The objective of this project was to evaluate various culturing and isolation techniques of Campylobacter jejuni and to develop methods to detect the organism in foods. The morphological, cultural and biochemical characteristics of C. jejuni were studied using developed microbiological methods. A variety of media, broths, microaerophilic atmospheres and diluents, now available, were tested for their applicability to detect low numbers of the organism in food samples. Direct plating, filtration, double incubation enrichment, milk separation enrichment and swabbing methods were used to recover C. jejuni from seeded milk and fowl samples. As few as 16 organisms per ml of milk could be recovered using the double incubation enrichment. Raw milk samples from retail supermarkets and the Oregon State University Dairy Herd were tested for the presence of C. jejuni with the double incubation enrichment. No positive confirmation of the organism was made, although suspect microorganisms were observed microscopically. The survival of C. jejuni in foods and effect of sanitizers was studied. Raw and underprocessed foods pose the greatest risks as vehicles of Campylobacter infections. If contaminated foods are held at refrigeration temperatures C. jejuni could survive. Properly sanitized dairy equipment poses no apparent health problem and water should have a residual chlorine level of greater than 5 ppm to be safe. / Graduation date: 1982

Probiotics die over time during processing, storage and digestion, resulting in reduced health benefits to the consumer. Microencapsulation of microorganisms is an effective way to improve probiotic viability by restricting cell exposure to extreme conditions through the gastrointestinal tract until release in the colon. In this work, appearance and survival of encapsulated probiotic species from two genera was explored. Lactococcus lactis and Bifidobacterium longum were suspended in calcium alginate microbeads by spraying droplets of alginate-probiotic mixture into calcium chloride solution. This produced uniformly shaped transparent microbeads with high encapsulation yield. Encapsulating Lactococcus lactis extended viability during dry room temperature storage. Encapsulating Bifidobacterium longum revealed high variation between eight different strains from subspecies longum and infantis. Coating alginate particles with chitosan did not improve viability and, viability of free and encapsulated bifidobacteria decreased when exposed to simulated gastric and intestinal conditions. Data from these studies suggest microencapsulating probiotic cells is an invaluable process to extending cell viability. Future research should optimize current formulations to improve encapsulation yield and cell survival during processing, storage, and gastrointestinal transit.

Recent increases in chronic cardiovascular diseases, such as hypertension, have put pressure on the food industry to reduce sodium levels. Dairy products, though full of vital nutrients, are perceived as being high in sodium. However, the reduction of salt in dairy products could potentially alter the microbial stability, as well as cause unfavorable changes in flavor. In order to reduce the sodium level, while maintaining acceptable flavor and microbial stability, salt replacers and alternative antimicrobial agents may need to be introduced into the food matrix. To identify potential antimicrobials for use in reduced sodium dairy products, this study evaluated the efficacy of eight commercially available antimicrobials in TSA, milk agar, and cheese agar. Antimicrobials included MicroGard 100, MicroGard 430, Nisaplin, NovaGard CB1, Protect-M, PuraQ Verdad RV75, SEA-i F75 and VMY1P. Antimicrobials were also tested in combination with six commercial sodium reduction agents (potassium chloride, Puracal PP/USP, Purasal Hi Pure P Plus, PuraQ Verdad NV10, SaltWise 0029 and SaltWise 1029) to if there were any interference with antimicrobial activity. Antimicrobials with and without sodium reduction agents were added to the agar systems, then a five-strain cocktail of Listeria monocytogenes, Salmonella or Escherichia coli O157:H7 was spread plated at three concentrations: 101, 102 and 104 CFU/plate. Samples were then incubated at 35°C and observed for growth after 24 and 48h. SEA-i F75 was the most effective antimicrobial in each of the agars tested. Additionally, no interactions were observed between SEA-i F75 and any of the sodium replacement agents. SEA-i F75 was selected for use in a final challenge study using six formulations of LMPS mozzarella cheese: regular sodium control cheese (1.7% NaCl, no antimicrobial added); low sodium control cheese (0.7% NaCl, no antimicrobial added); low sodium treated cheese (0.7% NaCl, treatment with SEA-i F75); low sodium cheese with KCl as salt replacer (0.7% NaCl, 1.0% KCl, treatment with SEA-i F75); low sodium cheese with Alta 2345 as salt replacer (0.7% NaCl, 0.25% Alta 2345, treatment with SEA-i F75); and low sodium cheese with Salona as salt replacer (0.7% NaCl, 0.95% Salona, treatment with SEA-i F75). Fifteen gram cheese pieces from each formulation were dipped in an antimicrobial solution containing 0.25% SEA-i F75 then inoculated with L. monocytogenes, Salmonella, or E. coli O157:H7 at a target inoculum concentration of 102-103 CFU/g and incubated at either 4° or 12°C. In all trials, over all formulations and temperatures tested, initial decreases in counts, followed by organism recovery were observed. Therefore, SEA-i F75 was not effective at reducing the counts of pathogenic bacteria in LMPS mozzarella cheese. Results from this study highlight the effect of the food matrix, and its components on antimicrobial efficacy. Future research includes examining the effect of one of the other antimicrobials in LMPS mozzarella cheese.

Campylobacter jejuni is a prominent food-borne pathogen and causative agent of Campylobacteriosis infection. This infection arises primarily from the consumption of foodstuffs/beverages previously contaminated with this microorganism. Despite current food control measures currently employed, this pathogen remains problematic despite its fastidious nature. To help control the spread of Campylobacter many hurdles are used in the food industry, including natural products such as modified atmosphere and salt. Other natural antimicrobials are shown to possess antibacterial activity. Chitosan, a natural antimicrobial, has promising uses in food production. However, little is known about the transcriptional differences of C. jejuni upon chitosan exposure. Molecular responses may allow for generation of adaptive responses in an attempt to combat such a stressor, possibly altering phenotypic and virulence potential. The aims of this study were to establish the adaptive response of C. jejuni to low molecular weight chitosan by assessing the phenotypic and molecular differences arising from the chitosan challenge. By exposing C. jejuni NTCT11168 to suboptimal levels of chitosan, based on MIC determination on a model system, an adaptive strain of C. jejuni has been developed. This newly developed isolate, referred to as the ‘adapted’ C. jejuni NCTC11168 was found to have enhanced antimicrobial tolerance to chitosan, with a 3.83 fold increase in MIC relative to the parental wild-type C. jejuni NCTC11168 cells (0.012% - 0.046% (w/v) respectively). Antimicrobial activity of chitosan was found to be pH dependant. Differences in motility were also apparent between the ‘adapted’ and wild-type strains. Consistent increases in motility were noted in the ‘adapted’ cells, especially in relation to wild-type after 24, 48 and 72 hours (p < 0.05). Biofilm formation was also enhanced especially in the ‘adapted’ isolate relative to the wild-type NCTC11168 cells. This was indicated by significant increases, most notably after 3 days formation (p < 0.05). Microarray analysis revealed significant alterations in transcriptional levels in response to chitosan exposure. Protein-encoding genes, believed to be associated with energy metabolism, solute/ion uptake/acquisition and efflux systems, were found to be differentially regulated when grown in the presence of low molecular weight chitosan (p < 0.05). Overlapping between initial comparisons revealed several genes, which were differentially regulated. Most notably, PEB3 (major antigenic peptide), Cj0017c (disulphide bond formation) and Cj0294 (thiamine biosynthesis) genes were found to be up regulated in ‘adapted’ C. jejuni cells when compared to the wild-type (p < 0.05). Whilst significant down regulation was found in the protein-coding genes, Cj0025c (putative sodium:dicarboxylate symporter) and Cj1608 (two-component regulator). These results indicate that regulation of these genes may contribute to enhanced phenotypic responses observed in the ‘adapted’ isolate of C. jejuni NCTC11168. These findings are likely to account for increased growth and survival of ‘adapted’ C. jejuni NCTC11168 when challenged to this antimicrobial, when compared to the wild-type. The findings in this study provide useful information as to how C. jejuni NCTC11168 can develop an adaptive tolerance response to chitosan and that alterations in transcriptional aspects may facilitate this, contributing to an enhanced phenotypic response and virulence potential.

Compatibility of mixed cultures of Streptococcus lactis and Streptococcus cremoris we e studied by the phage tracer technique in 10% nonfat dry milk and a whey-based phage inhibitory medium. Methods of membrane dialysis and differential enumeration were tried and abandoned. Dominance occurred in nonfat dry milk and in the whey-based medium during a continuous fermentation period within a pH range 6.8 to 5.2. The dominance pattern of two-strain combinations in 10% nonfat dry milk was different from that in the whey-based media. Dominance varied from one strain to another in no consistent sequence in some combinations. Dominance patterns of combinations were compared with patterns of stimulation and inhibition.

No description available.

Graduation date: 1958

Graduation date: 1972

The metachromatic agar-diffusion test of Lachica, Hoeprich, and Genigeorgis (1971) was modified by adding 17 ml of the agar medium to a 100 by 15 mm plastic petri dish, using 5 µl samples and 3 mm wells. The test detected 0.5 ng/ml of thermonuclease in 3 hr at 37 C. This was equivalent to 106 to 10 7 cells/ml of Staphylococcus aureus strains 196 E, 14458, and 19095 grown in Brain Heart Infusion (BHI) broth. The test sensitivity was decreased ten fold in raw and pasteurized milk. One-half gram of cheese was dissolved in 4.5 ml of 0.1 M sodium citrate for quantitative studies. When staphylococcal (>8 x 10 /ml) contaminated raw milk was pasteurized and made into cheese, more thermonuclease activity/ml was noted in the curd than whey. During prolonged storage at 4 C, thermonuclease activity decreased in raw and pasteurized milk and laboratory made cheese. Bacillus subtilus, Streptococcus faecalis var liquefaciens, Streptococcus thermophilus, and Streptococcus cremoris strain ML8 reduced thermonuclease activity during growth incubation in BHI broth. Thermonuclease was detected directly in staphylococcal contaminated 7 laboratory aged cheese (>10 /gm) without prior purification, concentration, or extraction procedures.

The multibillion dollar agricultural industry is an important part of the United States economy, and the management of factors that affect crop and human health is imperative to maintaining this economic sector. The fungi Botrytis cinerea, Fusarium pallidoroseum, and Fusarium moniliforme are the causative agents of several plant diseases and can cause significant crop loss both before and after harvest in commodities such as strawberries, lettuce, citrus, and grains. Fungicides are employed to control these phytopathogens, but the use of these chemicals has led to an increase in fungicide resistance and may negatively affect the environment and human health. In addition to plant pathogens, foodborne pathogens also have a substantial impact on the agricultural industry. Foodborne disease outbreaks involving Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7 not only cause considerable economic losses, but can also result in devastating health problems for consumers. The increase in fungicide resistance and number of produce-related foodborne disease outbreaks warrants investigation into additional methods of microbial control for use in the agricultural industry. Many bacterial species, including Lactic Acid Bacteria (LAB) and Bacillus species, produce antifungal and antimicrobial compounds, thus the use of biological control agents pre- and postharvest could augment current methods of pathogen management. The purpose of this study was to screen 22 bacterial isolates for inhibitory activity against the fungal phytopathogens Botrytis cinerea, Fusarium pallidoroseum, and Fusarium moniliforme and the foodborne pathogens Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7 in vitro, then evaluate antimicrobial efficacy of select isolates against the foodborne pathogens on fresh produce. To evaluate antifungal activity, the bacterial isolates were individually spot-inoculated onto Tryptic Soy Agar, Potato Dextrose Agar, or MRS agar, depending on isolate growth requirements and then a plug of fungal-colonized agar was placed onto the center of the isolate-inoculated plate. Plates were incubated at 24°C for 10 days; fungal growth was evaluated daily, beginning on Day 3. Nine of the 22 isolates screened inhibited all three fungi; inhibition by these isolates ranged from 51-62% for B. cinerea, 60-68% for F. pallidoroseum, and 40-61% for F. moniliforme. Isolates were also screened for biosurfactant activity using the drop-collapse test. Biosurfactant production was detected in seven of the nine isolates. Bacillus megaterium, Bacillus coagulans, Bacillus thuringiensis BT2 and three Bacillus amyloliquefaciens isolates demonstrated strong biosurfactant activity and suppression of all three fungi, and therefore are recommended for further study. Antimicrobial activity of the isolates was assessed using two methods: LAB isolates were screened using a seeded-overlay method and all other isolates were evaluated by spot inoculating the isolate on pathogen-seeded TSA. Three LAB isolates and six Bacillus isolates suppressed L. monocytogenes, Salmonella, and E. coli O157:H7 in vitro. Based on the results of the screening, three LAB isolates—Lactobacillus plantarum, Pediococcus acidilactici, and Pediococcus pentosaceus—were selected for further evaluation and use in challenge studies on fresh produce. The role of organic acids in pathogen inhibition was evaluated by incubating L. monocytogenes, Salmonella, and E. coli O157:H7 cultures in the cell-free supernatant (CFS; pH 3.81-4.27) or the neutralized cell-free supernatant (pH adjusted to 6.5 -7.0) of each isolate. When neutralized, the antimicrobial activity of the CFS of the three LAB isolates was greatly diminished, illustrating the role of lactic acid in the inhibition of pathogen growth. To assess antimicrobial efficacy on Iceberg lettuce, a cocktail of the three LAB isolates (7-8 log CFU/g) was sprayed onto lettuce spot-inoculated with L. monocytogenes (2-3 log CFU/g); lettuce was incubated at 10°C for 14 d. L. monocytogenes levels were 1.84 log lower on LAB-treated lettuce than on untreated lettuce at the end of incubation. Because the LAB cocktail suppressed the growth of L. monocytogenes on lettuce, testing on fresh produce continued using DF1, which was a powdered product comprised of the three LAB isolates and media components. Because DF1 caused substantial browning of Iceberg lettuce after 2 d, Gala apples were chosen to evaluate the antimicrobial activity of DF1 against L. monocytogenes, Salmonella, and E. coli O157:H7. The effect of DF1 on L. monocytogenes, Salmonella, and E. coli O157:H7 on Gala apples was determined by spraying a Gala apple spot-inoculated with pathogen (6-7 log CFU/plug) with approximately 3 mL of a 20% DF1 solution, then incubating at 20°C for 5 d. After 5 d incubation, L. monocytogenes, Salmonella, and E. coli O157:H7 levels on DF1-treated apples were approximately 4, 2, and 2 log higher than the control, respectively. Based on the results of these experiments, DF1 is not the optimal formulation for the biocontrol of foodborne pathogens on fresh produce. This study identified several bacterial isolates with potential for use in the biocontrol of plant and foodborne pathogens. Further investigation is required to assess possible use in the agricultural industry, including characterization of bioactive compounds, optimization of biocontrol product formulation, and evaluation of the commercial viability of the biocontrol product