Quantification of the emetic toxin cereulide in food products by liquid chromatography-mass spectrometry using synthetic cereulide as a standard.

Bacillus cereus produces the emetic toxin cereulide, a cyclic dodecadepsipeptide that can act as a K(+) ionophore, dissipating the transmembrane potential in mitochondria of eukaryotic cells. Because pure cereulide has not been commercially available, cereulide content in food samples has been expressed in valinomycin equivalents, a highly similar cyclic potassium ionophore that is commercially available. This research tested the biological activity of synthetic cereulide and validated its use as a standard in the quantification of cereulide contents in food samples. The synthesis route consists of 10 steps that result in a high yield of synthetic cereulide that showed biological activity in the HEp-2 cell assay and the boar sperm motility assay. The activity is different in both methods, which may be attributed to differences in K(+) content of the test media used. Using cereulide or valinomycin as a standard to quantify cereulide based on liquid chromatography-mass spectrometry (LC-MS), the concentration determined with cereulide as a standard was on average 89.9% of the concentration determined using valinomycin as a standard. The recovery experiments using cereulide-spiked food products and acetonitrile as extraction solute showed that the LC-MS method with cereulide as a standard is a reliable and accurate method to quantify cereulide in food, because the recovery rate was close to 100% over a wide concentration range.

Characterization and Exposure Assessment of Emetic Bacillus cereus and Cereulide Production in Food Products on the Dutch Market.

The emetic toxin cereulide, which can be produced by Bacillus cereus, can be the cause of food poisoning upon ingestion by the consumer. The toxin causes vomiting and is mainly produced in farinaceous food products. This article includes the prevalence of B. cereus and of cereulide in food products in The Netherlands, a characterization of B. cereus isolates obtained, cereulide production conditions, and a comparison of consumer exposure estimates with those of a previous exposure assessment. Food samples (n = 1,489) were tested for the presence of B. cereus; 5.4% of the samples contained detectable levels (>10(2) CFU/g), and 0.7% contained levels above 10(5) CFU/g. Samples (n = 3,008) also were tested for the presence of cereulide. Two samples (0.067%) contained detectable levels of cereulide at 3.2 and 5.4 µg/kg of food product. Of the 481 tested isolates, 81 produced cereulide and/or contained the ces gene. None of the starch-positive and hbl-containing isolates possessed the ces gene, whereas all strains contained the nhe genes. Culture of emetic B. cereus under nonoptimal conditions revealed a delay in onset of cereulide production compared with culture under optimal conditions, and cereulide was produced in all cases when B. cereus cells had been in the stationary phase for some time. The prevalence of cereulide-contaminated food approached the prevalence of contaminated products estimated in an exposure assessment. The main food safety focus associated with this pathogen should be to prevent germination and growth of any B. cereus present in food products and thus prevent cereulide production in foods.

Use of Reference Materials (Spray-Dried Milk Artificially Contaminated with Salmonella typhimurium ) to Validate Detection Methods for Salmonella.

Reference materials consisting of 0.28 g of spray-dried milk artificially contaminated with Salmonella typhimurium and contained in gelatin capsules (mean contamination level 4.2 salmonellae per capsule) were used to validate detection of Salmonella species in food. For this, 10 samples of 25 g of mayonnaise, milk powder, minced meat, or poultry meat were spiked with reference materials without repair and with reference materials after repair, respectively. It was found that the physiological condition of Salmonella present in the reference material had no influence on the recovery rate of the organism. The recovery rates of Salmonella were tested by applying various methods [ISO-6579 method, SALMONELLA-TEK™ (Organon Teknika), the modified semisolid Rappaport Vassiliadis agar (MSRV), and the Salmonella rapid test (Oxoid)]. The recovery rates found (averaged on the types of foods tested) were 67% for the Salmonella rapid test, 83% for the MSRV, 86% for the ISO-6579, and 91% for the SALMONELLA-TEK. The use of reference materials is recommended to validate detection of Salmonella species.

Influence of Reconstitution on Isolation and Enumeration of Listeria monocytogenes from Milk Powder Used for Reference Samples.

To test the performance of the Listeria isolation methods, reference samples consisting of gelatin capsules filled with spray-dried milk powder containing Listeria have been developed. During the spray-drying process the Listeria cells are exposed to heat stress and are susceptible to osmotic stress during the reconstitution procedure. To limit the effect of osmotic shock, the milk powder has to be encapsulated in gelatin in order to guarantee slow dissolution. Furthermore, the capsules have to be preen-riched in a nonselective medium. The practical consequences of these findings are discussed.