Staphtox predictor - A dynamic mathematical model to predict formation of Staphylococcus enterotoxin during heating and fermentation of meat products.

Existing growth models for S. aureus predict growth in relation to temperature, aw/NaCl and pH, and the assessment of probable Staphylococcus enterotoxin (SE) formation is based solely on the number of S. aureus. However, during the production of meat products such as fermented sausages and semi-processed hams, growth of S. aureus is a critical control point in HACCP plans. There is a need to develop a model that evaluates the safety of the product regarding SE formation in relation to the product composition, changes in pH or temperature during the processing and the number of S. aureus in the final product. The objective of the present work is to develop a mathematical model that predicts both the increase in the number of S. aureus and whether SE formation is possible in different meat product processes. A total of 78 experiments were carried out in a meat model system. The experiments covered a range of different temperatures (10-40 °C), pH (4.6-6.0), water phase salt (WPS) (2.2-5.6%) and Sodium nitrite concentrations (0-150 ppm). The meat model system was inoculated with approximately 103 CFU/g of a multi-strain cocktail and incubated at the different temperatures. The cocktail consisted of three strains of S. aureus producing the Staphylococcus enterotoxins A to D (SEA to SED) and a methicillin-resistant strain producing SEG, SEI, SEM, SEN, SEO and SEU. Enumeration of S. aureus was performed several times during the incubation, SE was extracted from samples with >5 log CFU/g, and the SEA-E content was analysed by an ELISA method. Maximum growth rates and lag times calculated from microbiological data, together with temperature, pH, WPS and Sodium nitrite, were used to develop a SE and a growth model. The growth model was developed by training a neural network and the SE model based on logistic regression. The SE and growth models were validated on separate data sets (N = 200 SE model, N = 63 growth model) including both dynamic and static conditions. The SE model predicted all occurrences of toxin formation in the validation data sets. The growth model is a fail-safe model and the prediction errors are comparable to laboratory reproducibility. In conclusion, the models are applicable for predicting the increase in S. aureus and for evaluating if SE formation is likely during processing of meat products. The models are available to producers and other interested parties at www.dmripredict.dk.

Sulphation of lithocholic acid in the colon-carcinoma cell line CaCo-2.

High levels of bile acids in the colon may correlate with an increased risk of colon cancer, but the underlying mechanisms are not known. Proteoglycan structures have been shown to change when human colon cells differentiate in vitro. The expression of [(35)S]sulphated molecules was used as a phenotypic marker to study the effects of bile acids on the human-colon-carcinoma cell line CaCo-2. [(35)S]sulphated compounds were isolated from the medium of cell fractions of cells metabolically labelled with [(35)S]sulphate in the absence and presence of cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid (LA). Labelled molecules were analysed by gel chromatography, HPLC and SDS/PAGE in combination with chemical and enzymic methods. The expression of (35)S-labelled proteoglycans was not affected by any of the bile acids tested. However, the level of sulphated metabolites increased 7-18-fold in different experiments during a 22 h labelling period in the presence of an LA concentration of 10 microg/ml (26.6 nmol/ml) compared with controls. Further analyses showed that this was due, at least in part, to the sulphation of LA itself. This sulphation of LA was a rapid process followed by secretion back to the medium. Brefeldin A did not reduce the sulphation of LA, indicating that this conversion takes place in the cytosol, rather than in the Golgi apparatus of the CaCo-2 cells. LA in colon may be sulphated efficiently by the colonocytes to reduce the toxic effects of this particular bile acid. Sulphation may possibly be an important protective mechanism in the colon.

Differentiation-associated modulation of heparan sulfate structure and function in CaCo-2 colon carcinoma cells.

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O-sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.