Alicyclobacillus acidoterrestris is a spore-forming food spoilage bacterium. Its spore is problematic to the juice industry because of its ability to grow in low pH environments and survive pasteurization processes. The purpose of this study was to investigate the effect of the non-thermal technology, high pressure homogenization (HPH) and the antimicrobial compound, dimethyl dicarbonate (DMDC), on inactivation of A. acidoterrestris, in a broth system. Vegetative cells and spores of five strains of A. acidoterrestris (N-1100, N-1108, N-1096, SAC and OS-CAJ) were screened for their sensitivity to HPH (0, 100, 200 and 300 MPa) in Bacilllus acidoterrestris thermophilic (BAT) broth. Strain SAC (most resistant) and OS-CAJ (least resistant) were further tested for their sensitivity to 250 ppm DMDC. This was followed by evaluation of combined effects of HPH and DMDC against strain SAC. Effects of HPHand DMDC treatment combinations (no DMDC, 250 ppm DMDC added 12 h before, 2 h before, immediately before, and immediately after 300 MPa HPH treatment) on spores of SAC over a 24-h period were evaluated. After all treatments, samples were serially diluted and surface plated onto BAT agar, and the populations were determined after incubation at 44 &degC for 48 h. All HPH and DMDC treatments significantly (P<0.05) inhibited growth of vegetative cells, spores were less affected by these treatments. HPH caused a 1-to 2-log reduction in vegetative cell populations at 300 MOa for four strains, but only about 0.5-log reduction of SAC strain. Spores of all five strains were not significantly reduced by HPH. DMDC also slowed growth of vegetative cells significantly. For vegetative cells of SAC and OS-CAJ, 250 ppm DMDC reduced the population by about 2 log whereas spore population was reduced by less than 0.5 log. The addition of DMDC together with HPH slightly enhanced the inactivation effect over a 24-h period as compared with treatment with HPH alone. These results demonstrate that HPH and DMDC show promise for aiding in control of growth of vegetative cells of A. acidoterrestris. However, neither treatment alone or in combination, is very effective against spores.

Chill brines used in ready-to-eat meat processing may be an important source of post-processing contamination by Listeria monocytogenes. The purpose of this study was to determine the efficacy of ultraviolet light (UV) in combination with antimicrobials to reduce L. monocytogenes in fresh and used chill brines. Three different antimicrobials were used in combination with UV; citric acid (CA, 0.2 and 0.5%), dimethyl dicarbonate (DMDC, 250 and 500 ppm), and hydrogen peroxide (HP, 2000 and 4000 ppm). For fresh brine studies, brine (8.0% w/v NaCl) was prepared and inoculated with a cocktail of three L. monocytogenes strains (approximately 6 log CFU/mL). Brine was treated with UV alone, antimicrobials alone, and combination of UV and antimicrobials. Moreover, to observe the effect of treatment temperature and brine circulation through the UV system on survival of listeriae cells, inoculated brine was circulated through the system without any treatment that served as control for all the treatments. For UV treatment, inoculated brine solution was exposed to UV in an Ultraviolet Water Treatment Unit (Model: AMD 150B/1/2T D; Aquionics Inc., Peak output: 254 nm) fitted with an inline chiller to maintain brine temperature of -1°C. Samples were withdrawn at regular intervals for 120 minutes. When L. monocytogenes population was no longer detectable via direct plating on MOX, enrichment was performed and suspect colonies were confirmed using API-Listeria. For antimicrobial-only (i.e., no UV) treatments, a specific concentration of antimicrobial was added in inoculated brine and samples were taken for 120 minutes. For the brine that received combination of UV and antimicrobial treatments, UV was turned on once a specific concentration of antimicrobial was added in inoculated brine and samples were withdrawn at regular intervals for 120 minutes. When treated with UV alone, L. monocytogenes population decreased from approximately 6 log CFU/mL to below the detection limit (i.e., 1 log CFU/mL) in 15 minutes with the reduction rate of 0.87 log CFU/mL per minute. However, cells were detectable by enrichment through 120 minutes. The highest rate of decline (0.90 log CFU/mL per minute) was achieved by the combination of UV and 500 ppm DMDC (UV+500 ppm DMDC), which was not significantly different from the reduction rates of UV and UV+0.5% CA. UV+500 ppm DMDC reduced L. monocytogenes to the detection limit in 15 minutes and the organism was not detected by enrichment after 60 minutes. Though the reduction rate of UV+0.5% CA was not significantly lower than the rate of UV+500 ppm DMDC (P>0.05), the former treatment resulted in non-detectable levels more quickly (45 minutes) than the latter (60 minutes). Thus, based on enrichment studies UV+0.5% CA was the most effective treatment in reducing the population of L. monocytogenes in fresh brine. Moreover, when brine was treated with 0.5% CA alone the population decreased to below detection limit in 15 minutes with the rate significantly lower than UV+500 ppm DMDC and UV+0.5% CA (P<0.05). However, L. monocytogenes was not detectable by enrichment from 60 minutes. To summarize, through enrichment studies we observed that UV+0.5% CA, UV+500 DMDC, and 0.5% CA Control were more effective than other treatments in reducing the listeriae population to a non-detectable level. Spent brine is recycled brine that was obtained from a frankfurter processor after its maximum usage. Results of spent brine studies showed that when brine was treated with UV+4000 ppm HP and UV+2000 ppm HP, L. monocytogenes population decreased to the detection limit in 45 minutes and was not detected by enrichment from 120 minutes. These treatments were observed to be the most effective treatments with a reduction rate of 0.12 log CFU/mL per minute. The reduction rate of some other treatments such as, UV+250 and 500 ppm DMDC, UV+0.2% and 0.5% CA, and UV alone was not significantly different from UV+4000 and 2000 ppm HP. However, the population was detected through enrichment up to 120 minutes in all other treatments. The results of these studies indicate that combinations of UV and antimicrobial may be more effective than either treatment alone (except 0.5% CA treatment) to process fresh chill brines. However, the antimicrobials and UV were less effective for controlling L. monocytgoenes in spent brine; presumably due to the presence of organic matter. / Ph. D.

Hazard Analysis Critical Control Point (HACCP) regulations for juice manufacture require the application of a process that will result in a 5-log reduction (99.999%) of the pertinent pathogen in the juice being processed. The use of ultraviolet (UV) light, as an alternative to traditional thermal processing, has been adopted by some juice processors as a means of meeting the HACCP 5-log performance standard. However, little research had been performed to determine the effect of UV when used in combination with antimicrobial agents that are commonly added to juice products. Therefore, the objectives of this work were (1) to determine if chemical preservatives and ultraviolet light have a combined effect on the reduction of Escherichia coli in apple cider, and (2) to determine the influence of adding chemical preservatives at different points in the processing of juice (i.e., either prior to or after ultraviolet light processing) on the reduction of Escherichia coli in apple cider. In this study, refrigerated (4°C) pasteurized apple cider that contained no added preservatives was inoculated with E. coli ATCC 25922, a surrogate strain for E. coli O157:H7, and exposed to UV (peak output: 254 nm). The following chemical preservatives were added to apple cider either prior to or after UV exposure: dimethyl dicarbonate (75 and 150 ppm), hydrogen peroxide (75 and 150 ppm), potassium sorbate (1000 and 2000 ppm), and sodium benzoate (1000 and 2000 ppm). Following UV exposure and chemical preservative application, inoculated juices were stored at 4°C for 72 hours. Samples were collected prior to and immediately after UV exposure and at 24, 48, and 72 hours of storage. At each sampling point, juice portions (0.1 ml) were serially diluted in peptone diluent (0.1%) and surface plated onto Tryptic Soy Agar (TSA). Counts of the bacterial colonies were made 48 hours after incubating plates at 35°C. Overall, reductions of E. coli were greater in cider treated with preservatives after UV processing than when preservatives were added prior to UV processing (P < 0.05). Furthermore, dimethyl dicarbonate and hydrogen peroxide were more effective than potassium sorbate and sodium benzoate in reducing E. coli populations in conjunction with UV (P < 0.05). When added prior to UV exposure, potassium sorbate was the least effective, allowing for the greatest survival (P < 0.05). This study describes the use of UV in conjunction with hydrogen peroxide and dimethyl dicarbonate as an effective method for producing a 5-log or greater reduction of E. coli O157:H7 in apple cider. / Master of Science