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Title:   Author  item
                                            Rasooly, Reuvenitem
                                            Do, Paulaitem
                                            Levin, Carolitem
                                            Friedman, Mendel
  Submitted to: Applied and Environmental Microbiology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 2/16/2010
                                    Publication  Date: 6/2/2010
                                    Citation: Rasooly, R., Do, P.M., Levin, C.E., Friedman, M. 2010. Inhibition of Shiga Toxin 2 (Stx2) in Apple Juices and its Resistance to Pasteurization. Applied and Environmental Microbiology. Vol. 75(5):M296-M301.Interpretive Summary: Shiga-like toxins are produced by E. coli O157:H7. This bacterium is a pathogen of major importance for food safety, causing foodborne illnesses, ranging from mild diarrhea to a life threatening complication known as hemolytic uremic syndrome. The bacterium produces a family of related toxins with two major groups, verocytotoxin 1 (Stx1) and verocytotoxin 2 (Stx2). Stx 2 is reportedly several orders of magnitude more toxic than Stx1. In the present study, we discovered that freshly prepared juice from locally purchased Red Delicious apples, but not juice from Golden Delicious apples, inactivated the biological activity of the toxin. However, both Golden Delicious juice and water with added 0.3% polyphenol-rich grape pomace, a byproduct of wine production, also inactivated the Shiga toxin. Additional studies with immunomagnetic beads with specific antibodies against the toxin revealed that only part of the added Stx2 in apple juice appears to be irreversibly bound to apple juice and grape pomace constituents. The results suggest that food-compatible and safe anti-toxin compounds can be used to inactivate Shiga toxins in apple juice and possibly also in other liquid and solid foods.
                                Technical Abstract: In the present study, we evaluated Shiga toxin (Stx2) activity in apple juices by measuring a decrease in dehydrogenase activity of Vero cells with the microculture tetrazolium (MTT) assay. Freshly prepared juice from Red Delicious apples and Golden Delicious apples inhibited the biological activity of the bacterial toxin Stx2 produced by E. coli O157:H7 strains. Studies with immunomagnetic beads bearing specific antibodies against the toxin revealed that Stx2 activity was restored when removed from the apple juice. SDS gel electrophoresis revealed no difference (P < 0.05) in the densities or molecular weights between Stx2 in either PBS or apple juices. These results suggest that Stx2 may be reversibly bound to small molecular weight constituents in the juice. The Stx2 toxin was not inactivated on exposure to heat programs (63 degree C for 30 min, 72 degree C for 15 s, 89 degree C for 1 s) commonly used to pasteurize apple juice, but lost all activity when exposed to 100 degree C for 5 min. The results suggest that pasteurization of apple juice used to inactivate E. coli O157:H7 has no effect on Stx2, and that food-compatible and safe antitoxin compounds can be used to inhibit the biological activity of the Shiga toxin.


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