Microcalorimetric Investigations of Reversible Staphylococcal Enterotoxin Unfolding.

Staphylococcal food poisoning (SFP) is a common food-borne illness often associated with contamination during food handling. The genes for Staphylococcal enterotoxin (SE) isoforms SEA and SEB are frequently detected in human nasal Staphylococcus aureus isolates and these toxins are commonly associated with SFP. Past studies described the resistance of preformed SE proteins to heat inactivation and their reactivation upon cooling in foods. Full thermodynamic analyses for these processes have not been reported, however. The thermal stabilities of SEA, SEB, and SEH and reversibility of unfolding in simple buffers were investigated at pH 4.5 and pH 6.8 using differential scanning calorimetry (DSC). SEA and SEB unfolding was irreversible at pH 6.8 and at least partially reversible at pH 4.5 while SEH unfolding was irreversible at pH 4.5 and reversible at pH 6.8. Additional studies showed maximum refolding for SEB at pH 3.5-4.0 and diminished refolding at pH 4.5 with increasing ionic strength. SE-stimulated secretion of interferon-gamma by human peripheral blood mononuclear cells was used to assess residual SE biological activity following heat treatments using conditions matching those used for DSC studies. The biological activities of SEB and SEH exhibited greater resistance to heat inactivation than that of SEA. The residual activities of heat-treated SEB and SEH were measurable but diminished further in the presence of reconstituted nonfat dry milk adjusted to pH 4.5 or pH 6.8. To different extents, the pH and ionic strengths typical for foods influenced the thermal stabilities of SEA, SEB, and SEH and their potentials to renature spontaneously after heat treatments.

Immunomagnetic Capture of Big Six Shiga Toxin-Producing Escherichia coli Strains in Apple Juice with Detection by Multiplex Real-Time PCR Eliminates Interference from the Food Matrix.

Having reliable methods for detecting Shiga toxin-producing Escherichia coli (STEC) in foods is an important food safety goal. The majority of STEC outbreaks have involved either the O157:H7 serotype or one of six non-O157 serogroups, O26, O45, O103, O111, O121, and O145, termed "The Big Six." We have compared detection by PCR of the Shiga toxin genes stx1a and stx2a from STEC bacteria isolated from unclarified apple juice by simple centrifugation with the use of an immunocapture technique to minimize contaminants (such as pectin and polyphenols that may copurify with DNA) that may interfere with DNA amplification efficiencies and limit sensitivity. An internal control for successful immunocapture, DNA extraction, and PCR amplification was generated by introducing the pmRaspberry plasmid into an stx null strain, yielding an E. coli O45 pmRaspberry derivative that can be added to food samples directly. Using serial dilutions of a representative Big Six STEC in apple juice, our immunocapture method resulted in a 50% probability of detection value of 3.34, 2.25, and 4.25 CFU for detection by multiplex real-time PCR, growth on solid agar, and multiplex endpoint PCR, respectively. The time to result was 6.5 h, 9.5 h, and 1.5 days for immunocapture of Big Six STECs and detection by multiplex real-time PCR, endpoint PCR, and growth on solid agar, respectively. A set of 52 Big Six STEC isolates and 30 non-Big Six STEC strains was used to establish the inclusivity and exclusivity of the method. Finally, the ability to detect Big Six STEC contamination reliably was confirmed at 4.5 and 45 CFU/25-mL portions of refrigerated apple juice.

Influence of yogurt fermentation and refrigerated storage on the stability of protein toxin contaminants.

Dairy products sold in a ready-to-eat form present the risk that adulterants persisting through manufacturing, storage, and distribution would reach consumers. Pathogenic microbes, including shigatoxigenic strains of Escherichia coli and the toxins they produce, are common food safety hazards associated with dairy products. Ricin and abrin are plant-derived ribosome-inactivating protein toxins related to the shiga-like toxins produced by E. coli. Limited information exists on the effects of manufacturing processes on the stabilities of these heat-resistant ribosome-inactivating proteins in the presence of foods. The goal of this study was to determine how typical yogurt manufacturing and storage processes influence ribosome-inactivating protein toxins. Ricin and abrin were added to skim or whole milk and batch pasteurized. Complete inactivation of both toxins was observed after 30 minutes at 85 °C. If the toxins were added after pasteurization, the levels of ricin and abrin in yogurt and their cytotoxic activities did not change significantly during fermentation or refrigerated storage for 4 weeks. The activities of ricin and abrin were inhibited by skim milk, nonfat yogurt, whole milk, and whole milk yogurt. The results showed minimal effects of the toxins on yogurt pH and %titratable acidity but inhibitory effects of yogurt on toxin activity.

Thermal inactivation reaction rates for ricin are influenced by pH and carbohydrates.

Ricin is a lethal protein toxin produced by the castor bean plant. Ricin is known to possess significant heat resistance. Therefore, we placed it in a variety of foods to study the influence of the food matrix on behavior of a thermally stable protein toxin. First order rate constants for the thermal inactivation of ricin in foods and simple buffers were measured using cytotoxicity assays. We observed greater thermal stability at 75 °C for the cytotoxic activity of ricin when it was placed in a yogurt-containing fruit drink compared to its stability when placed in the other foods tested. We found that galactose and high molecular weight exopolysaccharides present in various dairy products contributed to the thermal stability of ricin. Differential scanning calorimetry also showed enhanced thermal stability for ricin at pH 4.5. Our results demonstrate the importance of considering pH and the presence of stabilizing ligands in the thermal inactivation of protein toxins in foods.

Chemical inactivation of protein toxins on food contact surfaces.

We compared the kinetics and efficacies of sodium hypochlorite, peracetic acid, phosphoric acid-based detergent, chlorinated alkaline detergent, quaternary ammonium-based sanitizer, and peracetic acid-based sanitizer for inactivating the potential bioterrorism agents ricin and abrin in simple buffers, food slurries (infant formula, peanut butter, and pancake mix), and in dried food residues on stainless steel. The intrinsic fluorescence and cytotoxicity of purified ricin and abrin in buffers decreased rapidly in a pH- and temperature-dependent manner when treated with sodium hypochlorite but more slowly when treated with peracetic acid. Cytotoxicity assays showed rapid and complete inactivation of ricin and crude abrin in food slurries and dried food residues treated 0-5 min with sodium hypochlorite. Toxin epitopes recognized by ELISA decayed more gradually under these conditions. Higher concentrations of peracetic acid were required to achieve comparable results. Chlorinated alkaline detergent was the most effective industrial agent tested for inactivating ricin in dried food residues.