Thermal and chemical inactivation of ricin and Shiga toxins in orange juice.

Abstract

The potential use of ricin and Shiga toxins (Stxs) as bioterror weapons in the food supply is a major concern for homeland security. Denaturation effects of thermal and chemical treatments are expected to reduce the toxicity of ricin and Shiga toxins in water solutions, but their effectiveness and stabilities in food matrices are largely unknown. The objective of this project was the identification of heat and chemical treatments capable of inactivating ricin and Shiga toxins in orange juice so that large quantities can be safely disposed in the event of an intentional attack. Diluted ricin was mixed with orange juice for inactivation studies. Thermal stability was determined in capillary tubes using a water bath at high temperatures typical of pasteurization. For chemical inactivation, sodium hypochlorite (NaOCl), sodium hydroxide (NaOH) and peracetic acid (PA) were added alone or in combination to samples with or without thermal treatment. The ricin concentration in samples was determined using an ELISA. The Arrhenius model was used to evaluate temperature dependence. Enterohemorrhagic Escherichia coli strains were used to produce Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Shiga toxins were added into phosphate buffered saline (PBS) or orange juice to study the inactivation effects. The same inactivation method was also used for heat treatment of Stxs. The concentration of Stxs was determined by an ELISA and a cytotoxicity assay was conducted to confirm the inactivation. Kinetics studies were done to evaluate inactivation parameters. Heat inactivation of ricin followed first-order kinetics. The half-life (t1/2) of ricin at 72, 80, 85 and 90°C were 72.6, 9.0, 2.0 and 0.5 min, respectively. The Z value was 8.8°C indicating high temperature sensitivity. When the concentration of each chemical was increased to a sufficient amount, the detection limit of the ELISA kit was reached when measuring ricin inactivated within 5 s at room temperature. A significant synergism between NaOCl and NaOH and considerable efficacy with treatment with PA alone were observed. The heat inactivation of Stxs in PBS and orange juice also followed first-order reaction kinetics. Both Shiga toxins in PBS and orange juice would reach the concentration that was not detectable with ELISA within 30 s at 90°C and 120 s at 85°C. The Z values for Stx1 and Stx2 were 6.7 and 7.2°C in PBS as well as 8.7 and 6.9°C in orange juice, respectively. This study delivered the first series of time/temperature/concentration conditions that would serve as the basis for recommendations for treating orange juice subjected to intentional adulteration with ricin or Shiga toxins in an orange juice plant with typical pasteurization equipment so it can be safely disposed into the environment.