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.

Prevalence, attachment ability and strength of the biological control agent Bacillus thuringiensis on tomato.

Bacillus thuringiensis (Bt) is commonly used as a biological control agent (BCA) to control insect pests in edible plant production and can as such be introduced into the food chain of fresh produce. Using standard food diagnostics Bt will be detected and reported as presumptive B. cereus. Tomato plants are often sprayed with Bt biopesticides for insect control, thus these Bt BCAs can also reach the tomato fruits and persist until consumption. In this study, vine tomatoes from the retail in Belgium (Flanders) were investigated for the occurrence and residual numbers of presumptive B. cereus and Bt. Of 109 tomato samples, 61 (56%) were tested positive for presumptive B. cereus. Of the presumptive B. cereus isolates (n = 213) recovered from these samples, 98% were identified as Bt by the production of parasporal crystals. Further quantitative real-time PCR assays on a subselection of Bt isolates (n = 61) showed that 95% of Bt isolates were indistinguishable from Bt biopesticide strains that are approved to be used on crops in the EU. Furthermore, the attachment strength of tested Bt biopesticide strains showed easier wash-off properties if using the commercial Bt granule formulation than the unformulated lab-cultured Bt or B. cereus spore suspensions.

Detection of toxins involved in foodborne diseases caused by Gram-positive bacteria.

Bacterial toxins are food safety hazards causing about 10% of all reported foodborne outbreaks in Europe. Pertinent to Gram-positive pathogens, the most relevant toxins are emetic toxin and diarrheal enterotoxins of Bacillus cereus, neurotoxins of Clostridium botulinum, enterotoxin of Clostridium perfringens, and a family of enterotoxins produced by Staphylococcus aureus and some other staphylococci. These toxins are the most important virulence factors of respective foodborne pathogens and a primary cause of the related foodborne diseases. They are proteins or peptides that differ from each other in their size, structure, toxicity, toxicological end points, solubility, and stability, types of food matrix to which they are mostly related to. These differences influence the characteristics of required detection methods. Therefore, detection of these toxins in food samples, or detection of toxin production capacity in the bacterial isolate, remains one of the cornerstones of microbial food analysis and an essential tool in understanding the relevant properties of these toxins. Advanced research has led into new insights of the incidence of toxins, mechanisms of their production, their physicochemical properties, and their toxicological mode of action and dose-response profile. This review focuses on biological, immunological, mass spectrometry, and molecular assays as the most commonly used detection and quantification methods for toxins of B. cereus, C. botulinum, C. perfringens, and S. aureus. Gathered and analyzed information provides a comprehensive blueprint of the existing knowledge on the principles of these assays, their application in food safety, limits of detection and quantification, matrices in which they are applicable, and type of information they provide to the user.

Shift in performance of food safety management systems in supply chains: case of green bean chain in Kenya versus hot pepper chain in Uganda.

BACKGROUND: This study investigates the level of design and operation of food safety management systems (FSMS) of farmers and export traders in Kenya and Uganda. FSMS diagnostic tools developed for the fresh produce chain were used to assess the levels of context riskiness, FSMS activities and system output in primary production (n = 60) and trade (n = 60). High-risk context characteristics combined with basic FSMS are expected to increase the risk on unsafe produce. RESULTS: In Uganda both farmers and export traders of hot peppers operate in a high- to moderate-risk context but have basic FSMS and low systems output. In Kenya, both farmers and export traders of green beans operate in a low- to moderate-risk context. The farmers have average performing FSMS, whereas export trade companies showed more advanced FSMS and system output scores ranging from satisfactory to good. CONCLUSION: Large retailers supplying the EU premium market play a crucial role in demanding compliance with strict voluntary food safety standards, which was reflected in the more advanced FSMS and good system output in Kenya, especially traders. In Kenya, a clear shift in more fit-for-purpose FSMS and higher system output was noticed between farms and trade companies. In the case of Uganda, traders commonly supply to the less demanding EU wholesale markets such as ethnic specialty shops. They only have to comply with the legal phytosanitary and pesticide residue requirements for export activities, which apparently resulted in basic FSMS and low system output present with both farmers and traders. © 2015 Society of Chemical Industry.

Performance of Drying Technologies to Ensure Microbial Safety of Dried Fruits and Vegetables.

Dried fruits, vegetables, herbs, and spices are produced in and sourced from many countries worldwide, but they have been increasingly reported to be involved in outbreaks and alerts due to the presence of foodborne pathogens such as Salmonella. These dried products are mainly produced by solar drying and conventional air drying, but a wide range of drying technologies are available. From a technological point of view the general trend is to optimize and standardize the drying process to ensure high-quality products to be offered. Drying technologies are mainly evaluated for their performance to reduce water activity at low energy cost while maintaining good sensorial quality of the dried product. However, as low water activity foods are increasingly recognized to support microbial survival and dried products are often consumed as they are, or are used as ingredients in many ready-to-eat foods, there is increasing attention to the microbiological quality and safety aspects of these products. This review presents traditional and emerging technologies to dry fruits, vegetables, herbs, and spices and discusses their potential to inactivate bacteria and viruses throughout the drying process. Overall, the microbial inactivation effect of the presented technologies has not yet been thoroughly assessed, even for traditional methods like solar drying, conventional air drying, or freeze-drying. Emerging technologies such as dielectric (assisted) drying and low-pressure superheated steam drying have been shown to reduce microbial populations; however, the number of studies is still low. Very few studies have focused on viral inactivation during drying processes.

Microbial safety and sanitary quality of strawberry primary production in Belgium: risk factors for Salmonella and Shiga toxin-producing Escherichia coli contamination.

Strawberries are an important fruit in Belgium in both production and consumption, but little information is available about the presence of Salmonella and Shiga toxin-producing Escherichia coli (STEC) in these berries, the risk factors in agricultural production, and possible specific mitigation options. In 2012, a survey was undertaken of three soil and three soilless cultivation systems in Belgium. No Salmonella spp. were isolated. No STEC was detected in the strawberry samples (0 of 72), but STEC was detected by PCR in 11 of 78 irrigation water and 2 of 24 substrate samples. Culture isolates were obtained for 2 of 11 PCR-positive irrigation water samples and 2 of 2 substrate samples. Multivariable logistic regression analysis revealed elevated generic E. coli numbers (the odds ratio [OR] for a 1 log increase being 4.6) as the most important risk factor for STEC, together with the berry-picking season (elevated risk in summer). The presence of generic E. coli in the irrigation water (≥1 CFU per 100 ml) was mainly influenced by the type of irrigation water (collected rainfall water stored in ponds was more often contaminated than groundwater pumped from boreholes [OR = 5.8]) and the lack of prior treatment (untreated water versus water subjected to sand filtration prior to use [OR = 19.2]). The follow-up study in 2013 at one of the producer locations indicated cattle to be the most likely source of STEC contamination of the irrigation water.

Selection criteria for water disinfection techniques in agricultural practices.

This paper comprises a selection tool for water disinfection methods for fresh produce pre- and postharvest practices. A variety of water disinfection technologies is available on the market and no single technology is the best choice for all applications. It can be difficult for end users to choose the technology that is best fit for a specific application. Therefore, the different technologies were characterized in order to identify criteria that influence the suitability of a technology for pre- or postharvest applications. Introduced criteria were divided into three principal components: (i) criteria related to the technology and which relate to the disinfection efficiency, (ii) attention points for the management and proper operation, and (iii) necessities in order to sustain the operation with respect to the environment. The selection criteria may help the end user of the water disinfection technology to obtain a systematic insight into all relevant aspects to be considered for preliminary decision making on which technologies should be put to feasibility testing for water disinfection in pre- and postharvest practices of the fresh produce chain.

Pre- and postharvest preventive measures and intervention strategies to control microbial food safety hazards of fresh leafy vegetables.

This review includes an overview of the most important preventive measures along the farm to fork chain to prevent microbial contamination of leafy greens. It also includes the technological and managerial interventions related to primary production, postharvest handling, processing practices, distribution, and consumer handling to eliminate pathogens in leafy greens. When the microbiological risk is already present, preventive measures to limit actual contamination events or pathogen survival are considered intervention strategies. In codes of practice the focus is mainly put on explaining preventive measures. However, it is also important to establish more focused intervention strategies. This review is centered mainly on leafy vegetables as the commodity identified as the highest priority in terms of fresh produce microbial safety from a global perspective. There is no unique preventive measure or intervention strategy that could be applied at one point of the food chain. We should encourage growers of leafy greens to establish procedures based on the HACCP principles at the level of primary production. The traceability of leafy vegetables along the chain is an essential element in ensuring food safety. Thus, in dealing with the food safety issues associated with fresh produce it is clear that a multidisciplinary farm to fork strategy is required.

Bacillus cereus Adhesion to Simulated Intestinal Mucus Is Determined by Its Growth on Mucin, Rather Than Intestinal Environmental Parameters.

Adhesion of pathogenic bacteria to intestinal mucus, the protective layer of the gastrointestinal epithelium, is often considered a virulence factor. The ability of food-poisoning Bacillus cereus strains to attach to mucus and the factors affecting this interaction have not yet been investigated. Therefore, the role of adhesion in pathogenesis of B. cereus still remains unknown. In the present study, an in vitro assay based on mucin agar was used to simulate adhesion of B. cereus to mucus. Bacterial-associated factors (e.g., strain specificity and microbial competition) known to influence adhesion to different surfaces and a variety of environmental conditions (e.g., pH and oxygen) encountered in the gastrointestinal tract were investigated. The effect of these parameters on B. cereus NVH 0500/00 mucin adhesion was generally limited even in the presence of microbial competition. This suggests that B. cereus NVH 0500/00 is a versatile pathogen. Inoculation of 4 to 5 log colony-forming units (CFU) per milliliter. B. cereus NVH 0500/00 resulted in 5-6 log CFU/mL mucin-associated bacteria after a short incubation period. This indicates that this pathogenic strain could grow in the presence of mucin agar. This growth may potentially mask the effect of the studied conditions. Yet, extensive attachment of B. cereus to mucin is not necessarily a prerequisite for virulence, because other pathogenic strains do not adhere with the same efficiency to mucin. Nevertheless, adhesion may contribute to the disease by providing close contact to nutrient sources, such as mucin, which would not only result in bacterial proliferation, but also in disruption of the protective host mucus surface.

Comparison of sample types and analytical methods for the detection of highly campylobacter-colonized broiler flocks at different stages in the poultry meat production chain.

Exclusion of broiler batches, highly colonized with Campylobacter (>7.5 log10 colony-forming units/g), from the fresh poultry meat market might decrease the risk of human campylobacteriosis. The objective of this study was to compare different sample types (both at the farm and the slaughterhouse) and methods (direct culture, quantitative real-time polymerase chain reaction [qPCR], propidium monoazide [PMA]-qPCR) applied for the quantification of the Campylobacter colonization level. In addition, the applicability of the lateral flow-based immunoassay, Singlepath(®) Direct Campy Poultry test (Singlepath(®) test), was evaluated as a rapid method for the qualitative detection of Campylobacter in highly colonized broiler batches. Campylobacter counts differed significantly between sample types collected at farm level (cecal droppings, feces, boot swabs) and at slaughterhouse level (cecal content, fecal material from crates). Furthermore, comparison of Campylobacter counts obtained by different methods (direct culture, qPCR, PMA-qPCR) in cecal droppings revealed significant differences, although this was not observed for cecal-content samples. Evaluation of the Singlepath(®) test on cecal droppings and cecal-content samples revealed an acceptable level of sensitivity and specificity. In conclusion, cecal droppings and cecal content are proposed as the most representative sample types for quantification of Campylobacter colonization level of broilers at farm and slaughterhouse, respectively. Direct culture and qPCR are equally sensitive for quantification of Campylobacter in fresh cecal-content samples. PMA treatment before qPCR inhibits the signal from dead Campylobacter cells. Consequently, when samples are extensively stored and/or transported, qPCR is preferred to direct culture and PMA-qPCR. Furthermore, the Singlepath(®) test offers a convenient alternative method for rapid detection of Campylobacter in highly colonized broiler batches.

Fate of Foodborne Viruses in the "Farm to Fork" Chain of Fresh Produce.

Norovirus (NoV) and hepatitis A virus (HAV) are the most important foodborne viruses. Fresh produce has been identified as an important vehicle for their transmission. In order to supply a basis to identify possible prevention and control strategies, this review intends to demonstrate the fate of foodborne viruses in the farm to fork chain of fresh produce, which include the introduction routes (contamination sources), the viral survival abilities at different stages, and the reactions of foodborne viruses towards the treatments used in food processing of fresh produce. In general, the preharvest contamination comes mainly from soli fertilizer or irrigation water, while the harvest and postharvest contaminations come mainly from food handlers, which can be both symptomatic and asymptomatic. Foodborne viruses show high stabilities in all the stages of fresh produce production and processing. Low-temperature storage and other currently used preservation techniques, as well as washing by water have shown limited added value for reducing the virus load on fresh produce. Chemical sanitizers, although with limitations, are strongly recommended to be applied in the wash water in order to minimize cross-contamination. Alternatively, radiation strategies have shown promising inactivating effects on foodborne viruses. For high-pressure processing and thermal treatment, efforts have to be made on setting up treatment parameters to induce sufficient viral inactivation within a food matrix and to protect the sensory and nutritional qualities of fresh produce to the largest extent.

Application of long-range and binding reverse transcription-quantitative PCR to indicate the viral integrities of noroviruses.

This study intends to establish and apply methods evaluating both viral capsid and genome integrities of human noroviruses (NoVs), which thus far remain nonculturable. Murine norovirus 1 (MNV-1) and human NoV GII.4 in phosphate-buffered saline suspensions were treated with heat, UV light, or ethanol and detected by reverse transcription-quantitative PCR (RT-qPCR), long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR. For MNV-1 heated at 60°C for 2 and 30 min, limited reductions of genomic copies (<0.3-log) were obtained by RT-qPCR and long-range RT-qPCR, while the cell-binding pretreatments obtained higher reductions (>1.89-log reduction after 60°C for 30 min by binding long-range RT-qPCR). The human NoV GII.4 was found to be more heat resistant than MNV-1. For both MNV-1 and human NoV GII.4 after UV treatments of 20 and 200 mJ/cm(2), no significant difference (P > 0.05) was observed between the dose-dependent reductions obtained by the four detection methodologies. Treatment of 70% ethanol for 1 min was shown to be more effective for inactivation of both MNV-1 and human NoV GII.4 than the heat and UV treatments used in this study. Subsequently, eight raspberry and four shellfish samples previously shown to be naturally contaminated with human NoVs by RT-qPCR (GI and GII; thus, 24 RT-qPCR signals) were subjected to comparison by this method. RT-qPCR, long-range RT-qPCR, binding RT-qPCR, and binding long-range RT-qPCR detected 20/24, 14/24, 24/24, and 23/24 positive signals, respectively, indicating the abundant presence of intact NoV particles.

Microbiological sampling plan based on risk classification to verify supplier selection and production of served meals in food service operation.

Food service operations are confronted with a diverse range of raw materials and served meals. The implementation of a microbial sampling plan in the framework of verification of suppliers and their own production process (functionality of their prerequisite and HACCP program), demands selection of food products and sampling frequencies. However, these are often selected without a well described scientifically underpinned sampling plan. Therefore, an approach on how to set-up a focused sampling plan, enabled by a microbial risk categorization of food products, for both incoming raw materials and meals served to the consumers is presented. The sampling plan was implemented as a case study during a one-year period in an institutional food service operation to test the feasibility of the chosen approach. This resulted in 123 samples of raw materials and 87 samples of meal servings (focused on high risk categorized food products) which were analyzed for spoilage bacteria, hygiene indicators and food borne pathogens. Although sampling plans are intrinsically limited in assessing the quality and safety of sampled foods, it was shown to be useful to reveal major non-compliances and opportunities to improve the food safety management system in place. Points of attention deduced in the case study were control of Listeria monocytogenes in raw meat spread and raw fish as well as overall microbial quality of served sandwiches and salads.

Molecular Methods in Food Safety Microbiology: Interpretation and Implications of Nucleic Acid Detection.

Because of increasing demand for rapid results, molecular techniques are now applied for the detection of microorganisms in foodstuffs. However, interpretation problems can arise for the results generated by molecular methods in relation to the associated public health risk. Discrepancies between results obtained by molecular and conventional culture methods stem from the difference in target, namely nucleic acids instead of actively growing microorganisms. Nucleic acids constitute 5% to 15% of the dry weight of all living cells and are relatively stable, even after cell death, so they may be present in a food matrix after the foodborne microorganisms have been inactivated. Therefore, interpretation of the public health significance of positive results generated by nucleic acid detection methods warrants some additional consideration. This review discusses the stability of nucleic acids in general and highlights the persistence of microbial nucleic acids after diverse food-processing techniques based on data from the scientific literature. Considerable amounts of DNA and RNA (intact or fragmented) persist after inactivation of bacteria and viruses by most of the commonly applied treatments in the food industry. An overview of the existing adaptations for molecular assays to cope with these problems is provided, including large fragment amplification, flotation, (enzymatic) pretreatment, and various binding assays. Finally, the negligible risks of ingesting free microbial nucleic acids are discussed and this review ends with the future perspectives of molecular methods such as next-generation sequencing in diagnostic and source attribution food microbiology.

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 nonbiofilm 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.

Prevalence and growth potential of Listeria monocytogenes in innovative, pre-packed, plant-based ready-to-eat food products on the Belgian market.

In recent years, pre-packed ready-to-eat (RTE) food products on the Belgian market have shifted to a more plant-based composition due to a variety of reasons, including consumer concerns about health, animal welfare, and sustainability. However, similar to animal-based RTE foods, plant-based RTE foods can be susceptible to the presence and outgrowth of Listeria monocytogenes (L. monocytogenes). Three innovative, pre-packed, plant-based RTE food product categories on the Belgian market were identified based upon data gaps regarding the prevalence and growth potential of this pathogen. These were vegetarian and vegan deli sandwich slices (category 1), fresh-cut (mixes of) leafy vegetables (category 2), and multi-ingredient salad bowls (category 3). Reports on associated listeriosis outbreaks and recalls were collected and a comprehensive literature review on the prevalence of L. monocytogenes (i.e. detection in 25 g food) was performed. In addition, the prevalence of L. monocytogenes was also determined through an exploratory retail survey of ca. 50 different RTE products of each category. A batch was considered positive if L. monocytogenes was detected in a food item, either on the day of purchase, at the end of shelf life, or both. During the retail survey, L. monocytogenes was not detected in category 2 (0 out of 51 batches), while 1 out of 51 and 6 out of 48 batches were found positive for respectively category 1 and 3. The observed L. monocytogenes concentration did not exceed 10 CFU/g at any point in time in any batch. Furthermore, challenge tests were performed to determine the growth potential of L. monocytogenes in nine pre-packed, plant-based RTE food products (two to four different products of each category, and three different batches per product). After inoculation, products were stored for half of their shelf life at 7 °C and half of their shelf life at 9 °C (simulation of resp. retail and consumer storage). In six of the nine challenge tests executed, growth of L. monocytogenes was supported (i.e. growth potential ≥0.50 log10 CFU/g during shelf life). The highest growth potential was observed for fresh-cut iceberg lettuce (3.60 log10 CFU/g in 9 days), but a large variation regarding the growth potential of L. monocytogenes was noted both between and within the three studied pre-packed, plant-based RTE food product categories. This variation was mainly caused by differences in product composition, physicochemical product characteristics, present (competitive) microbiota such as lactic acid bacteria, applied preservation techniques, and shelf life.

Machine Learning and Imputation to Characterize Human Norovirus Genotype Susceptibility to Sodium Hypochlorite.

Human norovirus (HuNoV) is the leading cause of foodborne illness in the developed world and a major contributor to gastroenteritis globally. Its low infectious dose and environmental persistence necessitate effective disinfection protocols. Sodium hypochlorite (NaOCl) bleach is a widely used disinfectant for controlling HuNoV transmission via contaminated fomites. This study aimed to evaluate the susceptibility of HuNoV genotypes (n = 11) from genogroups I, II, and IV to NaOCl in suspension. HuNoV was incubated for 1 and 5 min in diethyl pyrocarbonate (DEPC) treated water containing 50 ppm, 100 ppm, or 150 ppm NaOCl, buffered to maintain a pH between 7.0 and 7.5. Neutralization was achieved by a tenfold dilution into 100% fetal bovine serum. RNase pre-treatment followed by RT-qPCR was used to distinguish between infectious and non-infectious HuNoV. Statistical methods, including imputation, machine learning, and generalized linear models, were applied to process and analyze the data. Results showed that NaOCl reduced viral loads across all genotypes, though efficacy varied. Genotypes GI.1, GII.4 New Orleans, and GII.4 Sydney were the least susceptible, while GII.6 and GII.13 were the most susceptible. All NaOCl concentrations above 0 ppm were statistically indistinguishable, and exposure duration did not significantly affect HuNoV reduction, suggesting rapid inactivation at effective concentrations. For instance, some genotypes were completely inactivated within 1 min, rendering extended exposure unnecessary, while other genotypes maintained the initial concentration at both 1 and 5 min, indicating a need for longer contact times. These findings underscore the critical role of HuNoV genotype selection in testing disinfection protocols and optimizing NaOCl concentrations. Understanding HuNoV susceptibility to NaOCl bleach informs better disinfection strategies, aiding public health and food safety authorities in reducing HuNoV transmission and outbreaks.

Salmonella Prevalence in Raw Cocoa Beans and a Microbiological Risk Assessment to Evaluate the Impact of Cocoa Liquor Processing on the Reduction of Salmonella.

Salmonella in raw cocoa beans (n = 870) from main sourcing areas over nine months was analyzed. It was detected in 71 (ca. 8.2%) samples, with a contamination level of 0.3-46 MPN/g except for one sample (4.1 × 104 CFU/g). Using prevalence and concentration data as input, the impact of thermal treatment in cocoa processing on the risk estimate of acquiring salmonellosis by a random Belgian chocolate consumer was calculated by a quantitative microbiological risk assessment (QMRA) approach. A modular process risk model from raw cocoa beans to cocoa liquor up to a hypothetical final product (70-90% dark chocolate tablet) was set up to understand changes in Salmonella concentrations following the production process. Different thermal treatments during bean or nib steam, nib roasting, or liquor sterilization (achieving a 0-6 log reduction of Salmonella) were simulated. Based on the generic FAO/WHO Salmonella dose-response model and the chocolate consumption data in Belgium, salmonellosis risk per serving and cases per year at population level were estimated. When a 5 log reduction of Salmonella was achieved, the estimated mean risk per serving was 3.35 × 10-8 (95% CI: 3.27 × 10-10-1.59 × 10-7), and estimated salmonellosis cases per year (11.7 million population) was 88 (95% CI: <1-418). The estimated mean risk per serving was 3.35 × 10-9 (95% CI: 3.27 × 10-11-1.59 × 10-8), and the estimated salmonellosis cases per year was 9 (95% CI: <1-42), for a 6 log reduction. The current QMRA model solely considered Salmonella reduction in a single-step thermal treatment in the cocoa process. Inactivation obtained during other process steps (e.g. grinding) might occur but was not considered. As the purpose was to use QMRA as a tool to evaluate the log reduction in the cocoa processing, no postcontamination from the processing environment and ingredients was included. A minimum of 5 log reduction of Salmonella in the single-step thermal treatment of cocoa process was considered to be adequate.

Towards a risk-based food safety management system in the fresh produce supply chain in Da Nang, Viet Nam.

Food safety has emerged as a paramount concern for both Vietnamese consumers and the government. However, limited data are available on food safety management systems in Viet Nam. This study identified significant gaps in good agricultural and hygienic practices along the fresh produce chain (farmers and traditional wholesalers/market sellers) in the region of Da Nang, Viet Nam. This was achieved through a survey on good agricultural and hygienic practices for farmers (n = 100) and sellers (n = 100), which researchers further supplemented by microbiological analysis for E. coli, Salmonella spp., and Listeria monocytogenes on leafy greens, water in contact with produce and contact surfaces (hands). The results indicated that 86.0 % of farmers and 54.0 % of sellers received food safety training in the last 3 years; and women dominated both vegetable cultivation but also trading. Farm-level deficiencies included inadequate handwashing practices, lack of documentation for manure application schedules, improper washing and drying of harvest tools, failure to keep containers elevated off the ground, improper storage of vegetables, and inadequate covering of containers, with respectively 34.0 %, 30.3 %, 12.1 %, 41.7 % and 7.9 % of farmers executing the practice as prescribed by the WHO/FAO '5 keys of growing safer fruits and vegetables'. As for sellers, the most dominant gaps (<50.0 % compliance) were the way of handwashing and the practice of keeping containers elevated off the ground before, during, and after harvesting. The microbiological analysis confirmed that, in a total of 36 fresh produce samples including mustard greens, cucumber, lettuce, and crown daisy, the number of samples positive for E. coli, Salmonella spp., and L. monocytogenes were 12, 2, and 10 respectively. Samples of hands and the irrigation water showed high contamination with E. coli. Based on identified gaps, risk communication tools were developed and distributed amongst farmers, sellers, and Da Nang food safety management authority (governmental organisation performing inspections in the traditional food markets). As intervention, two farmers and two sellers were trained in safe agricultural practices for the cultivation of fresh vegetables (managerial intervention) and instructed to use tap water as irrigation water instead of uncontrolled surface water (technological intervention). A post-assessment was conducted, including redoing the survey on good practices and microbiological analysis. The outcome of these interventions showed positive results in terms of good agricultural and hygienic practices resulting in improved hygiene levels and safety of the fresh produce. The findings from this research have the potential to provide a model for the development of a science-based risk management strategy in alternative food chains or geographic areas in emerging countries.

Physicochemical quality and chemical safety of chlorine as a reconditioning agent and wash water disinfectant for fresh-cut lettuce washing.

Chlorine was assessed as a reconditioning agent and wash water disinfectant in the fresh-cut produce industry. Artificial fresh-cut lettuce wash water, made from butterhead lettuce, was used for the experiments. In the reconditioning experiments, chlorine was added to artificial wash water inoculated with Escherichia coli O157 (6 log CFU/ml). Regression models were constructed based on the inactivation data and validated in actual wash water from leafy vegetable processing companies. The model that incorporated chlorine dose and chemical oxygen demand (COD) of the wash water accurately predicted inactivation. Listeria monocytogenes was more resistant to chlorine reconditioning in artificial wash water than Salmonella spp. and Escherichia coli O157. During the washing process with inoculated lettuce (4 log CFU/g), in the absence of chlorine, there was a rapid microbial buildup in the water that accumulated to 5.4 ± 0.4 log CFU/100 ml after 1 h. When maintaining a residual concentration of 1 mg/liter free chlorine, wash water contamination was maintained below 2.7, 2.5, and 2.5 log CFU/100 ml for tap water and artificial process water with COD values of 500 and 1,000 mg O2/liter, respectively. A model was developed to predict water contamination during the dynamic washing process. Only minor amounts of total trihalomethanes were formed in the water during reconditioning. Total trihalomethanes accumulated to larger amounts in the water during the wash water disinfection experiments and reached 124.5 ± 13.4 µg/liter after 1 h of execution of the washing process in water with a COD of 1,000 mg O2/liter. However, no total trihalomethanes were found on the fresh-cut lettuce after rinsing.