The use of antibiotics in animal feed is a large cause of concern due to bacterial resistance, which occurs when bacteria change after exposure to antibiotics and become less affected by the drug. Consequently, the desire to find a replacement for these antibiotics has garnered interest in both farmers and consumers. The ideal substance would display antimicrobial activity without promoting bacterial resistance and would still function as a growth promoter in animals. One antimicrobial that fits this criterion is carvacrol, a compound found in oregano extract. Carvacrol exhibits antimicrobial activity in a number of bacteria, including E.coli O157:H7 and S. enteritidis. In this study, the effectiveness of carvacrol nanoemulsions against E.coli and S. enteritidis growth in a micro-broth dilution assay was tested, as well as the use of alamarBlueTM dye reagent as a bacterial viability indicator. The carvacrol nanoemulsion was diluted in TSB to obtain 2000, 1000, 500, 250, 125, and 62.5 ppm, then aliquots of 100 μl of each dilution were added to a 96-well microtiter plate. Test group wells were inoculated with 100 μl of microorganism while control cells remained uninoculated, and the plate was incubated for 24h at 37 °C. After incubation, 10μl of alamarBlueTM was added to each well, and the microtiter plate was incubated for one hour. After one hour, the color changes were analyzed both visually and via fluorescence. Additionally, a traditional plating assay in which samples were plated on tryptic soy agar was done alongside the microtiter plate assay. It was found that the minimum inhibitory concentration of carvacrol needed to inhibit E. coli growth was 500ppm, and to inhibit S. enteritidis growth was 800ppm. These results show that carvacrol may be suitable as a possible replacement for antibiotic in animal feed in the future.

The proteolytic system of Lactococcus lactis starter cultures influences both flavor and the characteristic body and texture of cheese. The ability to further understand and control how different components of this proteolytic system work together to hydrolyze milk proteins would be of immense importance to the dairy industry. The goal of this research was to characterize Lactococcus lactis subsp. lactis starter bacteria with varying prt operon compositions by proteinase specificity, aminopeptidase and lipase activities, growth, and influence on cheese flavor. By using a cheese slurry system, a statistical model to predict milk protein hydrolysis patterns was developed. Lactococcus lactis subsp. lactis C20 has five plasmids of 55 (pJK550), 48 (pJK480), 43 (pJK430), 3.7 (pJK037), and 2.1 (pJK021) kilo bases. Two of these plasmids (pJK550 and pJK430) are necessary for full proteolytic capability, i.e., clotting milk in 16 h at 20°C. Plasmid pJK550 codes for a proteinase that catalyses the first step in casein degradation. Plasmid pJK430 codes for an oligopeptide transport system, which further transports peptides across the membrane for bacterial metabolism. Strains were constructed containing twelve different combinations of proteolytic phenotypes, such as Lac+PrtP+Opp+, Lac+PrtP+Opp-, Lac+PrtP-Opp+, Lac+Prt-Opp-, Lac-PrtP+Opp+, Lac-PrtP+Opp-, Lac-Prt-Opp+, and Lac-Prt-Opp-. The proteinase specificities of these strains toward milk proteins were dependent on the genotypes present. Genetically all strains showed a P1-type proteinase. Enzymatically C20 had group g proteinase specificity, whereas the rest of the strains containing the proteinase gene showed mixed group specificity. a a-Casein was only slightly hydrolyzed by all strains. B-Casein had a variable pattern, as did mixed casein and milk. K-Casein hydrolysis showed similar degradation patterns in all strains except CB06, which varied in its profile from the other strains. Sensory evaluation showed that culture had a significant effect on rancidity but not on acidity or bitterness. It also showed that the proteolytic system was associated with lipase activity in these strains. A statistical prediction model was developed that allowed strains to be classified according to their amino acid hydrolysis patterns. Mixed casein solution proved to be the best substrate for this analysis. Relationships among strains were seen more easily with canonical analysis and distance tables than by looking only at amino acid hydrolysis patterns.

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Listeria monocytogenes is a major concern for the food industry. It is one of the major agents causing listeriosis. The objective of the first part of this study is to evaluate the effect of hydration level on attached listeria monocytogenes on stainless steel/High density polyethylene transferred to food products. Attached cells were prepared on stainless steel/High density polyethylene. Transfer experiments were conducted from inoculated surface material slides to cold smoked salmon fillets. This experiment was repeated 6 times. The results were analyzed with an analysis of variance by SAS. The differences between the different RH% and surface conditions were not statistically significant. There was variability in between packages, brands and over the course of storage after opening, and likely contributed to the variability of transfer observed in this set of experiments. The objective of the second part of the research is to study the effect of hydration level on the detachment of Listeria monocytogenes biofilm growing on stainless steel by using Atomic force microscope. Biofilms were grown on stainless steel in drip flow bioreactor at 32 °C for 72 h. Then biofilms were equilibrated over saturated salt solution at 20 °C for 48 h before the Atomic Force Microscope experiment. The results showed that cohesive energy value of the biofilm increased with biofilm depth. Only square shaped displaced 2.5X2.5 μm region were visualized after serious of raster scanning under high load which means that moisture condition of Listeria monocytogenes biofilm can significantly affect the cohesiveness between of Listeria monocytogenes biofilm.

Bacillus cereus is a ubiquitously found foodborne pathogen that is frequently associated with two types of illness: emesis and diarrhea. Two heat labile enterotoxins have been associated with the diarrheal syndrome whereas a heat stable acid stable peptide toxin has been associated with the emetic syndrome. In the U.S, B. cereus is responsible for 1-2% of the total outbreaks from bacteria. Although there are reports of isolation and characterization of this pathogen from various food stuffs all around the world, there are no reports on the levels, toxin producing ability, or growth characteristics from U.S retail rice. Considering that rice is grown mostly in developing countries and most of the rice in the U.S is imported, there is a high chance of the rice being contaminated with B. cereus spores. Therefore, the major objective of this thesis was to characterize B. cereus spores from U.S retail rice. The levels were determined and further the enterotoxigenic ability and the growth characteristics along with the physical characteristics of the isolates were studied. Among the 178 samples analyzed, Spores of Bacillus species were found in 94 (52.8%) of the rice samples with an average concentration of 32.6 CFU/g (3.6-460 CFU/g). Eighty nine of the 94 isolates were tested positive for one of the two enterotoxins produced by B. cereus. none of the 94 isolates tested positive for the emetic gene. All the isolates generally grew well in cooked rice. Levels of 106/g were detected in cooked rice after 22h at 200C and after 34h at 170C whereas at 120C the counts did not go above 104/g even after 48h. A significant difference in the heat resistance of the emetic and the diarrheal strains was found. The emetic but not the diarrheal type grew well at inoculum levels of 102/g and 103/g level following cooking. So these results suggest although the diarrheal type are more predominant in U.S retail rice, the chances of foodborne illness arising from the diarrheal strains is low. B. cereus, B, thuringiensis and B. mycoides were investigated for their physical characteristics. Appendages were not found on B. mycoides. By contrast, all the isolates had exosporia. The isolates were characterized to be moderately to highly hydrophobic and all the isolates had a net negative charge. Judging by their physical characteristics, it can be concluded that these spores may have a high affinity for adhering to inert surfaces.

Produce is responsible for 46% of all foodborne illnesses in the USA. Salmonella enterica causes 19,000 hospitalizations each year, and has been associated with produce. Presently, chlorine based sanitizers are most often used, however organic matter reduces its antimicrobial activity. Bacteriophage treatments are an all-natural, alternative method for pathogen inactivation. The objective of this study was to determine the efficacy of a five-strain bacteriophage treatment against a S. enterica cocktail in simulated wash waters at different temperatures. Bacteriophage and S. enterica were enumerated in simulated wash water solutions. One set of experiments studied bacteriophage and S. enterica growth in TSB+vegetable solutions. Bacteriophage behavior was not statistically different (p < 0.05) in spinach, romaine, or iceberg lettuce across different concentrations of organic matter. S. enterica reduction was approximately 2 log over 135 minutes for vegetable solutions and for the TSB control. S. enterica reduction was only 0.5 log in water solutions. The next set of experiments studied bacteriophage and S. enterica growth in vegetable solutions. Spinach wash water and tryptone soy broth solutions (TSB) at 20 °C and 37 °C. S. enterica was not reduced in spinach solution studies at 20 °C and 37 °C or at broth solutions at 20 °C. However, S. enterica was effectively reduced 4 log in broth solutions at 37 °C up to 7.5 hours, but grew to high levels after 24 hours. These results indicate that bacteriophage could not effectively control bacteria levels in produce wash water, and may need to be optimized.

The Food Safety Modernization Act (FSMA), specifically the Produce Safety Rule (PSR), requires growers to clean and sanitize food-contact surfaces to protect against produce contamination. The ATP monitoring device is a potential sanitation tool to monitor the efficacy of an on-farm cleaning and sanitation program that could help growers meet regulatory expectations mandated by PSR. The ATP device uses bioluminescence to detect all ATP (found in bacteria and produce matter cells) from a swabbed surface. Little work has been done to test the efficacy of these tools under postharvest conditions. The present study evaluated ATP measurement for postharvest surface cleanliness evaluation. Concentrations of leafy greens (spinach, romaine, red cabbage) (with/without L. innocua) were used as organic matter inocula onto stainless steel, HDPE plastic, and bamboo wood coupons to represent postharvest surfaces. The ATP levels on the coupons were measured using swabs and ATP monitoring device. Results showed that the concentration of L. innocua and leafy greens on a surface had a highly significant effect on the ATP device reading (PL. innocua at 4.5 log CFU/coupon where the ATP device could no longer detect ATP from L. innocua. The type of leafy green on a food-contact surface did not affect the ATP reading (P=0.88). Leafy greens with L. innocua had a higher ATP reading when compared to saline and L. innocua, demonstrating the presence of leafy green matter contributes to ATP reading when combined with L. innocua. The different food-contact surfaces had different ATP readings (P=0.03) and the ATP device did not detect bacterial or leafy green ATP from bamboo wood surfaces (P=0.16). Based upon our results, ATP measurement is an appropriate tool to measure produce or bacterial contamination on stainless steel or HDPE plastic surfaces, however it is not recommended for wood surfaces.

The fresh-cut produce industry has seen expansive growth in recent years, to meet consumer demand ready-to-eat (RTE) salads have included the use of non-traditional ingredients. Uncommon ingredients include beet greens, kale, broccoli stalk, and Brussels sprouts, since these ingredients have not historically been consumed raw, potential food safety issues should be reassessed. Current processing technologies include produce washes that can reduce microbial levels but do not eradicate all populations. The lack of a kill step in produce processing emphasizes the need to minimize pathogen contamination during production and growth during a product’s shelf life. Listeria monocytogenes, a leading cause of foodborne illness related deaths, continues to challenge the industry with recent outbreaks and recalls of fresh-cut produce. These events present the need to better understand L. monocytogenes growth potential in RTE produce during storage and distribution. Traditional salad greens have been researched extensively, however, limited knowledge is available on new inclusions. While temperature is known to strongly influence microbial growth, the effects of physical abuse during storage and distribution are unknown. The purpose of this study was to characterize L. innocua’s, a surrogate for L. monocytogenes, growth behavior in processed beet greens, kale, broccoli stalk, and Brussels sprouts when exposed to simulated physical and thermal abuses during storage and distribution. To evaluate L. innocua growth during storage and distribution produce samples were obtained from a local processor in retail packaging and surface inoculated. The samples were conditioned at 4℃ for 18h prior to being exposed to a series of physical abuses (compression, drop, and vibration) typical of storage and distribution. After abuse, produce was incubated at 4 or 8°C and sampled post-abuse through 16 and 11 days, respectively. Samples were enumerated for L. innocua, aerobic and psychrotrophic microorganisms, and lactic acid bacteria. To monitor growing conditions in each vegetable, product pH, water activity, and headspace (gas analysis), were observed at each time pull. The study found physical abuse had no significant effect on L. innocua, or microbiota growth regardless of vegetable or incubation temperature (P > 0.05). Vegetable intrinsic factors (pH, Aw, and headspace) did not seem to interfere in L. innocua or background microbiota growth during incubation. All vegetables supported L. innocua growth under 8℃. Growth of L. innocua was greatest in beet greens, followed by kale, broccoli stalk, and Brussels sprouts in descending order. Significant growth of L. innocua at 4 and 8ᵒC was seen on day 6 and 4 in beet greens, 11 and 6 in Brussels sprouts, 16 and 4 in kale, and 16 and 6 in broccoli stalk (P < 0.05). Overall, these results show the studied RTE vegetables can support L. monocytogenes growth during storage and distribution, especially under abusive temperatures, demonstrating the importance of prevention strategies during processing and refrigeration throughout RTE produce shelf life.

Hard cider is an alcoholic drink made from fermented crushed fruit, typically apples. The popularity of this fermented alcoholic beverage has been on the rise within the last decade. Historically, hard cider has been deemed safe due to the presence of ethanol and the low pH. Although there is lack of scientific evidence to prove that hard cider will and can be safe from foodborne pathogens. Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes are three predominate foodborne bacterial pathogens of concern in the food and beverage industry. Escherichia coli O157:H7 in particular has been associated with fresh produce and more specifically apples, and apple products such as apple juice. The purpose of this study was to determine the bactericidal effects of pH, ethanol, and malic acid on Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes to evaluate the safety parameters for safe hard cider production and storage. The fate of foodborne pathogens in cider was determined during hard cider fermentation, in a cider model system, and in commercial cider. Escherichia coli O157:H7, Salmonella spp, and Listeria monocytogenes did not survive a 5-day fermentation period resulting in a > 7 log CFU/mL reduction of each pathogen with no significant change in pH. The final ABV of the cider at the end of the 5-day fermentation was 4.4%. In the cider model system, the lower the pH and higher the ABV the quicker die off was observed, at pH 2.8, 3.0, 3.2, and 3.4 with 7, 8, and 9% ethanol concentration there was a 6.6 log reduction in E. coli O157:H7 population after 1 day. By the 7-day incubation period, no pathogens were detected at all pH and ABV combinations except for at pH 3.6 and 3.8 with 4% ethanol having ≤0.6 log CFU/mL of the population surviving. Similar E. coli O157:H7 inactivation patterns were observed in the model system and in the commercial ciders. The six commercial ciders observed had varying pH, ABV (%), and malic acid concentrations but successfully resulted in a > 6 log CFU/mL reduction in population of E. coli O157:H7 within 4 days of incubation. The ciders with the highest ABV’s, 8.7 and 9.6% observed a > 6 log reduction by 1 day. It was observed that at some point in time pH plays a bigger role in the presence of less ethanol, but it is clear that ethanol and pH work synergistically to kill of pathogens present in cider fermentation, a cider model, and commercial cider.

The FSMA Produce Safety Rule (PSR) requires citrus packers to more closely assess, manage, and monitor food safety risks. Although there have been no foodborne illness outbreaks and only one recall in fresh citrus, the risk of pathogens coming in on the fruit and cross contamination during washing still exists. Packhouses have dynamic washing systems and in-plant validations may be the only way to demonstrate compliance with the PSR. In-plant validations use surrogates in place of pathogens, and none have been identified or validated for citrus. The aim of this research was to identify a surrogate for use in fresh citrus packhouses. Potential surrogates were screened for free chlorine resistance, survival under commercial storage conditions, and shedding and attachment characteristics during simulated washing. E. faecium NRRL B-2354 and P. pentosaceus NRRL B-14009 were selected for further study. Resistance to chlorine was not significantly different between E. faecium and L. monocytogenes FSL J1-031 when exposed to 3 ppm free chlorine for 30, 60, 90, and 120 s at 20 and 100 ppm TSB (pE. faecium and P. pentosaceus behavior was significantly different than L. monocytogenes (p=0.05), indicating that neither is a suitable surrogate. In shedding and attachment, either the fruit (shedding) or water (attachment) was inoculated, washing was simulated, and organisms were enumerated from the water (shedding) or fruit (attachment). Both potential surrogates were statistically different than L. monocytogenes (pE. faecium can be used for L. monocytogenes shedding estimates and E. faecium and P. pentosaceus can be used for attachment estimates. Overall, this research suggests that E. faecium NRRL B-2354 can be considered as a surrogate for L. monocytogenes in whole, fresh citrus validation studies on chlorinated washes and – with appropriate adjustments – on shedding and attachment characteristics.