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The heat resistance was determined for Salmonella typhimurium and Listeria monocytogenes Scott A suspended in 2% UHT processed milk and in a biofilm matrix. Pure cultures at an initial concentration of 10⁵ / ml were used. Heat resistance was determined by two methods. One method was sealed borosilicate glass TDT tubes that were completely submerged in the heating menstrum. Biofilms were grown on Buna-n rubber o-rings (4.46 mm O.D. x 1.41 mm]. D.) for 36 hours. All other cultures used were in stationary phase of growth. The three treatments tested were: inoculated milk, sterile milk and a biofilm on an o-ring, and inoculated milk with a sterile o-ring. At the three temperatures tested (60, 63, 67°C), there was no significant difference (p>0.05) in D-values between treatments. There was a significant difference (p<0.001) between the D-values for Salmonella and Listeria. The second method used a laboratory scale HTST pasteurizer to determine the difference in heat resistance of the same organisms suspended in 2% milk vs. sloughed off pieces of biofilm in milk. Pure cultures of the organisms at an initial inoculum of 10⁵ / ml were used. Flow rates of the pasteurizer were adjusted to achieve two different F-values for each organism at a reference temperature of 71.7°C. Neither S. typhimurium nor L. monocytogenes Scott A was recovered from pasteurized samples of either treatment. The heating involved in come up and cool down of the transit lines was considered in determining F-value. Under commercial HTST processing, concentrations of 10⁵ / ml of S. typhimurium and L. monocytogenes Scott A would not survive pasteurization. The results also show that if pieces of biofilms (3.8 x 10⁻⁴ mm²- 8.8 x 10⁻³ mm²) were sloughed off gaskets in the processing lines they would not survive pasteurization. The heating characteristics of these two systems were so dissimilar they could not be compared. It should however be noted that in the TDT tubes it was necessary to obtain a slightly higher F-value before no growth was seen as compared with the pasteurizer. In the pasteurizer the laminar flow properties would contribute to a more uniform heating. The TDT tube experiences convection heating which can produce cold spots in the tubes and could explain the need for an increased F-value. / Master of Science

Ready-to-eat meat products have been implicated in several foodborne listeriosis outbreaks. Microbial contamination of these products can occur after the product has been thermally processed and is being rapidly chilled using salt brines. The objective of this study was to determine the effect of ultraviolet irradiation on the inactivation of Listeria monocytogenes and lactic acid bacteria in a model brine chiller system. Two concentrations of brines (7.9%w/w or 13.2%w/w) were inoculated with a ~6.0 log10 CFU/ml cocktail of L. monocytogenes or lactic acid bacteria and passed through the ultraviolet (UV) treatment system for 60 minutes. Three replications of each bacteria and brine combination were performed and resulted in at least a 4.5 log reduction in microbial numbers in all treated brines after exposure to ultraviolet light. Bacterial populations were significantly reduced after five minutes exposure to UV light in the model brine chiller as compared to the control, which received no UV light exposure (P<0.05). The maximum rate of inactivation for both microorganisms in treated brines occurred between minute 1 and 15 of ultraviolet exposure. Overall, results indicate that inline treatment of chill brines with ultraviolet light (UVC) shows promise in inactivating L. monocytogenes and lactic acid bacteria. Due to the low capital involved in initiating a continuous inline UV system, the use of ultraviolet energy may prove to be beneficial for effectively controlling pathogens in recycled chill brines without interrupting the chilling operation. An inline ultraviolet system could be used in a hazard analysis and critical control points plan. / Master of Science

Since 2006 there have been four Salmonella enterica and one Listeria monocytogenes foodborne outbreaks linked to whole cantaloupe fruit. No post-harvest intervention to reduce potential contamination on cantaloupe currently exists. The complex surface topography of netted cantaloupes aids bacterial attachment. This research evaluates the use of allyl isothiocyanate (AITC; a natural antimicrobial) to reduce populations of S. enterica Michigan and L. monocytogenes on the surface of cantaloupe. Fifty μl of S. Michigan or L. monocytogenes was inoculated onto whole ‗Athena‘ or ‗Hales Best Jumbo‘ (‗HBJ‘) cantaloupe fruit in 22 mm diameter circles and allowed to dry for 90 min. resulting in 6.60 log CFU/g. Cantaloupe received either AITC liquid or vapor, sterile deionized water, 200 ppm sodium hypochlorite per circle, or no treatment. All cantaloupes were stored in separate sealed glass desiccators for 1 or 24 h at 25°C or 35°C. To enumerate the bacteria following treatment, 22 mm sections of the rind were removed, homogenized and plated onto appropriate agar. Headspace analysis using Gas Chromatography-Mass Spectrometry (GC-MS) quantified the concentration of each AITC vapor treatment. The texture quality of the pericarp tissue of whole cantaloupes was evaluated after 24 h treatments, followed by two weeks of storage at 4°C. The concentration of vapor ranged from 3.4 to 19.6 μl AITC/L inside the desiccators. The liquid treatment reduced (P < 0.05) S. Michigan populations on ‗Athena‘ (3 log CFU/g) and L. monocytogenes on ‗HBJ‘ (2.6 log CFU/g). The longer exposure time to the AITC vapor (24 h versus 1 h) resulted in a greater reduction of both S. Michigan and L. monocytogenes on ‗Athena‘ and treatments at 35°C reduced microbial populations up to 4.5 times greater (P < 0.05). The highest vapor concentration reduced (P < 0.05) both pathogens at least 3.0 log CFU/g on ‗Athena‘ at 25°C. Generally, bacterial pathogens from the surface of ‗Athena‘ cantaloupe were reduced more than pathogens inoculated on the surface of ‗HBJ.‘ The application of AITC liquid or vapor is a natural alternative post-harvest treatment to 200 ppm free chlorine to reduce the level of bacterial contamination on cantaloupe surfaces for certified organic production. / Ph. D.

Listeria monocytogenes is the major pathogen in ready-to-eat meat products such as deli meats and frankfurters. Contamination can occur via the salt brines that are used to cool thermally processed meats. Both L. monocytogenes and lactic acid bacteria can grow and thrive under these brine conditions, and may become competitive with each other for available nutrients. The objective of this study was to determine the effect of a three strain cocktail of lactic acid bacteria Enterococcus faecalis, Carnobacterium gallinarum, and Lactobacillus plantarum on the survival of Listeria monocytogenes and Listeria innocua in brines stored under low temperatures up to 10 days. Three brine concentrations (0%, 7.9%, and 13.2% NaCl) were inoculated with ~7.0 log₁₀ cfu/ml of one of five cocktails (L. monocytogenes, L. innocua, lactic acid bacteria (LAB), L. monocytogenes + LAB, or L. innocua + LAB) and stored for 10 days at either 4°C or 12°C. Three replications of each brine/cocktail/temperature combination were performed. No reductions of L. monocytogenes were seen in 7.9 or 13.2% NaCl with LAB; however, reductions of L. monocytogenes were seen in the 0% NaCl with LAB (1.43 log at 4°C and 3.02 log at 12°C). Listeria innocua was significantly less resilient to environmental stresses than L. monocytogenes, both with and without LAB present (p<0.05). This research indicates these strains of lactic acid bacteria are not effective at reducing L. monocytogenes in brines at low temperatures. Furthermore, the use of L. innocua as a model for L. monocytogenes is not appropriate under these environmental conditions. / Master of Science

Bacterial biofilms attached to food contact surfaces are an ongoing concern for the food industry due to the resistance of bacteria within biofilms to detergents and sanitizers. Within food manufacturing facilities, stainless steel is a common food-contact surface in which microbial cell attachment and biofilm formation may occur. Identifying methods to prevent and remove biofilms during standard cleaning and sanitation practices could prove useful, as mature biofilms can release planktonic cells into an aqueous environment, causing continual low-level contamination. Dental studies involving delmopinol hydrochloride, a cationic surfactant, have found a preventative and dissociating affect on biofilms, where food applications have scarcely been researched. This study demonstrates the prevention and removal of Listeria monocytogenes 1/2a and S. enterica Agona biofilms on stainless steel with pre- and post-exposures of delmopinol hydrochloride. Stainless steel blanks (#304, 16 gauge, 2cm x 2cm, finish #4) were submerged in a 0.2% or 0.5% delmopinol solution before or after biofilm formation. Treatment times were 1, 5 or 10 minutes, whereas controls were not exposed to the delmopinol solution. Disinfected stainless steel blanks were spot-inoculated with 20µL of a 10⁹ CFU/mL liquid culture, and pre-exposed blanks were additionally submerged in delmopinol and dried prior to inoculation. Biofilms were exclusively formed on the finished and inoculated side by placing the surface face-down on TSA. After cell attachment and biofilm development for 24 hours at 25°C, blanks were rinsed with phosphate buffer. Post-exposed blanks were submerged in 0.2% or 0.5% delmopinol for 1, 5 or 10 minutes before all blanks were individually vortexed for 90 seconds to dislodge films. Bacterial populations were determined by surface plating onto TSA followed by incubation at 32°C for L. monocytogenes and 37°C for S. Agona for 48 hours. Treatments were in-duplicate and repeated three times for each microorganism. Pre-exposure of 0.2% delmopinol resulted in a significant decrease in L. monocytogenes concentration at 1, 5 and 10 minute exposures (P < 0.05). Pre-exposures with the 0.5% solution had no significant effect on L. monocytogenes biofilm populations (P > 0.05), whereas all post-exposures lead to a significant decline in biofilm concentrations (P < 0.0001). Post-exposures of 10 minutes exhibited a mean log₁₀ reduction of 5.59 and 6.40 log₁₀ for 0.2% and 0.5% delmopinol solutions, respectively. For S. Agona, 0.2% pre-exposure resulted in no significant log10 reduction (P > 0.05), while the 10 minute 0.5% pre-exposure exhibited a minimal reduction in bacterial growth (P < 0.05). Post-exposures of 10 minutes exhibited a mean log10 reduction of 7.65 and 7.75 log10 for 0.2% and 0.5% delmopinol solutions, respectively. For L. monocytogenes and S. Agona, post-exposure to delmopinol hydrochloride caused a notable log10 reduction. The removal effect of delmopinol on biofilms is significantly greater the preventative effect. / Master of Science in Life Sciences

The ability of Listeria monocytogenes, to attach to various food contact surfaces such as stainless steel, polypropylene, and rubber compounds is well documented. The retention of these or other pathogenic bacteria on food contact surfaces increases the risk of transmission to food products. The objective of this study was to compare several methods for quantitative recovery of Listeria monocytogenes from stainless steel surfaces. A cocktail of four serotypes of Listeria monocytogenes (Scott A (4b)), 1/2b, 3b, and 4b) were mixed in equivalent concentrations and inoculated onto type 304 stainless steel coupons in a 2cm x 2cm area. After a one hour exposure, coupons were sampled by one of the following methods: 1) swabbing using a pre-moistened Dacron swab, 2) rinsing with phosphate buffered saline, 3) direct contact onto a Tryptic Soy Agar containing 0.6% yeast extract (TSA+YE) plate surface for 10 seconds, 4) sonication in an ultrasonic water bath (40 kHz), 5) contact with the bristles of a sonicating brush head for 1 min, and 6) indirect contact (2-4 mm) with the bristles of a sonicating brush head for 1 min. Coupon rinses were plated onto TSA containing 0.6% yeast extract and incubated for 24 hours at 35°C. The three sonication methods yielded higher recovery than the other three methods (p < 0.05). Brushing the coupons with the sonicating brush head yielded a recovery level of 58% and indirect exposure to the sonicating brush head permitted a recovery level of 65% from the initial microbial load. The lowest cell recovery (~20%) was observed with the swab and direct agar contact methods. / Master of Science

Environmental sampling is a pathogen monitoring technique that has become important in the food industry. Many food processing companies have adopted environmental sampling as a way to verify good manufacturing practices and sanitation plans in their facilities. Environmental sampling is helpful because it gives better information on the source of product contamination than end product sampling. Two specific pathogens of concern to the food industry are Listeria monocytogenes and Salmonella enterica. Environmental samples are rarely analyzed immediately, but instead may be batched for later analysis or shipped to an off site testing facility. Multiple media on the market today is used for storage and transport of environmental samples. These various media types, differences in holding temperatures and time create variability in test sample conditions. Select time, temperature and media combinations were tested to determine their effect on Listeria monocytogenes and Salmonella enterica populations during transport and storage of samples. Cocktails of Listeria monocytogenes and Salmonella enterica were added separately to sample tubes containing D/E Neutralizing Broth, Neutralizing Buffer or Copan SRK Solution. Bacterial counts at 0, 12, 24 and 48 hours post inoculation were compared. Neutralizing Buffer and Copan SRK Solution maintained consistent bacterial populations at all temperatures. At 10° and 15°C, D/E Broth supported bacterial growth. This study helps validate the use of D/E Neutralizing Broth, Neutralizing Buffer and Copan SRK Solution for environmental sample transport and storage at proper holding temperatures. At temperatures >10°C Neutralizing Buffer or Copan SRK solution should be used if quantifying microbial recovery. / Master of Science

Meat and poultry slaughtering and processing practices have been associated with the microbial contamination with Listeria spp. Ready-to-eat poultry products have been considered as a primary agent associated with Listeria monocytogenes illness outbreaks. Developing environmental monitoring programs (EMPs) that are based on product and/or process risk level analysis is a useful approach to reduce contamination in poultry processing plants and enhance food safety. Sampling criteria that is based on product risk levels and process control in ready-to-eat poultry processing facilities was developed to allow users to design and conduct appropriate sampling plans to target Listeria spp. After developing the criteria, an internet-based environmental monitoring program ("EZSafety") was developed to allow poultry producers to enhance their sample collection and analysis of test results over time and conduct appropriate sampling plans for Listeria spp. and other microbiological indicators. The frontend of the program website was built using React Native (an open-source JavaScript library for building user interfaces). The backend of the program website was built using Node.js which executes JavaScript code outside a web browser. MongoDB was used as a document-oriented database for the website. The program was evaluated by 20 food safety professionals to assess its ability to develop appropriate sampling plans to target Listeria spp. The majority of these participants believed that EZSafety has several tools that are effective for targeting Listeria spp. and other indicators and enhancing environmental monitoring. Additionally, most participants agreed that EZSafety is organized and user-friendly. EMPs can play a significant role in improving the detection rate and the prevention of Listeria spp. and other indicators in poultry processing plants. / Master of Science in Life Sciences / Meat and poultry slaughtering and processing practices have been associated with the microbial contamination with a bacterium known as Listeria. Cooked poultry products during the manufacturing process have been considered as a primary agent associated with Listeria monocytogenes (disease causing type of bacteria) sickness outbreaks. Developing environmental monitoring plans to detect and prevent this bacterium in poultry processing establishments is a useful approach to reduce contamination and enhance food safety. Several guidelines and baselines were developed to allow users to design and conduct appropriate environmental monitoring plans to target this bacterium. After developing these guidelines and baselines, an internet-based environmental monitoring program ("EZSafety") was developed to allow poultry processors to enhance their sample collection and analysis of test results over time. The program was developed using several kinds of computer platforms (JavaScript, React Native, and MongoDB) . These open-source platforms were used to design, develop, and store the program over the internet. In order to validate its usefulness, the program was evaluated by 20 users who are majored in food safety and familiar with poultry processing plants hygiene to assess its ability to suggest appropriate monitoring plans. Most of the participants believed that EZSafety has several tools that are effective for targeting Listeria and other kinds of bacteria and enhancing environmental monitoring plans. Additionally, most participants agreed that EZSafety is organized and user-friendly. Such automated monitoring programs can play a significant role in enhancing the detection rate and the prevention of Listeria and other organisms in poultry processing facilities.

Crab meat is important to the economy of coastal Virginia. The objectives of this study were to complete a shelf-life study on two different packaging styles of fresh crab meat and to test the inhibition capabilities of Carnobacterium piscicola against the pathogen, Listeria monocytogenes. In a shelf-life study, a 12 ounce food grade polyethylene traditional snap-lid container of fresh crab meat was compared to an 8 ounce SimpleStep® trays with Cryovac™ film of equally fresh crab meat sealed with 10,000 cc/m2/24hr oxygen transmission rate (OTR) film. Eleven g samples were used for the microbial shelf-life study conducted at 4°C for 12 days. Aerobic plate counts of crab meat indicated microbial growth from the SimpleStep® trays with Cryovac™ film in 10,000 cc/m2/24hr OTR versus the polyethylene snap-lid was not significant (P>0.05). In objective two, 25 g samples of fresh and pasteurized blue crab (Callinectes sapidus) meat were inoculated with 0.1ml of each, C. piscicola and L. monocytogenes. Three different concentrations of the inoculation levels were studied on select days at both 4°C and 10°C. Microbial spoilage was defined as 107 CFU/g. In fresh crab meat, at both 4°C and 10°C, crab meat spoilage occurred at 7 days or less. In the pasteurized crab meat, at 4°C and 10°C, spoilage did not occur prior to 26 days, and studies were terminated at 28 days of storage. The growth of the two organisms in fresh crab meat was found to be significant for the differing concentration levels and sampling days (P<0.05). The growth of the two organisms in pasteurized crab meat was significant for different concentration levels, sampling days and temperature (P<0.05). In both fresh and pasteurized crab meat, regardless of the inoculation ratios, the L. monocytogenes and C.piscicola followed similar growth trends, but L. monocytogenes was higher in the 2:2 CFU/g concentration and lower at the 6:2 CFU/g concentration level. Although C. piscicola did not completely inhibit L. monocytogenes growth at any concentration ratio, some inhibition was observed. / Master of Science