Expert study to select indicators of the occurrence of emerging mycotoxin hazards.

This article describes a Delphi-based expert judgment study aimed at the selection of indicators to identify the occurrence of emerging mycotoxin hazards related to Fusarium spp. in wheat supply chains. A panel of 29 experts from 12 European countries followed a holistic approach to evaluate the most important indicators for different chain stages (growth, transport and storage, and processing) and their relative importance. After three e-mailing rounds, the experts reached consensus on the most important indicators for each of the three stages: wheat growth, transport and storage, and processing. For wheat growth, these indicators include: relative humidity/rainfall, crop rotation, temperature, tillage practice, water activity of the kernels, and crop variety/cultivar. For the transport and storage stage, they include water activity in the kernels, relative humidity, ventilation, temperature, storage capacity, and logistics. For wheat processing, indicators include quality data, fraction of the cereal used, water activity in the kernels, quality management and traceability systems, and carryover of contamination. The indicators selected in this study can be used in an identification system for the occurrence of emerging mycotoxin hazards in wheat supply chains. Such a system can be used by risk managers within governmental (related) organizations and/or the food and feed industry in order to react proactively to the occurrence of these emerging mycotoxins.

Inactivation rates of Cronobacter spp. and selected other bacterial strains in powdered infant formulae stored at different temperatures.

The aim of this study was to determine the survival of two strains of Cronobacter (Enterobacter sakazakii) and six other bacterial strains inoculated into dry powdered infant formula (PIF) stored for 22 weeks at several temperatures between 7 and 42 degrees C. The experimental setup involved a relatively high initial concentration of bacteria, around 10(4) CFU/g of powder, and enumeration of survivors with a minimum detection level of 100 CFU/g. For all strains tested, it was found that the number of bacterial cells decreased faster with increasing temperature. Cronobacter spp. cells generally survived better at high temperatures (37 and 42 degrees C) than the other bacteria, while such a difference in survival was not apparent at other temperatures. To describe the effect of temperature on survival, both the Weibull distribution model and the log-linear model were tested. At 22 degrees C, decline rates of 0.011 and 0.008 log units per day were found for Cronobacter sakazakii ATCC 29544 and Cronobacter strain MC10, respectively. Assuming a linear relationship between log-transformed D-values and temperature, z-values estimated for C. sakazakii ATCC 29544 and Cronobacter MC10 were 13.3 and 23.5 degrees C, respectively. Such differences found in resistance among Cronobacter spp. would be relevant to consider when establishing quantitative risk assessments on consumer risks related to PIF.

Growth of Cronobacter spp. under dynamic temperature conditions occurring during cooling of reconstituted powdered infant formula.

Reconstituted infant formulae are excellent growth media for Cronobacter spp. (formerly Enterobacter sakazakii) and other microorganisms that may be present in such products. Immediate consumption or rapid cooling and storage at a low temperature are therefore recommended as control measures to prevent microbial growth. Placing a container filled with reconstituted liquid formula in the refrigerator, however, does not mean that the temperature of the liquid is directly the same as the set-point of the refrigerator. This study describes the temperature profiles and methods to predict lag time and possible growth of Cronobacter spp. during the cooling process in three types of containers. The overall heat transfer coefficients (alpha) were determined and were shown to have a very large variability in both household refrigerators and an air-ventilated refrigerator equipped with a fan. A mathematical model was built to predict the growth of Cronobacter spp. under dynamic temperature conditions using three models for the lag time. The various estimations for the lag time had a remarkably strong impact on the predicted growth. The assumption of a constant k-value (k = lag time x specific growth rate = lambda x micro = 2.88) fitted the experimental data best. Predictions taking into account the large variability in heat transfer showed that proliferation of Cronobacter spp. during cooling may be prevented by limiting the volume to be cooled to portion size only, or by reconstituting at temperatures of 25 degrees C or lower. The model may also be used to predict growth in other situations where dynamic temperature conditions exist.

Effects of preculturing conditions on lag time and specific growth rate of Enterobacter sakazakii in reconstituted powdered infant formula.

Enterobacter sakazakii can be present, although in low levels, in dry powdered infant formulae, and it has been linked to cases of meningitis in neonates, especially those born prematurely. In order to prevent illness, product contamination at manufacture and during preparation, as well as growth after reconstitution, must be minimized by appropriate control measures. In this publication, several determinants of the growth of E. sakazakii in reconstituted infant formula are reported. The following key growth parameters were determined: lag time, specific growth rate, and maximum population density. Cells were harvested at different phases of growth and spiked into powdered infant formula. After reconstitution in sterile water, E. sakazakii was able to grow at temperatures between 8 and 47 degrees C. The estimated optimal growth temperature was 39.4 degrees C, whereas the optimal specific growth rate was 2.31 h(-1). The effect of temperature on the specific growth rate was described with two secondary growth models. The resulting minimum and maximum temperatures estimated with the secondary Rosso equation were 3.6 degrees C and 47.6 degrees C, respectively. The estimated lag time varied from 83.3 +/- 18.7 h at 10 degrees C to 1.73 +/- 0.43 h at 37 degrees C and could be described with the hyperbolic model and reciprocal square root relation. Cells harvested at different phases of growth did not exhibit significant differences in either specific growth rate or lag time. Strains did not have different lag times, and lag times were short given that the cells had spent several (3 to 10) days in dry powdered infant formula. The growth rates and lag times at various temperatures obtained in this study may help in calculations of the period for which reconstituted infant formula can be stored at a specific temperature without detrimental impact on health.

A simple and rapid cultural method for detection of Enterobacter sakazakii in environmental samples.

A method was developed to detect and identify Enterobacter sakazakii in environmental samples. The method is based on selective enrichment at 45+/-0.5 degrees C in lauryl sulfate tryptose broth supplemented with 0.5 M NaCl and 10 mg/liter vancomycin (mLST) for 22 to 24 h followed by streaking on tryptone soy agar with bile salts. When exposed to light during incubation at 37 degrees C, E. sakazakii produces yellow colonies within 24 h; identification was confirmed by testing for alpha-glucosidase activity and by using API 20E strips. All of the E. sakazakii strains tested (n = 99) were able to grow in mLST at 45+/-0.5 degrees C, whereas 35 of 39 strains of potential competitors, all belonging to the Enterobacteriaceae, were suppressed. A survey was carried out with 192 environmental samples from four different milk powder factories. Using this new protocol, E. sakazakii was isolated from almost 40% of the samples, whereas the reference procedure (enrichment in buffered peptone water, isolation on violet red bile glucose agar, and biochemical identification of randomly chosen colonies) only yielded 26% positive results. This selective method can be very useful for the rapid and reliable detection of E. sakazakii in environmental samples.

A new protocol for the detection of Enterobacter sakazakii applied to environmental samples.

Enterobacter sakazakii is a motile, peritrichous, gram-negative rod that was previously known as a yellow pigmented Enterobacter cloacae. It is documented as a rare cause of outbreaks and sporadic cases of life-threatening neonatal meningitis, necrotizing enterocolitis, and sepsis. E. sakazakii has been isolated from milk powder-based formulas, and there is thus a need to investigate whether and where E. sakazakii occurs in these manufacturing environments. For this purpose, a simple detection method was developed based on two features of E. sakazakii: its yellow pigmented colonies when grown on tryptone soy agar and its constitutive alpha-glucosidase, which is detected in a 4-h colorimetric assay. Using this screening method, E. sakazakii strains were isolated from three individual factories from 18 of 152 environmental samples, such as scrapings from dust, vacuum cleaner bags, and spilled product near equipment. The method is useful for routine screening of environmental samples for the presence of E. sakazakii.