An identification procedure for foodborne microbial hazards.

A stepwise and interactive identification procedure for foodborne microbial hazards has been developed in which use is made of several levels of detail ranging from rough hazard identification to comprehensive hazard identification. This approach allows one to tackle the most obvious hazards first, before focusing on less obvious hazards. The interactive character of the identification procedure is based on the use of several knowledge sources. Combination of knowledge sources, expressed in the use of knowledge rules, supports the user in systematically selecting hazards which may pose a real risk to the consumer. Due to the structured method and the clear definitions of the knowledge rules, the procedure is transparent and may be changed if necessary. The hazard identification procedure has been implemented as a computer program, resulting in a decision-supporting identification system. It provides a way to efficiently assess those hazards which may cause harm if not brought under control during processing. The procedure forms a basis for quantitative risk assessments.

Application of predictive microbiology to estimate the number of Bacillus cereus in pasteurised milk at the point of consumption.

A procedure is presented to quantitatively estimate the growth of a particular organism in a food product during chilled storage using predictive microbiology. This results in a quantification of the contribution of every individual process step to the total number of organisms, which may be a useful tool to support decisions on existing process lines as well as in process and product design. It is demonstrated that predictive microbiology will only estimate to within orders of magnitude of bacterial growth. This helps to pinpoint the most important aspects of a line. The calculations can be helpful to set critical limits and to detect hazards by performing 'what if' analyses. The procedure is explained for the growth of Bacillus cereus in milk. It is indicated, that with the current information, the effect of time/temperature can be estimated. However, to make an accurate exposure analysis, more information will be needed.

Modelling Bacterial Growth of Lactobacillus curvatus as a Function of Acidity and Temperature.

Models that describe the effect of acidity, temperature, and the combined effect of these variables on the growth parameters of Lactobacillus curvatus are developed and validated. Growth parameters (lag time, specific growth rate, and maximum population density) were calculated from growth data at different temperature-acidity combinations. Experiments were set up to assess the quantitative effects of temperature and acidity on the growth parameters rather than for parameter estimation solely. The effect of acidity is monitored at several constant temperature values. Models are set up and fitted to the data. The same procedure is used at constant acidity values to model the effect of temperature. For lag time, specific growth rate, and maximum population density, the effect of temperature could be multiplied with the effect of acidity to obtain combinatory models that describe the effect of both controlling factors on the growth parameters. Lag time measurements showed large deviations, and therefore the lag time models developed can only be used to estimate the order of magnitude of lag time.

Modelling growth rates of Listeria innocua as a function of lactate concentration.

The effect of sodium lactate (NaL) concentration on growth of Listeria innocua in a yeast-extract/peptone broth at pH 5.5, 6.0, 6.5 and 7.0 at 4, 10, 20 and 30 degrees C was modelled with the modified Gompertz model. NaCl was used as a reference to distinguish between the water activity effect and the specific inhibitory effect of NaL. Minimum inhibitory concentrations (MIC) of NaCl appeared to be significantly higher than MIC values of NaL, indicating that NaL had a specific inhibitory effect on growth of L. innocua. The MIC values of NaL and NaCl were not much influenced by the temperature. The pH of the growth medium was shown to have influence on the MIC values of NaL but not on the MIC values of NaCl. Total growth inhibition of L. innocua at low pH (5.5) took place at lower NaL concentrations (217 mM) than at neutral pH (1071-1339 mM), indicating that the undissociated lactic acid plays a role in the mechanism of inhibition. However, MIC values for undissociated acid increased with decreasing pH from 0.8 mM at pH 7 to 5 mM at pH 5.5. It is therefore likely that besides acidification of the cytoplasm due to diffusion of undissociated acid into the cell, other mechanisms are involved. Growth rates at NaL concentrations between 0 and the MIC value decreased progressively with increasing concentrations down to 0 at the MIC value, and were strongly influenced by both temperature and pH. Growth rates in the presence of NaCl were influenced by the temperature only. It was shown that a modified Monod equation with three parameters was effective for description of growth rates of L. innocua at NaL and NaCl concentrations over the whole experimental range.

Evaluation of data transformations and validation of a model for the effect of temperature on bacterial growth.

The temperature of chilled foods is an important variable for controlling microbial growth in a production and distribution chain. Therefore, it is essential to model growth as a function of temperature in order to predict the number of organisms as a function of temperature and time. This article deals with the correct variance-stabilizing transformation of the growth parameters A (asymptotic level), mu (specific growth rate), and lambda (lag time). This is of importance for the regression analysis of the data. A previously gathered data set and model for the effect of temperature on the growth of Lactobacillus plantarum (M. H. Zwietering, J. T. de Koos, B. E. Hasenack, J. C. de Wit, and K. van 't Riet, Appl. Environ. Microbiol. 57:1094-1101, 1991) is extended with new data. With the total data set (original and new data), a variance-stabilizing transformation is selected in order to determine which transformation should precede fitting. No transformation for the asymptote data, a square root for the growth rate, and a logarithmic transformation for the lag time were found to be appropriate. After these transformations, no significant correlation was found between the variance and the magnitude of the variable. Model corrections were made and model parameters were estimated by using the original data. With the new data, the models were validated by comparing the lack of fit of the models with the measurement error, using an F test. The predictions of the models for mu and lambda were adequate. The model for A showed a systematic deviation, and therefore a new model for A is proposed.

Modeling of bacterial growth with shifts in temperature.

The temperature of chilled foods is an important variable for the shelf life of a product in a production and distribution chain. To predict the number of organisms as a function of temperature and time, it is essential to model the growth as a function of temperature. The temperature is often not constant in various stages of distribution. The objective of this research was to determine the effect of shifts in temperature. The suitability and usefulness of several models to describe the growth of Lactobacillus plantarum with fluctuating temperatures was evaluated. It can be assumed that temperature shifts within the lag phase can be handled by adding relative parts of the lag time to be completed and that temperature shifts within the exponential phase result in no lag phase. With these assumptions, the kinetic behavior of temperature shift experiments was reasonably well predicted, and this hypothesis was accepted statistically in 73% of the cases. Only shifts of temperature around the minimum temperature for growth showed very large deviations from the model prediction. The best results were obtained with the assumption that a temperature shift (within the lag phase as well as within the exponential phase) results in an additional lag phase. This hypothesis was accepted statistically in 93% of the cases. The length of the additional lag phase is one-fourth of the lag time normally found at the temperature after the shift.

Minimum inhibitory concentration (MIC) of sodium lactate for pathogens and spoilage organisms occurring in meat products.

Pathogens and spoilage organisms occurring in meat products were screened in laboratory media, according to a method of Eklund (1983), in order to determine the specific inhibitory effect of lactate on growth of these microorganisms under optimum growth conditions (pH 6.5, 20 degrees C). In general, Gram-positive bacteria were more sensitive towards lactate than Gram-negative bacteria. It was shown especially, that strains that were able to grow at water activities of 0.95 and below in the presence of NaCl (Staphylococcus aureus, Listeria monocytogenes, Brochothrix thermosphacta) were inhibited by sodium lactate (NaL). It appeared, that yeasts were resistant to large amounts of NaL (> 10% w/v). However, NaL had a specific inhibitory effect on growth of these organisms when compared to the effect of NaCl. These results indicate that lactate addition to food products with a pH near neutrality offers good prospects for shelf life prolongation.

Inhibition of the growth of yeasts in fermented salads.

Salads composed of vegetables and/or meat in an oil-in-water emulsion were prepared by fermentation for 7 h at 42 degrees C or 45 degrees C with strains of Lactobacillus spp. Their stability towards spoilage yeasts was studied using Saccharomyces cerevisiae, Saccharomyces exiguus and Torulaspora delbrueckii, isolated from salads, as well as Pichia membranaefaciens and Zygosaccharomyces bailii. Salads fermented with good lactic starters usually had pH values of < or = 4.2 and lactic acid concentrations of 0.28 to 0.43% (w/w). High numbers of spoilage yeasts (and production of large volumes of CO2) were not attained in these salads, provided the initial concentration of spoilage yeasts was sufficiently low (< or = 100 CFU/g). Inhibition of spoilage yeasts in lactic fermented salads is probably due to lactic acid, the low storage temperature and the low residual oxygen concentration.

Faecal micro-organisms on the hands of carriers: Escherichia coli as model for Salmonella.

To evaluate the public health hazard caused by Salmonella carriers as food handlers, a study was carried out to gather more quantitative data about faecal contamination of hands after stools. Faecal E. coli was used as a model bacterium for Salmonella. In total 92 subjects cooperated in this study. The hand was sampled before toilet use, and also after stools, with or without washing of the hands. Besides E. coli the number of Enterobacteriaceae was also determined. It appeared that hands may be contaminated with Enterobacteriaceae, regardless of toilet use, for a well before as after stools about 60% of the sampled hands carried a detectable number of Enterobacteriaceae. That is why the presence of Enterobacteriaceae on hands is not a good indicator for toilet hygiene. In 4% of the samples before stools and in 25% of the samples taken after stools E. coli could be detected (> 20 CFU/sample). The average 10log CFU of E. coli in the positive samples taken before and after stools were about the same: 2.30 per sample. Hand washing after stools reduced the numbers of Enterobacteriaceae and E. coli on the hands. It was concluded that symptomless Salmonella excretors in the period starting two weeks after infection form only a low risk in carrying over Salmonella by their hands to food. Especially if normal hygiene is practiced like washing hands after stools, the number of contaminated hands will be very low and furthermore the number of faecal micro-organisms will be very small.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose and sucrose fermenting capacity of homofermentative lactic acid bacteria used as starters in fermented salads.

Salads, i.e., vegetables and/or meat in an oil-in-water emulsion with a low pH due to the addition of acids, are popular in western countries. The shelf life of these salads is influenced mainly by storage temperature and the use of preservatives, i.e., sorbic and benzoic acid. Lactic acid fermentation could be an interesting novel approach to salad preparation. As part of an investigation on fermented salads, lactic acid bacteria from the genera Lactobacillus, Pediococcus and Streptococcus were screened for their capacity to ferment glucose and sucrose in a model system and in a standard salad. The temperature optimum of these strains was in the range of 34-48 degrees C. Most of these strains produced about equal amounts of L(+)- and D(-)-lactic acid. To determine the specific sugar fermenting capacity (mmol lactic acid produced per min per g dry weight) of non-growing cells of lactic acid bacteria, a semi-automated model system was used, based on monitoring the pH decrease in a phosphate buffer during conversion of sugars to lactic acid. The strains tested showed acid production rates on glucose varying between 0.03 and 0.39 mmol of acid produced per min per g dry weight (average 0.23). These glucose-grown cells showed varying, but lower fermentation rates on sucrose (0-0.25 mmol/min/g d.w., average 0.04) than sucrose-grown cells (0-0.33 mmol/min/g d.w., average 0.19). Results obtained with the model system were in good agreement with those of fermentation tests, carried out with a standard salad. Inhibition of spoilage bacteria in the standard salad could be achieved by using starter cultures with high fermenting capacity.

Somatic embryogenesis and regeneration of flowering plants in rose.

Somatic embryos of the cut rose cultivars 'Domingo' and 'Vickey Brown' were obtained from callus derived from leaf explants on half strength Murashige and Skoog medium with low concentrations of kinetin and 1-naphthyl acetic acid or 2-naphthyloxyacetic acid. Somatic embryos were first observed after 6 to 12 weeks of culture on callus formed at the basis or midrib of the leaf. Embryos could be grown to phenotypically true to type greenhouse plants.

Some aspects of bacterial contamination of hands of workers in food service establishments.

In 28 kitchens of restaurants and 10 kitchens of institutions, the hands of 280 persons were sampled in order to determine the role which hands play in contaminating food during preparation. The presence of salmonellae and the number of Enterobacteriaceae and Staphylococcus aureus in these samples was examined. No salmonellae could be isolated. However, large numbers (greater than 10(5)/hand) of Enterobacteriaceae and Staphylococcus aureus occurred on about 8% of the hands. "Normal" washing of the hands resulted in a lower number of transient micro-organisms. Since washing did not influence the number of Staphylococcus aureus on the hands, this organism seems to behave more as a resident organism. Prepared foods such as salads, fried meat and bread were often handled with relatively highly contaminated hands.

Microbiological aspects of washing hands in slaughter-houses.

The hand hygiene of workers in a number of chicken-, pig-, calf- and cattle slaughter-houses was investigated. The number of E. coli and salmonellae on hands was determined before and after washing hands in order to measure the washing effect on the number of these faecal bacteria. All workers examined carried E. coli on their hands during work. The average logarithmic E. coli count on hands before washing was about 5.0 in chicken-and calf-slaughter-houses, about 3.5 in pig-and 3.0 in cattle-slaughter-houses. Hand washing decreased the E. coli count per hand by about 1.5 log cycle. Salmonellae were isolated from the hands in chicken- (59 pos./145 samples), pig-(42 pos./116 samples) and calf-slaughter-houses (11 pos./68 samples). Even after washing salmonellae were found to be present. Generally 'normal' washing causes a considerable reduction in the number of transient bacteria on hands. The presence of E. coli and Salmonella after washing, however, indicates that the effectiveness of the procedure needs to be improved.

Some aspects of microbial contamination of hands of workers in food industries.

With regard to food hygiene, the question is posed whether carriers of pathogenic organisms, like Salmonella can contaminate a product by their hands. This might especially be possible in case of bad toilet hygiene. Experiments were carried out in 13 food and 3 non-food establishments. An inquiry was made into habits of toilet use and hand washing. The following microbiological examinations were performed on workers' hands: total colony-forming-units per hand, and the presence of Salmonella, E. coli, Enterobact., Staph. aureus, faecal Streptococci and Cl. perfringens. Approximately 42% of workers in the food industry and approximately 64% of workers in other industries seems to use the factory toilet regularly for defaecation. Because of the presence of contaminated raw materials in food industries, it is impossible to get direct proof that faecal contamination of the hands is due to toilet use. However, there is indirect evidence that contamination of workers' hands by raw materials, especially of animal origin, is of much more importance than the consequences of toilet use. This is shown by the occurrence of Salmonella on the hands (5-36% pos.) in only those factories where raw materials of animal origin are handled, by the low incidence of E. coli on the hands in food industries where "clean" materials (vegetables, biscuits, chocolate) are handled, and by the total absence of E. coli on the hands of workers in two non-food industries.

Epidemiological studies on Salmonella in a certain area ("Walcheren Project"). IV. The incidence of Salmonella in the sewage system, in the faeces of man and pets as well as in shops, kitchens and lavatories in the village of Aagtekerke.

As part of the epidemiological investigations on Salmonella ion the former island of Walcheren, the contamination of sewage water in the village of Aagtekerke was studied over a prolonged period of time. These studies showed that this sewage water was frequently contaminated by large numbers of Salmonella. In the present study efforts were made to find the source of this contamination and for this purpose the incidence of Salmonella in the sewage system, in the faeces of man and pets as well as in shops, kitchens and lavatories in the village of Aagtekerke was studied for a period of three weeks in June 1977. Salmonella was only isolated from a few samples of faecal material of human and animal origin, and from the sewage system (including the inlet water and effluents of the sewage works). Serotyping showed that the strains isolated from human faeces were similar to those found to be present in the sewage system. This fact taken in conjunction with the results of the bacterial counts, suggests that the sewage system was only contaminated by a small number of carriers. The reduction of contamination observed in the sewage system during the period of investigation could be evidence that Salmonella organisms cannot survive by themselves in an environment of this type, at least not at the temperatures recorded when collecting samples from the sewage water. Growth of organisms under more favourable conditions cannot be ruled out. A questionnaire about the dietary habits and kitchen hygiene of the local population showed that contamination within households, originating with the food, is a real possibility.

Cross-contamination during the preparation of frozen chickens in the kitchen.

A study was made of the extent to which frozen broilers, contaminated with indicator organisms, can cause cross-contamination in the kitchen. In 60 kitchens a number of relevant objects were sampled during the preparation of contaminated frozen broilers. The results show that cross-contamination occurred in a high proportion of the kitchens examined. In many instances the indicator organism was still present on various objects even after rinsing, 'clearing' or washing up. In view of the possible risk of a cross-contamination with Salmonella spp. the importance of instructing food preparers is emphasized. No salmonellas could be found in the sinks of the 60 kitchens examined.

[Epidemiological studies on Salmonella in a particular area ('Walcheren Project'). IV. The incidence of Salmonella in the sewage system, in faeces of man and pets as well as in shops, kitchens and lavatories in the village of Aagtekerke (author's transl)]. / Epidemiologisch Salmonella-onderzoek in een bepaald gebied ('Project Walcheren'). IV. Het voorkomen van Salmonella in het rioolstelsel, in faeces van mensen en huisdieren, in winkels en in keukens en toiletten in het dorp Aagtekerke.

As part of the epidemiological investigations on Salmonella on the island of Walcheren, the contamination of sewage water in the village of Aagtekerke was studied over a prolonged period. These studies showed that this sewage water was frequently contaminated by large numbers of Salmonella. In the present study, efforts were made to find an answer to the question of the origins of this contamination. For this purpose, the incidence of Salmonella in the sewage system, in faeces of man and pets as well as in shops, kitchens and lavatories in the village of Aagtekerke was studied for a period of three weeks in June 1977. In addition to the findings in the sewage system (including the inlet water and effluents of the sewage works) which were positive for Salmonella, this organism was only isolated from a few faecal samples of human and animal origin. Serotyping showed that the strains isolated from human faeces were similar to those found to be present in the sewage system. This fact, in conjunction with the results of the bacterial counts. suggests that the sewage system was only contaminated by the small number of carriers. The reduction of contamination observed in the sewage system during the period of investigation could be evidence of the fact that Salmonella organisms cannot survive by themselves in an environment of this type, at least not at the temperatures recorded on collecting samples from the sewage water. Growth of organisms under more favourable conditions cannot be ruled out. An inquiry on dietary habits and kitchen hygiene of the local population showed that contamination within households. Origination with the food, is a real possibility.