escherichia coli strains after repetitive exposure to far-UVC and blue LED light.

AIMS: The aim of this study was to investigate dual far-UVC (Ultraviolet-C) (222 nm) and blue LED (Light Emitting Diode) (405 nm) light on the inactivation of extended spectrum ß-lactamase-producing Escherichia coli (ESBL-Ec) and to determine if repetitive exposure to long pulses of light resulted in changes to light tolerance, and antibiotic susceptibility. METHODS AND RESULTS: Antimicrobial efficiency of dual and individual light wavelengths and development of light tolerance in E. coli was evaluated through a spread plate method after exposure to light at 25 cm. Dual light exposure for 30 min resulted in a 5-6 log10 CFU mL-1 reduction in two ESBL-Ec and two antibiotic-sensitive control E. coli strains. The overall inhibition achieved by dual light treatment was always greater than the combined reductions (log10 CFU) observed from exposure to individual light wavelengths (combined 222-405 nm), indicating a synergistic relationship between blue LED and far-UVC light when used together. Repetitive long pulses of dual and individual far-UVC light exposure resulted in light tolerance in two ESBL-Ec strains but not the antibiotic-sensitive E. coli strains. Subsequent passages of repetitive light-treated ESBL-Ec strains continued to exhibit light tolerance. Antibiotic susceptibility was determined through a standard disk diffusion method. No changes were observed in the antibiotic susceptibility profiles for any of the four strains after exposure to either dual or individual wavelengths. CONCLUSIONS: Dual light exposure was effective in the disinfection of ESBL-Ec in solution; however, antibiotic-resistant E. coli were able to develop light tolerance after repetitive exposure to light.

A Comprehensive Review of Variability in the Thermal Resistance (D-Values) of Food-Borne Pathogens-A Challenge for Thermal Validation Trials.

The thermal processing of food relies heavily on determining the right time and temperature regime required to inactivate bacterial contaminants to an acceptable limit. To design a thermal processing regime with an accurate time and temperature combination, the D-values of targeted microorganisms are either referred to or estimated. The D-value is the time required at a given temperature to reduce the bacterial population by 90%. The D-value can vary depending on various factors such as the food matrix, the bacterial strain, and the conditions it has previously been exposed to; the intrinsic properties of the food (moisture, water activity, fat content, and pH); the method used to expose the microorganism to the thermal treatment either at the laboratory or commercial scale; the approach used to estimate the number of survivors; and the statistical model used for the analysis of the data. This review focused on Bacillus cereus, Cronobacter sakazakii, Escherichia coli, Listeria monocytogenes, and Clostridium perfringens owing to their pathogenicity and the availability of publications on their thermal resistance. The literature indicates a significant variation in D-values reported for the same strain, and it is concluded that when designing thermal processing regimes, the impact of multiple factors on the D-values of a specific microorganism needs to be considered. Further, owing to the complexity of the interactions involved, the effectiveness of regimes derived laboratory data must be confirmed within industrial food processing settings.

Efficacy of Dry Heat Treatment against Clostridioides difficile Spores and Mycobacterium tuberculosis on Filtering Facepiece Respirators.

The COVID-19 pandemic has required novel solutions, including heat disinfection of personal protective equipment (PPE) for potential reuse to ensure availability for healthcare and other frontline workers. Understanding the efficacy of such methods on pathogens other than SARS-CoV-2 that may be present on PPE in healthcare settings is key to worker safety, as some pathogenic bacteria are more heat resistant than SARS-CoV-2. We assessed the efficacy of dry heat treatment against Clostridioides difficile spores and Mycobacterium tuberculosis (M. tb) on filtering facepiece respirator (FFR) coupons in two inoculums. Soil load (mimicking respiratory secretions) and deionized water was used for C. difficile, whereas, soil load and PBS and Tween mixture was used for M. tb. Dry heat treatment at 85 °C for 240 min resulted in a reduction equivalent to 6.0-log10 CFU and 7.3-log10 CFU in C. difficile spores inoculated in soil load and deionized water, respectively. Conversely, treatment at 75 °C for 240 min led to 4.6-log10 CFU reductions in both soil load and deionized water. C. difficile inactivation was higher by >1.5-log10 CFU in deionized water as compared to soil load (p < 0.0001), indicating the latter has a protective effect on bacterial spore inactivation at 85 °C. For M. tb, heat treatment at 75 °C for 90 min and 85 °C for 30 min led to 8-log10 reduction with or without soil load. Heat treatment near the estimated maximal operating temperatures of FFR materials (which would readily eliminate SARS-CoV-2) did not achieve complete inactivation of C. difficile spores but was successful against M. tb. The clinical relevance of surviving C. difficile spores when subjected to heat treatment remains unclear. Given this, any disinfection method of PPE for potential reuse must ensure the discarding of any PPE, potentially contaminated with C. difficile spores, to ensure the safety of healthcare workers.

Effect of Hurdle Approaches Using Conventional and Moderate Thermal Processing Technologies for Microbial Inactivation in Fruit and Vegetable Products.

Thermal processing of packaged fruit and vegetable products is targeted at eliminating microbial contaminants (related to spoilage or pathogenicity) and extending shelf life using microbial inactivation or/and by reducing enzymatic activity in the food. The conventional process of thermal processing involves sterilization (canning and retorting) and pasteurization. The parameters used to design the thermal processing regime depend on the time (minutes) required to eliminate a known population of bacteria in a given food matrix under specified conditions. However, due to the effect of thermal exposure on the sensitive nutrients such as vitamins or bioactive compounds present in fruits and vegetables, alternative technologies and their combinations are required to minimize nutrient loss. The novel moderate thermal regimes aim to eliminate bacterial contaminants while retaining nutritional quality. This review focuses on the "thermal" processing regimes for fruit and vegetable products, including conventional sterilization and pasteurization as well as mild to moderate thermal techniques such as pressure-assisted thermal sterilization (PATS), microwave-assisted thermal sterilization (MATS) and pulsed electric field (PEF) in combination with thermal treatment as a hurdle approach or a combined regime.

Hyperspectral imaging and machine learning in food microbiology: Developments and challenges in detection of bacterial, fungal, and viral contaminants.

Hyperspectral imaging (HSI) is a robust and nondestructive method that can detect foreign particles such as microbial, chemical, and physical contamination in food. This review summarizes the work done in the last two decades in this field with a highlight on challenges, risks, and research gaps. Considering the challenges of using HSI on complex matrices like food (e.g., the confounding and masking effects of background signals), application of machine learning and modeling approaches that have been successful in achieving better accuracy as well as increasing the detection limit have also been discussed here. Foodborne microbial contaminants such as bacteria, fungi, viruses, yeast, and protozoa are of interest and concern to food manufacturers due to the potential risk of either food poisoning or food spoilage. Detection of these contaminants using fast and efficient methods would not only prevent outbreaks and recalls but will also increase consumer acceptance and demand for shelf-stable food products. The conventional culture-based methods for microbial detection are time and labor-intensive, whereas hyperspectral imaging (HSI) is robust, nondestructive with minimum sample preparation, and has gained significant attention due to its rapid approach to detection of microbial contaminants. This review is a comprehensive summary of the detection of bacterial, viral, and fungal contaminants in food with detailed emphasis on the specific modeling and datamining approaches used to overcome the specific challenges associated with background and data complexity.

Nature-Inspired Antimicrobial Surfaces and Their Potential Applications in Food Industries.

Antimicrobial resistance (AMR) is a growing global concern and has called for the integration of different areas of expertise for designing robust solutions. One such approach is the development of antimicrobial surfaces to combat the emerging resistance in microbes against drugs and disinfectants. This review is a compressive summary of the work done in the field of material science, chemistry, and microbiology in the development of antimicrobial materials and surfaces that are inspired by examples in nature. The focus includes examples of natural antimicrobial surfaces, such as cicada wings or nanopillars, dragonfly wings, shrimp shells, taro leaves, lotus leaves, sharkskin, gecko skin, and butterfly wings, along with their mechanism of action. Techniques, compositions, and combinations that have been developed to synthetically mimic these surfaces against bacterial/viral and fungal growth in food-processing areas have also been discussed. The applications of synthetic mimics of natural antimicrobial surfaces in food-processing environments is still a naïve area of research. However, this review highlights the potential applications of natural antimicrobial surfaces in the food-processing environment as well as outlines the challenges that need mitigations.

Genetic Factors Affect the Survival and Behaviors of Selected Bacteria during Antimicrobial Blue Light Treatment.

Antimicrobial resistance is a global, mounting and dynamic issue that poses an immediate threat to human, animal, and environmental health. Among the alternative antimicrobial treatments proposed to reduce the external use of antibiotics is electromagnetic radiation, such as blue light. The prevailing mechanistic model is that blue light can be absorbed by endogenous porphyrins within the bacterial cell, inducing the production of reactive oxygen species, which subsequently inflict oxidative damages upon different cellular components. Nevertheless, it is unclear whether other mechanisms are involved, particularly those that can affect the efficacy of antimicrobial blue light treatments. In this review, we summarize evidence of inherent factors that may confer protection to a selected group of bacteria against blue light-induced oxidative damages or modulate the physiological characteristics of the treated bacteria, such as virulence and motility. These include descriptions of three major photoreceptors in bacteria, chemoreceptors, SOS-dependent DNA repair and non-SOS protective mechanisms. Future directions are also provided to assist with research efforts to increase the efficacy of antimicrobial blue light and to minimize the development of blue light-tolerant phenotypes.

Applications of novel processing technologies to enhance the safety and bioactivity of milk.

Bioactive compounds in food can have high impacts on human health, such as antioxidant, antithrombotic, antitumor, and anti-inflammatory activities. However, many of them are sensitive to thermal treatments incurred during processing, which can reduce their availability and activity. Milk, including ovine, caprine, bovine, and human is a rich source of bioactive compounds, including immunoglobulins, vitamins, and amino acids. However, processing by various novel thermal and non-thermal technologies has different levels of impacts on these compounds, according to the studies reported in the literature, predominantly in the last 10 years. The reported effect of these technologies either covers microbial inactivation or the bioactive composition; however, there is a lack of comprehensive compilation of studies that compare the effect of these technologies on bioactive compounds in milk (especially, caprine and ovine) to microbial inactivation at similar settings. This research gap makes it challenging to conclude on the specific processing parameters that could be optimized to achieve targets of microbial safety and nutritional quality at the same time. This review covers the effect of a wide range of thermal and non-thermal processing technologies including high-pressure processing, pressure-assisted thermal sterilization, pulsed-electric field treatment, cold plasma, microwave-assisted thermal sterilization, ultra-high-pressure homogenization, ultrasonication, irradiation on the bioactive compounds as well as on microbial inactivation in milk. Although a combination of more than one technology could improve the reduction of bacterial contaminants to meet the required food safety standards and retain bioactive compounds, there is still scope for research on these hurdle approaches to simultaneously achieve food safety and bioactivity targets.

Hyperspectral imaging and deep learning for quantification of Clostridium sporogenes spores in food products using 1D- convolutional neural networks and random forest model.

Clostridium sporogenes spores are used as surrogates for Clostridium botulinum, to verify thermal exposure and lethality in sterilization regimes by food industries. Conventional methods to detect spores are time-consuming and labour intensive. The objectives of this study were to evaluate the feasibility of using hyperspectral imaging (HSI) and deep learning approaches, firstly to identify dead and live forms of C. sporogenes spores and secondly, to estimate the concentration of spores on culture media plates and ready-to-eat mashed potato (food matrix). C. sporogenes spores were inoculated by either spread plating or drop plating on sheep blood agar (SBA) and tryptic soy agar (TSA) plates and by spread plating on the surface of mashed potato. Reflectance in the spectral range of 547-1701 nm from the region of interest was used for principal component analysis (PCA). PCA was successful in distinguishing dead and live spores and different levels of inoculum (102 to 106 CFU/ml) on both TSA and SBA plates, however, was not efficient on the mashed potato (food matrix). Hence, deep learning classification frameworks namely 1D- convolutional neural networks (CNN) and random forest (RF) model were used. CNN model outperformed the RF model and the accuracy for quantification of spores was improved by 4% and 8% in the presence and absence, respectively of dead spores. The screening system used in this study was a combination of HSI and deep learning modelling, which resulted in an overall accuracy of 90-94% when the dead/inactivated spores were present and absent, respectively. The only discrepancy detected was during the prediction of samples with low inoculum levels (<102 CFU/ml). In summary, it was evident that HSI in combination with a deep learning approach showed immense potential as a tool to detect and quantify spores on nutrient media as well as on specific food matrix (mashed potato). However, the presence of dead spores in any sample is postulated to affect the accuracy and would need replicates and confirmatory assays.

Plasma-Activated Water (PAW) as a Disinfection Technology for Bacterial Inactivation with a Focus on Fruit and Vegetables.

Plasma-activated water (PAW) is generated by treating water with cold atmospheric plasma (CAP) using controllable parameters, such as plasma-forming voltage, carrier gas, temperature, pulses, or frequency as required. PAW is reported to have lower pH, higher conductivity, and higher oxygen reduction potential when compared with untreated water due to the presence of reactive species. PAW has received significant attention from researchers over the last decade due to its non-thermal and non-toxic mode of action especially for bacterial inactivation. The objective of the current review is to develop a summary of the effect of PAW on bacterial strains in foods as well as model systems such as buffers, with a specific focus on fruit and vegetables. The review elaborated the properties of PAW, the effect of various treatment parameters on its efficiency in bacterial inactivation along with its usage as a standalone technology as well as a hurdle approach with mild thermal treatments. A section highlighting different models that can be employed to generate PAW alongside a direct comparison of the PAW characteristics on the inactivation potential and the existing research gaps are also included. The mechanism of action of PAW on the bacterial cells and any reported effects on the sensory qualities and shelf life of food has been evaluated. Based on the literature, it can be concluded that PAW offers a significant potential as a non-chemical and non-thermal intervention for bacterial inactivation, especially on food. However, the applicability and usage of PAW depend on the effect of environmental and bacterial strain-based conditions and cost-effectiveness.

Development of Bacterial Spore Pouches as a Tool to Evaluate the Sterilization Efficiency-A Case Study with Microwave Sterilization Using Clostridium sporogenes and Geobacillus stearothermophilus.

In this study, novel spore pouches were developed using mashed potato as a food model inoculated with either Geobacillus stearothermophilus or Clostridium sporogenes spores. These spore pouches were used to evaluate the sterilization efficiency of Coaxially induced microwave pasteurization and sterilization (CiMPAS) as a case study. CiMPAS technology combines microwave energy (915 MHz) along with hot water immersion to sterilize food in polymeric packages. The spore pouches were placed at pre-determined specific locations, especially cold spots in each food tray before being processed using two regimes (R-121 and R-65), which consisted of 121 °C and 65 °C at 12 and 22 kW, respectively, followed by recovery and enumeration of the surviving spores. To identify cold spots or the location for inoculation, mashed potato was spiked with Maillard precursors and processed through CiMPAS, followed by measurement of lightness values (*L-values). Inactivation equivalent to of 1-2 Log CFU/g and >6 Log CFU/g for Geobacillus stearothermophilus and Clostridium sporogenes spores, respectively was obtained on the cold spots using R-121, which comprised of a total processing time of 64.2 min. Whereas, inactivation of <1 and 2-3 Log CFU/g for G. stearothermophilus and C. sporogenes spores, respectively on the cold spots was obtained using R-65 (total processing time of 68.3 min), whereas inactivation of 1-3 Log CFU/g of C. sporogenes spores was obtained on the sides of the tray. The results were reproducible across three processing replicates for each regime and inactivation at the specific locations were clearly distinguishable. The study indicated a strong potential to use spore pouches as a tool for validation studies of microwave-induced sterilization.

Identification of Cold Spots Using Non-Destructive Hyperspectral Imaging Technology in Model Food Processed by Coaxially Induced Microwave Pasteurization and Sterilization.

The model food in this study known as mashed potato consisted of ribose (1.0%) and lysine (0.5%) to induce browning via Maillard reaction products. Mashed potato was processed by Coaxially Induced Microwave Pasteurization and Sterilization (CiMPAS) regime to generate an F0 of 6-8 min and analysis of the post-processed food was done in two ways, which included by measuring the color changes and using hyperspectral data acquisition. For visualizing the spectra of each tray in comparison with the control sample (raw mashed-potato), the mean spectrum (i.e., mean of region of interest) of each tray, as well as the control sample, was extracted and then fed to the fitted principal component analysis model and the results coincided with those post hoc analysis of the average reflectance values. Despite the presence of a visual difference in browning, the Lightness (L) values were not significantly (p < 0.05) different to detect a cold spot among a range of 12 processed samples. At the same time, hyperspectral imaging could identify the colder trays among the 12 samples from one batch of microwave sterilization.

Differential gene expression for investigation of the effect of germinants and heat activation to induce germination in Bacillus cereus spores.

Differential gene expression was used to explore the mechanisms underpinning the differences in the impact of heat activation (70 °C for 30 min) on the germination of Bacillus cereus spores in the presence and absence of a germinant (L-alanine). The number of germinated cells, after heat activation plus L-alanine (3.5 ±â€¯0.02 log CFU/ml) in the spore only initial population was found to be higher than that in only heat activated spores (2.01 ±â€¯0.02 log CFU/ml). The concentration of DPA released by heat activated spores in the presence of L-alanine was 68.3 ±â€¯0.1 and 112.1 ±â€¯0.02 µg/ml after 30 and 60 min, compared to 96.5 and 166.2 ±â€¯0.01 µg/ml after 30 and 90 min, respectively released by spores subjected only to heat activation. Gene (BC0784) encoding for the spore germination protein, gerA operon was up-regulated with a log2-transformed fold change value of 1.2 due to heat activation in the presence of L-alanine. The GerA operon located in the inner membrane is known to be involved in the uptake of L-alanine by B. cereus and has been reported to be involved in L-alanine mediated germination. In addition the up-regulation of genes involved in the uptake of L-alanine is proposed to provide the answer to the synergistic effect of heat and L-alanine in inducing germination in B. cereus spores. In short, heat activation increases the ability of L-alanine to penetrate into the spore's inner membrane, where it can be recognized by the receptors for initiation of the germination pathway. In the current study, the majority of the ribosomal proteins were down-regulated (when spores were heat treated in presence of germinants) this process also appeared to slow down protein synthesis by restricting the protein translation machinery. Differential gene expression revealed the genes responsible for the pathways related to transport and recognition of L-alanine into the spore that could have led to the accelerated germination process along with partial shutting down of protein synthesis pathway and ABC transporters. Knowledge of gene regulation in spores during heat activation will help in the development of approaches to prevent spore germination, which could provide an additional safeguard against bacterial growth and toxin production in improperly cooled heat treated foods.

Effect of cold storage and different ions on the thermal resistance of B. cereus NZAS01 spores- analysis of differential gene expression and ion exchange.

Bacillus cereus spores in food are able to survive pasteurization, and if conditions are favourable, subsequently germinate, grow and produce toxins causing food poisoning. The objectives of this study were to firstly determine the impact of cold storage and ion uptake on the thermal resistance of B. cereus spores and secondly to use differential gene expression to help elucidate possible molecular mechanisms for the changes detected in their thermal resistance. B. cereus spores were held at 4 °C in either 0.05 or 0.5 M solutions of cations (Na+, Ca2+ Mg2+,K+, Zn2+) for 6 days and their D88-values were estimated. In the presence of sodium chloride (0.05 and 0.5 M), sodium phosphate buffer, (pH 7, 0.05 and 0.5 M) or zinc acetate (0.05 M), D88 values decreased by 8.8, 10.9, 11.2, 12.9, and 10.2 min respectively, with no evidence of germination (plating methods). Exposure of spores to Na+ in sodium phosphate buffer (pH 7, 0.05 and 0.5 M) or sodium chloride (0.05 and 0.5 M) resulted in the accumulation of Na+ (66.0 ±â€¯2.9, 193.1 ±â€¯4.6, 136.2 ±â€¯9.9 and 70.5 ±â€¯2.7 µg/g) by spores at the significant expense of K+ (10.8 ±â€¯0.5, 7.5 ±â€¯0.2, 8.1 ±â€¯0.4 and 3.6 ±â€¯0.4 µg/g respectively). The mechanism behind the loss of resistance in sodium phosphate buffer (0.05 M) was further investigated by monitoring the differential gene expression using mRNA sequencing. Genes encoding for uracil permease (BC_3890), Mg2+ P-type ATPase-like protein (BC_1581), ABC transporter ATP-binding protein (BC_0815), and 2-keto-3-deoxygluconate permease (BC_4841) were significantly (FDR value ≤0.05) upregulated. This upregulation indicated a possible increase in permeability, which is suggested to account for the increased uptake of sodium ions and the reduction measured in the spore's thermal resistance. This data suggests that during storage at 4 °C in the presence of sodium ions, spores should not be considered to be completely dormant.

Impact of temperature, nutrients, pH and cold storage on the germination, growth and resistance of Bacillus cereus spores in egg white.

B. cereus spores are a concern to the food industry, especially to the producers of heat sensitive food products like egg white or precooked and stored food such as fried rice. This study investigated the impact of nutrients, temperature (4, 8, 15 and 25 °C), pH (4, 5, 7 and 9), and cold storage on the germination, growth and resistance of B. cereus spores. In egg white held at 4 °C for 12 days spore germination was not apparent, however the addition of egg yolk (5%) resulted in a 2 Log colony forming units (CFU)/mL increase in vegetative cells (p < .05). Adding l-alanine (0.9 mg/mL) to egg white did not induce germination unless the spores were simultaneously heat activated at 70 °C for 30 min. On incubation at 15 or 25 °C in egg white, spore germination increased by 3.0 Log and 3.7 Log CFU/mL on day 12. The presence of 5% yolk further enhanced germination and subsequent sporulation during storage at 15 and 25 °C. Acidification (pH 4) of 10% egg white solution prevented germination at 4, 8, 15 and 25 °C. Spores held at 4 °C for 6 days in phosphate buffer (50 mM, pH 4) had visible deformations on their surface (scanning electron microscopy) and a significant reduction in D88, and D92 values of 13.9 and 8.2 min respectively. A better understanding of how spores sense and respond to changing environmental conditions will help in the development of processing strategies, involving multiple hurdles to ensure the prevention of germination and subsequent toxin production.

Bacillus Spores in the Food Industry: A Review on Resistance and Response to Novel Inactivation Technologies.

The importance and challenges presented by Bacillus spores in the food industry are briefly outlined with a focus on Bacillus cereus. The structure and the mechanism of resistance exhibited by Bacillus spores are described, and the steps involved in their germination are included. Novel technologies, using no or only mild heat treatments, to inactivate Bacillus spores are covered, including ultraviolet radiation, pulsed electric field, and high-pressure processing, both as stand-alone techniques or techniques as part of a hurdle approach.