Systematic risk ranking of microbiological hazards in infant foods.

Ensuring food safety, particularly for vulnerable groups, like infants and young children, requires identifying and prioritizing potential hazards in food chains. We previously developed a web-based decision support system (DSS) to identify specific microbiological hazards (MHs) in infant and toddler foods through a structured five-step process. This study takes the framework further by introducing systematic risk ranking (RR) steps to rank MH risks with seven criteria: process survival, recontamination, growth opportunity, meal preparation, hazard-food association evidence, food consumption habits of infants and toddlers in the EU, and MH severity. Each criterion is given a semi-quantitative or quantitative score or risk value, contributing to the final MH risk calculation via three aggregation methods: semi-quantitative risk scoring, semi-quantitative risk value, and outranking multi-criteria decision analysis (MCDA). To validate the criteria and ranking approaches, we conducted a case study to rank MH risks in infant formula, compared the results of the three risk ranking methods, and additionally evaluated the ranking results against expert opinions to ensure their accuracy. The results showed strong agreement among the three methods, consistently ranking Salmonella non-Typhi and Cronobacter spp. and Shiga-toxin-producing Escherichia coli as the top MH risks in infant formulae, with minor deviations. When MHs were ranked after an initial hazard identification step, all three methods produced nearly identical MH rankings, reinforcing the reliability of the ranking steps and the selected criteria. Notably, the risk value and MCDA methods provided more informative MH rankings compared to the risk scoring method. The risk value and risk scoring methods were implemented into an online tool, called the MIcrobiological hazards risk RAnking decision support system (Mira-DSS), available at https://foodmicrobiologywur.shinyapps.io/MIcrobial_hazards_RAnking/. In conclusion, our framework enables the ranking of MH risks, facilitating intervention comparisons and resource allocations to mitigate MH risks in infant foods, with potential applicability to broader food categories.

A secondary model for the effect of pH on the variability in growth fitness of Listeria innocua strains.

Variability in microbial growth is a keystone of modern Quantitative Microbiological Risk Assessment (QMRA). However, there are still significant knowledge gaps on how to model variability, with the most common assumption being that variability is constant. This is implemented by an error term (with constant variance) added on top of the secondary growth model (for the square root of the growth rate). However, this may go against microbial ecology principles, where differences in growth fitness among bacterial strains would be more prominent in the vicinity of the growth limits than at optimal growth conditions. This study coins the term "secondary models for variability", evaluating whether they should be considered in QMRA instead of the constant strain variability hypothesis. For this, 21 strains of Listeria innocua were used as case study, estimating their growth rate by the two-fold dilution method at pH between 5 and 10. Estimates of between-strain variability and experimental uncertainty were obtained for each pH using mixed-effects models, showing the lowest variability at optimal growth conditions, increasing towards the growth limits. Nonetheless, the experimental uncertainty also increased towards the extremes, evidencing the need to analyze both sources of variance independently. A secondary model was thus proposed, relating strain variability and pH conditions. Although the modelling approach certainly has some limitations that would need further experimental validation, it is an important step towards improving the description of variability in QMRA, being the first model of this type in the field.

Development of novel natto using legumes produced in Europe.

Natto is a traditional Japanese fermented product consisting of cooked soybeans fermented with Bacillus subtilis var. natto. We assessed three different B. subtilis strains and investigated their impact on product quality aspects, such as microbial quality, textural quality (poly-γ-glutamate strand formation), free amino acids (FAA), and volatile organic compounds (VOCs), but also the vitamin K1, K2 and B1 content, and presence of nattokinase. Using Bayesian contrast analysis, we conclude that the quality attributes were influenced by both the substrate and strain used, without significant differences in bacterial growth between strain or substrate. Overall, all the tested European legumes, except for brown beans, are adequate substrates to produce natto, with comparable or higher qualities compared to the traditional soy. Out of all the tested legumes, red lentils were the most optimal fermentation substrate. They were fermented most consistently, with high concentrations of vitamin K2, VOCs, FAA.

Optimal experimental design (OED) for the growth rate of microbial populations. Are they really more "optimal" than uniform designs?

Secondary growth models from predictive microbiology can describe how the growth rate of microbial populations varies with environmental conditions. Because these models are built based on time and resource consuming experiments, model-based Optimal Experimental Design (OED) can be of interest to reduce the experimental load. In this study, we identify optimal experimental designs for two common models (full Ratkowsky and Cardinal Parameters Model (CPM)) for a different number of experiments (10-30). Calculations are also done fixing one or more model parameters, observing that this decision strongly affects the layout of the OED. Using in silico experiments, we conclude that OEDs are more informative than conventional (equidistant) designs with the same number of experiments. However, OEDs cluster the experiments near the growth limits (Xmin and Xmax) resulting in impractical designs with aggregated experimental runs ~10 times longer than conventional designs. To mitigate this, we propose a novel optimality criterion (i.e., the objective function) that accounts for the aggregated time. The novel criterion provides a reduction in parameter uncertainty with respect to the conventional design, without an increase in the experimental load. These results underline that an OED is only based on information theory (Fisher information), so the results can be impractical when actual experimental limitations are considered. The study also emphasizes that most OED schemes identify where to measure, but do not give an indication on how many experiments should be made. In this sense, numerical simulations can estimate the parameter uncertainty that would be obtained for a particular experimental design (OED or not). These results and methodologies (available in Open Code) can guide the design of future experiments for the development of secondary growth models.