M.H. Zwietering

A decision support system for prediction of microbial spoilage in foods.

SummaryA method is developed to combine qualitative and quantitative information for the prediction of growth of microorganisms in foods. pH, water activity, temperature and oxygen availability of foods are coupled to growth characteristics of microorganisms. For that purpose, a database with characteristics of foods and a database of kinetic parameters of microorganisms are built. The first database has a tree structure, based on physical similarity of food products. This structure makes it possible to estimate information about a food product which is not listed by comparison with similar products at the same level of the tree or the level above. A method is developed to make an estimation of the microbial growth kinetics on the basis of models. This is done by introducing a growth factor, which can be calculated on the basis of readily available data from literature. Finally, qualitative knowledge is added. Since any bit of information can be changed, the system will give better predictions when more and more accurate information is added.

Future challenges to microbial food safety

Despite significant efforts by all parties involved, there is still a considerable burden of foodborne illness, in which micro-organisms play a prominent role. Microbes can enter the food chain at different steps, are highly versatile and can adapt to the environment allowing survival, growth and production of toxic compounds. This sets them apart from chemical agents and thus their study from food toxicology. We summarize the discussions of a conference organized by the Dutch Food and Consumer Products Safety Authority and the European Food Safety Authority. The goal of the conference was to discuss new challenges to food safety that are caused by micro-organisms as well as strategies and methodologies to counter these. Management of food safety is based on generally accepted principles of Hazard Analysis Critical Control Points and of Good Manufacturing Practices. However, a more pro-active, science-based approach is required, starting with the ability to predict where problems might arise by applying the risk analysis framework. Developments that may influence food safety in the future occur on different scales (from global to molecular) and in different time frames (from decades to less than a minute). This necessitates development of new risk assessment approaches, taking the impact of different drivers of change into account. We provide an overview of drivers that may affect food safety and their potential impact on foodborne pathogens and human disease risks. We conclude that many drivers may result in increased food safety risks, requiring active governmental policy setting and anticipation by food industries whereas other drivers may decrease food safety risks. Monitoring of contamination in the food chain, combined with surveillance of human illness and epidemiological investigations of outbreaks and sporadic cases continue to be important sources of information. New approaches in human illness surveillance include the use of molecular markers for improved outbreak detection and source attribution, sero-epidemiology and disease burden estimation. Current developments in molecular techniques make it possible to rapidly assemble information on the genome of various isolates of microbial species of concern. Such information can be used to develop new tracking and tracing methods, and to investigate the behavior of micro-organisms under environmentally relevant stress conditions. These novel tools and insight need to be applied to objectives for food safety strategies, as well as to models that predict microbial behavior. In addition, the increasing complexity of the global food systems necessitates improved communication between all parties involved: scientists, risk assessors and risk managers, as well as consumers.

Air-Liquid Interface Biofilms of Bacillus cereus: Formation, Sporulation, and Dispersion

ABSTRACT Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.

Growth and inactivation models to be used in quantitative risk assessments.

In past years many models describing growth and inactivation of microorganisms have been developed. This study is a discussion of the growth and inactivation models that can be used in a stepwise procedure for quantitative risk assessment. First, rough risk assessments are performed in which orders of magnitude for microbial processes are estimated by the use of simple models. This method provides an efficient way to find the main determinants of risk. Second, the main determinants of risk are studied more accurately and quantitatively. It is best to compare several models at this level, as no model is expected to be able accurately to predict microbial responses under all circumstances. By comparing various models the main determinants of risk are studied from several points of view, and risks can be assessed on a broad basis. If, however, process variations have a more profound effect on risk than the differences between models, it is most efficient to use the simplest model available. If relevant, the process variations can be stochastically described in the third level of detail. Stochastic description of the process parameters will however not change the conclusion on the usefulness of simple models in quantitative risk assessments. The proposed stepwise procedure that starts simply before going into detail provides a structured method of risk assessment and prevents the researcher from getting caught in too much complexity. This simplicity is necessary because of the complex nature of food safety. The principal aspects are highlighted during the procedure and many factors can be omitted since their quantitative effect is negligible.

A Quantitative Analysis of Cross-Contamination of Salmonella and Campylobacter spp. Via Domestic Kitchen Surfaces

Epidemiological data indicate that cross-contamination during food preparation in the home contributes noticeably to the occurrence of foodborne diseases. To help prevent such occurrences, the inclusion of a cross-contamination model in exposure assessments would aid in the development and evaluation of interventions used to control the spread of pathogenic bacteria. A quantitative analysis was carried out to estimate the probability of contamination and the levels of Salmonella and Campylobacter spp. on salads as a result of cross-contamination from contaminated chicken carcasses via kitchen surfaces. Data on the prevalence and numbers of these bacteria on retail chicken carcasses and the use of unwashed surfaces to prepare foods were collected from scientific literature. The rates of bacterial transfer were collected from laboratory experiments and literature. A deterministic approach and Monte Carlo simulations that incorporated input parameter distributions were used to estimate the contamination of the product. The results have shown that the probability of Campylobacter spp. contamination on salads is higher than that of Salmonella spp., since both the prevalence and levels of Campylobacter spp. on chicken carcasses are higher than those of Salmonella spp. It is realistic to expect that a fraction of the human exposure to Campylobacter spp., in particular, originates from cross-contamination in private kitchens during food handling. The number of human campylobacteriosis cases could be reduced either by reducing the degree of Campylobacter spp. contamination on chicken carcasses or by improving the hygiene in private kitchens. To eliminate the cross-contamination route, it is important to use separate surfaces or to properly wash the surfaces during the preparation of raw and cooked foods or ready-to-eat foods.

Complex microbiota of a Chinese “Fen” liquor fermentation starter (Fen-Daqu), revealed by culture-dependent and culture-independent methods

Daqu is a traditional fermentation starter that is used for Chinese liquor production. Although partly mechanized, its manufacturing process has remained traditional. We investigated the microbial diversity of Fen-Daqu, a starter for light-flavour liquor, using combined culture-dependent and culture-independent approaches (PCR-DGGE). A total of 190 microbial strains, comprising 109 bacteria and 81 yeasts and moulds, were isolated and identified on the basis of the sequences of their 16S rDNA (bacteria) and 26S rDNA and ITS regions (fungi). DGGE of DNA extracted from Daqu was used to complement the culture-dependent method in order to include non-culturable microbes. Both approaches revealed that Bacillus licheniformis was an abundant bacterial species, and Saccharomycopsis fibuligera, Wickerhamomyces anomalus, and Pichia kudriavzevii were the most common yeasts encountered in Fen-Daqu. Six genera of moulds (Absidia, Aspergillus, Mucor, Rhizopus, Rhizomucor and Penicillium) were found. The potential function of these microorganisms in starters for alcoholic fermentation is discussed. In general the culture-based findings overlapped with those obtained by DGGE by a large extent. However, Weissella cibaria, Weissella confusa, Staphylococcus saprophyticus, Enterobacter aerogenes, Lactobacillus sanfranciscensis, Lactobacillus lactis, and Bacillus megaterium were only revealed by DGGE.

Quantifying recontamination through factory environments - a review

Recontamination of food products can be the origin of foodborne illnesses and should therefore be included in quantitative microbial risk assessment (MRA) studies. In order to do this, recontamination should be quantified using predictive models. This paper gives an overview of the relevant modelling approaches that are available in the literature to quantify recontamination via factory environment. Different recontamination routes are described: recontamination via air, via processing equipment or via hand contact. Unfortunately, not many available models are directly applicable to the food industry; most models are developed for aquatic or environmental systems. Finally, a general systematic approach is proposed for modelling contamination from surfaces via air, hands or liquid into the product and ranges for the parameters are given.

Theobroma cacao L., “The Food of the Gods”: Quality Determinants of Commercial Cocoa Beans, with Particular Reference to the Impact of Fermentation

The quality of commercial cocoa beans, the principal raw material for chocolate production, relies on the combination of factors that include the type of planting material, the agricultural practices, and the post-harvest processing. Among these, the fermentation of the cocoa beans is still the most relevant since it is the process whereby the precursors of the cocoa flavor arise. The formation of these precursors depends on the activity of different microbial groups on the beans pulp. A comparison of fermentations in different countries showed that a well-defined microbial succession does not always take place and that the role of Bacillus spp. in this process remains unclear. Considering the overriding importance of the fermentation to achieve high quality commercial cocoa beans, we discuss the need of addressing the impact of the farming system, the ripeness state of the pods, and the role of microbial interactions on the fermentation in future research. In addition, the problem of high acidification cocoa beans, aspects dealing with the volatile fraction of the flavor, and the cocoa butter properties, all were identified as critical aspects that need further investigation. The standardization of the microbiological methods and the application of metagenomic approaches would magnify the knowledge in this domain.

Phenotypic and Transcriptomic Analyses of Mildly and Severely Salt-Stressed Bacillus cereus ATCC 14579 Cells

ABSTRACT Bacteria are able to cope with the challenges of a sudden increase in salinity by activating adaptation mechanisms. In this study, exponentially growing cells of the pathogen Bacillus cereus ATCC 14579 were exposed to both mild (2.5% [wt/vol] NaCl) and severe (5% [wt/vol] NaCl) salt stress conditions. B. cereus continued to grow at a slightly reduced growth rate when it was shifted to mild salt stress conditions. Exposure to severe salt stress resulted in a lag period, and after 60 min growth had resumed, with cells displaying a filamentous morphology. Whole-genome expression analyses of cells exposed to 2.5% salt stress revealed that the expression of these cells overlapped with the expression of cells exposed to 5% salt stress, suggesting that the corresponding genes were involved in a general salt stress response. Upregulation of osmoprotectant, Na+/H+, and di- and tripeptide transporters and activation of an oxidative stress response were noticeable aspects of the general salt stress transcriptome response. Activation of this response may confer cross-protection against other stresses, and indeed, increased resistance to heat and hydrogen peroxide could be demonstrated after preexposure to salt. A temporal shift between the transcriptome response and several phenotypic responses of severely salt-stressed cells was observed. After resumption of growth, these cells showed cellular filamentation, reduced chemotaxis, increased catalase activity, and optimal oxidative stress resistance, which corresponded to the transcriptome response displayed in the initial lag period. The linkage of transcriptomes and phenotypic characteristics can contribute to a better understanding of cellular stress adaptation strategies and possible cross-protection mechanisms.

Quantification of the Effects of Salt Stress and Physiological State on Thermotolerance of Bacillus cereus ATCC 10987 and ATCC 14579

ABSTRACT The food-borne pathogen Bacillus cereus can acquire enhanced thermal resistance through multiple mechanisms. Two Bacillus cereus strains, ATCC 10987 and ATCC 14579, were used to quantify the effects of salt stress and physiological state on thermotolerance. Cultures were exposed to increasing concentrations of sodium chloride for 30 min, after which their thermotolerance was assessed at 50°C. Linear and nonlinear microbial survival models, which cover a wide range of known inactivation curvatures for vegetative cells, were fitted to the inactivation data and evaluated. Based on statistical indices and model characteristics, biphasic models with a shoulder were selected and used for quantification. Each model parameter reflected a survival characteristic, and both models were flexible, allowing a reduction of parameters when certain phenomena were not present. Both strains showed enhanced thermotolerance after preexposure to (non)lethal salt stress conditions in the exponential phase. The maximum adaptive stress response due to salt preexposure demonstrated for exponential-phase cells was comparable to the effect of physiological state on thermotolerance in both strains. However, the adaptive salt stress response was less pronounced for transition- and stationary-phase cells. The distinct tailing of strain ATCC 10987 was attributed to the presence of a subpopulation of spores. The existence of a stable heat-resistant subpopulation of vegetative cells could not be demonstrated for either of the strains. Quantification of the adaptive stress response might be instrumental in understanding adaptation mechanisms and will allow the food industry to develop more accurate and reliable stress-integrated predictive modeling to optimize minimal processing conditions.