During ozone and heat treatments, juice samples were mixed at 150 rpm for the entire treatment time in a shaking water bath to ensure even distribution of inoculum and dispersal of ozone. All experiments were conducted in a fume hood. Excess gaseous ozone was passed into an ozone decomposer. For enumeration of pathogens, sample aliquots (1 ml) were transferred into test tubes containing 9 ml of D/E neutralizing broth (Difco, Becton Dickinson, Sparks, MD, USA) after each treatment

and homogenized using a vortex mixer (VM-10, Daihan Scientific Co., Ltd, RAD001 clinical trial Korea). After homogenization, samples were 10-fold serially diluted with 9 ml of 0.2% sterile buffered peptone water and 0.1 ml of the samples was spread plated onto selective media (E. coli O157:H7: Sorbitol MacConkey Agar (SMAC), Difco; S. Typhimurium: Xylose Lysine Desoxycholate Agar (XLD), Difco; and L. monocytogenes: Oxford Agar Base with antimicrobial supplement Osimertinib in vitro MB Cell (MOX), MB Cell). All plates were incubated at 37 °C for 24 h before counting.

Color was measured by using a Minolta colorimeter (model CR400; Minolta Co., Osaka, Japan). The values for L*, a* and b* were recorded to evaluate the color changes of apple juice after each treatment with heat or combination of ozone and heat. Untreated apple juice was used as the control. A 2 ml of sample was poured into the bottom half of the measurement equipment. The measuring head of the colorimeter was placed on top of the measurement Methisazone equipment. Before measurement, the treated juice was cooled to about 15 °C by dipping the bottle in crushed ice. The parameter L* is a measure of lightness, a* is an indicator of redness, and the parameter b* is a measure of yellowness. All measurements were performed in triplicate. To measure ozone concentration, distilled water was substituted for apple juice, as will

be explained in the Discussion section. The ozone concentration of distilled water treated with ozone in the same way as apple juice was measured by the indigo method (Bader and Hoigni, 1981). An indigo stock reagent was prepared by the following method (Gordon and Bubnis, 2002). Indigo stock solution was prepared by dissolving 770 mg of potassium indigotrisulfonate (Sigma Aldrich Co., LLC) into a 1 l flask containing 500 ml of distilled water and 1 ml of phosphoric acid (85%) and diluting to volume (1 l) with distilled water. Indigo reagent II was prepared in a 1 l flask by adding 100 ml of indigo stock solution, 10 g of sodium dihydrogen phosphate (NaH2PO4) and 7 ml of phosphoric acid (85%). This was stirred and diluted to volume (1 l) with distilled water. The solution was stored in the dark. After each treatment, the treated sample was cooled to about 15 °C and then 90 ml of the sample was transferred to a flask containing 10 ml of indigo reagent II. The absorbance was measured by a spectrophotometer at 600 nm.