Biofilm-forming ability of Bacillus thuringiensis strains from biopesticides on polystyrene and their attachment on spinach.

Bacillus thuringiensis-based commercial products as a biopesticide have been used for more than 60 years in agriculture. However, as one of the species in B. cereus group, B. thuringiensis has been considered as an emerging hazard with the potential to cause food toxico-infections. The present study aimed to evaluate the biofilm-forming ability of B. thuringiensis biopesticide strains and their attachment on spinach, compared to foodborne B. cereus strains. Biofilm formations of tested strains were found to be strain-specific and affected by the nutrient conditions more than the incubation time. Nutrient starvation conditions generally reduced the biofilm formation of tested B. thuringiensis and B. cereus strains, particularly B. thuringiensis ABTS-1857 strain was found as the non-biofilm former in starvation conditions. It is worth mentioning that B. thuringiensis SA-11 strain showed stronger biofilm-forming ability with more air-liquid interface biofilm than the other two B. thuringiensis biopesticide strains, but no such higher attachment of B. thuringiensis SA-11 to spinach was observed. These results indicate that B. thuringiensis SA-11 strain can enter the food processing lines by the attachment on spinach leaves, and it has the potential to form biofilms throughout the processing lines or the production environment when sufficient nutrients are available. However, more biofilm tests of B. thuringiensis biopesticide strains in the vegetable production chain should be performed. The dry formulation of commercial B. thuringiensis biopesticides enhanced their adhesion on spinach leaves, whereas the strength of adhesion was not improved by the formulation. In addition, 1-2 log reductions of spores after the intensive washing of spinach leaves in the lab were detected. However, the log reduction due to the actual washing done by the food processing companies in large-volume washing baths or by consumers at home would be limited and less than this lab simulation.

The Effect of Caco-2 Cells on Sporulation and Enterotoxin Expression by Foodborne Clostridium perfringens.

Clostridium perfringens enterotoxin (Cpe)-producing strains cause gastrointestinal infections in humans and account for the second-largest number of all foodborne outbreaks caused by bacterial toxins. The Cpe toxin is only produced during sporulation; this process might be affected when C. perfringens comes into contact with host cells. The current study determined how the cpe expression levels and spore formation changed over time during co-culture with Caco-2 cells (as a model of intestinal epithelial cells). In co-culture with Caco-2 cells, total C. perfringens cell counts first decreased and then remained more or less stable, whereas spore counts were stable over the whole incubation period. The cpe mRNA level in the co-culture with Caco-2 cells increased more rapidly than in the absence of Caco-2 cells (3.9-fold higher levels in coculture than in the absence of Caco-2 cells after 8 h of incubation). Finally, we found that cpe expression is inhibited by a cue released by Caco-2 cells (8.3-fold lower levels in the presence of supernatants of Caco-2 cells than in the absence of the supernatants after 10 h of incubation); as a consequence, the increased expression in co-culture with Caco-2 cells must be caused by a factor associated with the Caco-2 cells.

Rapid evolutionary tuning of endospore quantity versus quality trade-off via a phase-variable contingency locus.

The UV resistance of bacterial endospores is an important quality supporting their survival in inhospitable environments and therefore constitutes an essential driver of the ecological success of spore-forming bacteria. Nevertheless, the variability and evolvability of this trait are poorly understood. In this study, directed evolution and genetics approaches revealed that the Bacillus cereus pdaA gene (encoding the endospore-specific peptidoglycan-N-acetylmuramic acid deacetylase) serves as a contingency locus in which the expansion and contraction of short tandem repeats can readily compromise (PdaAOFF) or restore (PdaAON) the pdaA open reading frame. Compared with B. cereus populations in the PdaAON state, populations in the PdaAOFF state produced a lower yield of viable endospores but endowed them with vastly increased UV resistance. Moreover, selection pressures based on either quantity (i.e., yield of viable endospores) or quality (i.e., UV resistance of viable endospores) aspects could readily shift populations between PdaAON and PdaAOFF states, respectively. Bioinformatic analysis also revealed that pdaA homologs within the Bacillus and Clostridium genera are often equipped with several short tandem repeat regions, suggesting a wider implementation of the pdaA-mediated phase variability in other sporeformers as well. These results for the first time reveal (1) pdaA as a phase-variable contingency locus in the adaptive evolution of endospore properties and (2) bet-hedging between what appears to be a quantity versus quality trade-off in endospore crops.

Probing Long-Term Impacts: Low-Dose Polystyrene Nanoplastics Exacerbate Mitochondrial Health and Evoke Secondary Glycolysis via Repeated and Single Dosing.

Nanoplastics (NPs) are omnipresent in the environment and contribute to human exposure. However, little is known regarding the long-term effects of NPs on human health. In this study, human intestinal Caco-2 cells were exposed to polystyrene nanoplastics (nanoPS) in an environmentally relevant concentration range (102-109 particles/mL) under two realistic exposure scenarios. In the first scenario, cells were repeatedly exposed to nanoPS every 2 days for 12 days to study the long-term effects. In the second scenario, only nanoPS was added once and Caco-2 cells were cultured for 12 days to study the duration of the initial effects of NPs. Under repeated dosing, initial subtle effects on mitochondria induced by low concentrations would accrue over consistent exposure to nanoPS and finally lead to significant impairment of mitochondrial respiration, mitochondrial mass, and cell differentiation process at the end of prolonged exposure, accompanied by significantly increased glycolysis over the whole exposure period. Single dosing of nanoPS elicited transient effects on mitochondrial and glycolytic functions, as well as increased reactive oxygen species (ROS) production in the early phase of exposure, but the self-recovery capacity of cells mitigated these effects at intermediate culture times. Notably, secondary effects on glycolysis and ROS production were observed during the late culture period, while the cell differentiation process and mitochondrial mass were not affected at the end. These long-term effects are of crucial importance for comprehensively evaluating the health hazards arising from lifetime exposure to NPs, complementing the extensively observed acute effects associated with prevalent short-term exposure to high concentrations. Our study underlines the need to study the toxicity of NPs in realistic long-term exposure scenarios such as repeated dosing.

Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato.

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

The impact of indole and mucin on sporulation, biofilm formation, and enterotoxin production in foodborne Clostridium perfringens.

AIMS: Indole and mucin are compounds found in the host environment as they are produced by the host or by the host-associated microbiota. This study investigated whether indole and mucin impact Clostridium perfringens growth and sporulation, as well as enterotoxin production and biofilm formation. METHODS AND RESULTS: There was no impact on growth of Cl. perfringens for up to 400 µM indole and 240 mg/l mucin, and neither indole nor mucin affected sporulation. Reverse-transcriptase qPCR showed that mucin strongly upregulated the expression of Cl. perfringens enterotoxin (up to 121-fold increase), whereas indole had a much more modest effect (2-fold). This was also reflected in increased Cl. perfringens enterotoxin levels in mucin-treated Cl. perfringens (as assessed by a reversed passive latex agglutination assay). Finally, mucin and indole significantly increased biofilm formation of Cl. perfringens, although the effect size was relatively small (less than 1.5 fold). CONCLUSION: These results indicate that Cl. perfringens can sense its presence in a host environment by responding to mucin, and thereby markedly increased enterotoxin production.

Experimental accumulation and depuration kinetics and natural occurrence of microcystin-LR in basil (Ocimum basilicum L.).

Microcystin-LR (MC-LR) is a hepatotoxic metabolite that naturally occurs during some cyanobacterial blooms in eutrophic waterbodies, and irrigation of edible plants with MC-LR-contaminated water causes bioaccumulation of the toxin. However, sufficient information about accumulation and depuration mechanics in hydroculture-grown herb plants is still lacking. This work aimed at 1) investigating bioaccumulation and depuration of MC-LR in basil, 2) verifying the possible MC-LR detoxification mechanisms in the plant, and 3) detecting the natural occurrence of MC-LR in basil (n = 50) collected from the Belgian market. Basil plants grown in a hydroculture were exposed to MC-LR (5, 20, and 50 µg L-1) spiked in a Hoagland solution for seven days. MC-LR depuration was also studied by transferring the plants to a non-contaminated Hoagland solution after exposure to MC-LR for another seven days. MC-LR concentrations in Hoagland solution, basil leaves, and roots were quantified using a validated UHPLC-MS/MS method. In addition, ELISA and LC-HRMS (only basil leaves) were used for confirmation. The results showed an increase in the accumulated levels of MC-LR at higher exposure doses, with higher MC-LR levels in roots than in leaves for all the treatment conditions. For MC-LR depuration, significant reductions were observed in all the treatment conditions for roots only. No MC-LR conjugates, potentially related to metabolism, were detected by LC-HRMS. Finally, MC-LR was detected in one store-bought basil sample, representing the first occurrence of cyanotoxins in an edible crop from Belgium.

Current Knowledge of Individual and Combined Toxicities of Aflatoxin B1 and Fumonisin B1 In Vitro.

Mycotoxins are considered the most threating natural contaminants in food. Among these mycotoxins, aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are the most prominent fungal metabolites that represent high food safety risks, due to their widespread co-occurrence in several food commodities, and their profound toxic effects on humans. Considering the ethical and more humane animal research, the 3Rs (replacement, reduction, and refinement) principle has been promoted in the last few years. Therefore, this review aims to summarize the research studies conducted up to date on the toxicological effects that AFB1 and FB1 can induce on human health, through the examination of a selected number of in vitro studies. Although the impact of both toxins, as well as their combination, were investigated in different cell lines, the majority of the work was carried out in hepatic cell lines, especially HepG2, owing to the contaminants' liver toxicity. In all the reviewed studies, AFB1 and FB1 could invoke, after short-term exposure, cell apoptosis, by inducing several pathways (oxidative stress, the mitochondrial pathway, ER stress, the Fas/FasL signaling pathway, and the TNF-α signal pathway). Among these pathways, mitochondria are the primary target of both toxins. The interaction of AFB1 and FB1, whether additive, synergistic, or antagonistic, depends to great extent on FB1/AFB1 ratio. However, it is generally manifested synergistically, via the induction of oxidative stress and mitochondria dysfunction, through the expression of the Bcl-2 family and p53 proteins. Therefore, AFB1 and FB1 mixture may enhance more in vitro toxic effects, and carry a higher significant risk factor, than the individual presence of each toxin.

Bacillus cereus Sensu Lato Accelerate Cellular Bioenergetic Metabolism of Human Colorectal Adenocarcinoma Caco-2 Cell Line.

How foodborne enterotoxigenic Bacillus cereus rewires energy metabolism during intestinal tract infection is still not understood. In this study, we used the Seahorse XFe technology to simultaneously analyze oxygen consumption and acidification rates to estimate bioenergetic changes in the intestinal Caco-2 cell line after exposure to the B. cereus sensu lato (s.l.) enterotoxin-producing pathotypes, American Type Culture Collection (ATCC) 14579 (836), NVH0391-98 (828), and NVH0075/95 (825). Infection of Caco-2 led to a more energetic phenotype due to increased flux through oxidative phosphorylation and glycolysis. Strain 836 caused the most pronounced effects toward the specific energy phenotype, followed by strains 828 and 825. However, the metabolic potential of Caco-2 cells was most strongly induced by the 828 strain. Furthermore, infected cells manifested an increased adenosine triphosphate (ATP) production rate. Strain 828 caused the highest glycolytic and mitochondrial ATP production rates, followed by the 836 and 825 B. cereus s.l. strains. The glycolytic stress assay showed that strains 828 and 826 slightly increased compensatory glycolysis, providing a better understanding of the pathogenicity of this versatile pathogen. The results of this study underline that extracellular flux measurement can be used to accurately estimate bioenergetic perturbations of Caco-2 cells as a consequence of infection. Our findings enhance our understanding of how intestinal cells adjust their metabolism during infection with B. cereus s.l.

Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR.

Tropomyosin is the major and predominant allergen among shellfish. This study developed an ultrasensitive immuno-PCR method for the quantification of crustacean tropomyosin in foods. The method couples sandwich ELISA with the real-time PCR (rtPCR) amplification of marker DNAs. Monoclonal anti-TPM antibody was the capture antibody, polyclonal rabbit anti-shrimp tropomyosin antibody was the detection antibody, while natural shrimp tropomyosin served as the standard. A double-stranded amino-DNA was covalently conjugated to a secondary anti-rabbit antibody and subsequently amplified and quantified via rtPCR. The quantification sensitivity of immuno-PCR was 20-fold higher than analogous ELISA, with LOQ 19.8 pg/mL. The developed immuno-PCR method is highly specific for the detection of crustacean tropomyosin and is highly precise in a broad concentration range. Tropomyosin recovery in the spiked vegetable soup was 87.7-115.6%. Crustacean tropomyosin was also quantified in commercial food products. The reported immuno-PCR assay is the most sensitive method for the quantification of crustacean tropomyosin and is the first immuno-PCR-based assay for the quantification of food allergen and food protein in general. The described method could be easily adapted for the specific and ultrasensitive immuno-PCR-based detection of traces of any food allergen that is currently being quantified with ELISA, which is of critical importance for people with food allergies.

Cellular and bioenergetic effects of polystyrene microplastic in function of cell type, differentiation status and post-exposure time.

The ubiquity of microplastics (MPs) in food sources and personal care products increasingly raises concerns on human health. However, little is known about the duration of the effects of MPs and whether effects depend on cellular differentiation status. Herein, cellular and bioenergetic effects of MPs in different exposure scenarios on four types of human cell lines derived from lung (A549 and BEAS-2B), colon (Caco-2) and liver (HepG2) were investigated. These cell lines are models for the major exposure routes in the body (inhalation, ingestion and physiological transport through the liver by the portal vein). To this aim, different scenarios were implemented by exposing undifferentiated and differentiated cells to single dosing of 2-µm polystyrene (PS) (102-105 particles/mL) for 48 h and 12 days. The undifferentiated Caco-2 cells with short exposure (48 h) showed the highest uptake rate of PS yet without significant cellular and mitochondrial responses. The biological effects, with the exception of ROS production, were not influenced by differentiation states of A549 and Caco-2 cells although differentiated cells showed much weaker ability to internalize PS. However, PS had significantly long-term impacts on cellular and mitochondrial functions even after the initial exposure period. In particular, Caco-2 cells that were post-exposed for 12 days after single PS dosing suffered higher oxidative stress and exhibited mitochondrial dysfunction than that for short exposure. Correspondingly, we observed that PS particles still remained in cell membrane and even in nuclei with high retention rate by 14-d post exposure during which metabolism and exchange of internalization and release occurred in cells. This indicates PS could induce chronic stress and even harmful effects on human cells after single intake that persists for a long time. This study paves the way for assessing the influence of PS on human health at low particle concentrations and with multiple exposure scenarios.

Prototype of an Innovative Vacuum Dryer with an Ejector System: Comparative Drying Analysis with a Vacuum Dryer with a Vacuum Pump on Selected Fruits.

The following article describes new research about the design, construction and installation of the new prototype of a vacuum dryer with an ejector system. Moreover, the testing of this new prototype involved comparing the qualities of fruit dried in a vacuum drier with an ejector system to fruit dried in a convectional vacuum drier. The data obtained were then analyzed and presented. Due to their economic relevance and highly valuable nutritional value and sensory properties, sour cherries and apricots have been chosen to be the subjects for the testing. The most appropriate quality indicators for analyzing were moisture content, aw value, share and penetration force, total phenol, flavonoid and anthocyanin content and antioxidant activity (FRAP, DPPH and ABTS test). The main results of this study were achieved by designing, constructing, installing and testing the usage of the innovative prototype of a vacuum dryer with an ejector system in the laboratory of the Technology of fruit and vegetable products of the Faculty of Technology Novi Sad, University of Novi Sad. Based on our analyses of the obtained data, it was concluded that vacuum dryer with an ejector system are similar to vacuum dryer with a vacuum pump in terms of all tested physical, chemical and biological properties of dried samples. We observed similarities in some of the most important parameters, including product safety and quality, such as the aw value and the total phenol content, respectively. For example, in dried sour cherry, the aw values ranged from 0.250 to 0.521 with the vacuum pump and from 0.232 to 0.417 with the ejector system; the total phenol content ranged from 2322 to 2765 mg GAE/100 g DW with the vacuum pump and from 2327 to 2617 mg GAE/100 g DW with the ejector system. In dried apricot, the aw ranged from 0.176 to 0.405 with the vacuum pump and from 0.166 to 0.313 with the ejector system; total phenol content ranged from 392 to 439 mg GAE/100 g DW with the vacuum pump and from 378 to 428 mg GAE/100 g DW with the ejector system.

Aspergillus flavus and Fusarium verticillioides and Their Main Mycotoxins: Global Distribution and Scenarios of Interactions in Maize.

Maize is frequently contaminated with multiple mycotoxins, especially those produced by Aspergillus flavus and Fusarium verticillioides. As mycotoxin contamination is a critical factor that destabilizes global food safety, the current review provides an updated overview of the (co-)occurrence of A. flavus and F. verticillioides and (co-)contamination of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) in maize. Furthermore, it summarizes their interactions in maize. The gathered data predict the (co-)occurrence and virulence of A. flavus and F. verticillioides would increase worldwide, especially in European cold climate countries. Studies on the interaction of both fungi regarding their growth mainly showed antagonistic interactions in vitro or in planta conditions. However, the (co-)contamination of AFB1 and FB1 has risen worldwide in the last decade. Primarily, this co-contamination increased by 32% in Europe (2010-2020 vs. 1992-2009). This implies that fungi and mycotoxins would severely threaten European-grown maize.

UV-C and wet heat resistance of Bacillus thuringiensis biopesticide endospores compared to foodborne Bacillus cereus endospores.

Bacillus endospores (spores) are generally resistant to environmental and food processing-related stress including thermal and non-thermal processing in the food industry, such as pasteurization, and UV-C inactivation. Bacillus thuringiensis insecticidal crystals and spores as the active substances in commercial biopesticides can also be introduced to vegetable foods and their food processing environment due to pre-harvest treatment of edible crops. The resistance of B. thuringiensis biopesticide spores in comparison to the genetically closely related foodborne B. cereus against heat and UV-C treatment is investigated in this study. The results show that B. thuringiensis biopesticide spores with the commercial granulated product formulation are better protected and as such more resistant to both wet heat (D values at 90 °C: 50.1-79.5 min) and UV-C treatment (D values at 0.6 mW/cm2: 7.5-8.9 min) than the pure spore suspension. The enhanced UV-C resistance properties of B. thuringiensis-formulated spores also indicate that the B. thuringiensis spores in powder or granule formulation applied in the field might not be effectively inactivated by solar radiation (UV-A and UV-B) in a short period. Furthermore, the spores of one emetic B. cereus toxin-producing strain (LFMFP 254; a Belgian outbreak strain) were found more resistant to the wet heat at 90 °C (D90-value = 71.2 min) than other tested pure spore suspensions, although the spores of B. cereus 254 did not show different behavior against UV-C treatment. This result suggests that UV-C treatment can be applied as an effective inactivation method against B. cereus 254 spores.

Second order probabilistic assessment of chronic dietary exposure to aflatoxin M1 in Serbia.

Considering the genotoxic and cancerogenic nature of aflatoxin M1 (AFM1), its presence in milk and dairy products may pose health risks for consumers. The chronic exposure was calculated using a two-dimensional (second order) Monte Carlo model. Results of 13 722 milk and dairy product samples analysed in the 2015-2022 period were used. Milk and dairy products intake information was collected with a Food Frequency Questionnaire (FFQ) validated by a 24-h recall-based method. Risk characterization was done by calculation of the Margin of Exposure (MOE) and by calculation of AFM1 induced number of hepatocellular carcinoma (HCC) cases. Mean AFM1 Estimated Daily Intake (EDI) was highest in children at 0.336 (CI: 0.294-0.385) ng kg-1 bw day-1, followed by adolescents with 0.183 (CI: 0.164-0.204), then adult females with 0.161 (CI: 0.146-0.179) and finally adult males with lowest EDI of 0.126 (CI: 0.115-0.139) ng kg-1 bw day-1. MOE values based on mean EDI for all population groups were above risk associated threshold and the number of possible HCC cases was in the range of 0.0002-0.0021 cases per year for 105 individuals. The results suggest low health risks due to AFM1 exposure for the whole population. Still, this risk is not non-existent, especially for children as they have a higher ratio of the population exposed to risk associated AFM1 levels, with MOE values below risk indicating threshold starting at 77.5th percentile.

New insights into the combined toxicity of aflatoxin B1 and fumonisin B1 in HepG2 cells using Seahorse respirometry analysis and RNA transcriptome sequencing.

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are widely (co-)detected in food and known for their hepatotoxicity in humans. Still, their combined toxicity needs to be investigated, especially the impact on mitochondria. In our previous work, we examined the effect of short-term exposure to different doses of AFB1, FB1, and their binary mixture (MIX) on the bioenergetic status of HepG2 cells, a well-recognized in vitro model system for studying liver cell function. In the current work, we further investigated the (combined) effect of AFB1 and FB1 on the mitochondrial and glycolytic activity of HepG2 cells using Seahorse respirometry analysis and RNA transcriptome sequencing. The results showed that the co-exposure, especially at high doses, is more toxic due to a more inhibition of all parameters of mitochondrial respiration. However, FB1 contributes more to the MIX effects than AFB1. RNA transcriptome sequencing showed that the p53 signaling pathway, a major orchestrator of mitochondrial apoptosis, was differentially expressed. Moreover, the co-exposure significantly downregulated the genes encoding for Complexes I, II, III, and IV, representing the onset of the suppressed mitochondrial respiration in HepG2 cells.

Rare Sugar Metabolism and Impact on Insulin Sensitivity along the Gut-Liver-Muscle Axis In Vitro.

Rare sugars have recently attracted attention as potential sugar replacers. Understanding the biochemical and biological behavior of these sugars is of importance in (novel) food formulations and prevention of type 2 diabetes. In this study, we investigated whether rare sugars may positively affect intestinal and liver metabolism, as well as muscle insulin sensitivity, compared to conventional sugars. Rare disaccharide digestibility, hepatic metabolism of monosaccharides (respirometry) and the effects of sugars on skeletal muscle insulin sensitivity (impaired glucose uptake) were investigated in, respectively, Caco-2, HepG2 and L6 cells or a triple coculture model with these cells. Glucose and fructose, but not l-arabinose, acutely increased extracellular acidification rate (ECAR) responses in HepG2 cells and impaired glucose uptake in L6 cells following a 24 h exposure at 28 mM. Cellular bioenergetics and digestion experiments with Caco-2 cells indicate that especially trehalose (α1-1α), D-Glc-α1,2-D-Gal, D-Glc-α1,2-D-Rib and D-Glc-α1,3-L-Ara experience delayed digestion and reduced cellular impact compared to maltose (α1-4), without differences on insulin-stimulated glucose uptake in a short-term setup with a Caco-2/HepG2/L6 triple coculture. These results suggest a potential for l-arabinose and specific rare disaccharides to improve metabolic health; however, additional in vivo research with longer sugar exposures should confirm their beneficial impact on insulin sensitivity in humans.

Comparative digestion of thermally treated vertebrates and invertebrates allergen pairs in real food matrix.

The digestion stability of allergen pairs, tropomyosin, TM (fish and seafood allergen), and myosin light chain, MLC (chicken meat allergen) is compared among vertebrates and invertebrates in raw and cooked food matrix under standardized simulated in vitro gastrointestinal (GI) digestion. SDS-PAGE followed by multiple TM and MLC-specific antibodies in semidry WB revealed pepsin resistance of invertebrate TMs (abalone, oyster, shrimp) under diet-relevant conditions (raw, cooked). Vertebrate TMs (chicken, pork, beef) were less stable to digestion except that the raw chicken TM was observed pepsin resistant (not diet-relevant). Vertebrate (chicken) MLC was thermally stable. A new 28 kDa protein bound to anti-MLC antibody in cooked chicken and pork; could be the aggregates of MLC. Raw shrimp MLC showed pepsin resistance among invertebrates. A good correlation was observed between combined resistance of TM and MLC to GI digestion following the diet-relevant thermal treatment and reported protein allergenicity among vertebrates and invertebrates.

Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species.

In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52-48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19-100.00%) and sensitivity was 52.94% (95% CI, 35.13-70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67-65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.

Identification and characterization of Bacillus thuringiensis and other Bacillus cereus group isolates from spinach by whole genome sequencing.

Bacillus thuringiensis (Bt), used as a biological control agent (BCA), can persist on plants, and from there can be introduced into the final food product. In routine food safety diagnostics, these Bt residues cannot be distinguished from natural populations of Bacillus cereus present in plants and all are enumerated as "presumptive B. cereus." In this study, information on eventual use of Bt biopesticides, brand, application times and intervals provided by three food processing companies in Belgium, were integrated with quantitative data on presumptive B. cereus measured from fresh to frozen food products. This information together with data on genomic similarity obtained via whole genome sequencing (WGS) and cry gene profiling using a quantitative real-time PCR (qPCR) assay, confirmed that six out of 11 Bt isolates originated from the applied Bt biocontrol products. These identified Bt strains were shown to carry enterotoxin genes (nhe, hbl, cytK-2) and express Hbl enterotoxin in vitro. It was also noted that these Bt biopesticide strains showed no growth at standard refrigeration temperatures and a low or moderate biofilm-forming ability and cytotoxic activity. Our results also showed that the use of Bt as a BCA on spinach plants in the field led to higher residual counts of Bt in spinach (fresh or frozen) in the food supply chain, but the residual counts exceeding at present commonly assumed safety limit of 105 CFU/g was only found in one fresh spinach sample. It is therefore recommended to establish a pre-harvest interval for Bt biopesticide application in the field to lower the likelihood of noncompliance to the generic B. cereus safety limit. Furthermore, WGS was found to be the best way to identify Bt biopesticide isolates at the strain level for foodborne outbreaks and clinical surveillance. The developed qPCR assay for screening on the presence of cry genes in presumptive B. cereus can be applied as a rapid routine test as an amendment to the already existing test on Bt crystal proteins determined via phase-contrast microscopy.