Graduate Thesis Or Dissertation
 

Inactivation of Clostridium perfringens in Meat Products

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  • Clostridium perfringens type A strains are one of the main causative agents of gastrointestinal (GI) diseases in human and can cause both food poisoning (FP) and non-food-borne (NFB) diseases. Several factors contribute to the pathogenesis of C. perfringens type A strains including the wide presence of C. perfringens spores with highly resistant properties, production of C. perfringens enterotoxin (CPE), and the high growth rate of the bacterium. In addition to these intrinsic virulence factors, other external factors such as inadequate cooking, improper cooling, and abusive storage temperature of meat and poultry products can contribute to the majority of C. perfringens type A food poisoning outbreaks. The risk of contracting food-borne illnesses is usually reduced by commonly used food preservation method such as thermal processing. However, inactivating extremely heat-resistant spores is a major challenge and, thus, alternative strategies to conventional thermal processing technologies are in urgent demand. In the first study, the effectiveness of potassium sorbate and sodium benzoate were evaluated as growth inhibitors of vegetative cells and spores of C. perfringens type A FP and NFB isolates, in both laboratory medium and chicken meat model system. Our study showed that although the permissive levels of sorbate and benzoate used in food products did not affect spore germination, the outgrowth of germinated spores was effectively arrested. Lowering the pH of the medium increased the inhibitory effects of sorbate and benzoate against germination of spores of NFB isolates, and outgrowth of spores of both FP and NFB isolates. Furthermore, sorbate and benzoate inhibited vegetative growth of C. perfringens isolates. Although sorbate and benzoate showed inhibitory activities against C. perfringens in the rich medium, no such effect was observed in cooked chicken meat stored under extremely abusive conditions. Therefore, caution should be taken when applying these organic salts to meat products to reduce or eliminate C. perfringens spores. In the second study, the natural polymer chitosan with low, medium, and high molecular weight was examined to assess its inhibitory effect against C. perfringens FP isolates in a variety of aspects including spore germination, outgrowth, and vegetative growth in laboratory medium or chicken meat model system. Chitosan with all three different molecular weights at concentration of 0.1 mg/ml at pH 4.5 could inhibit the germination of spores of FP isolates. However, higher level (0.25 mg/ml) of chitosan was required to effectively arrest outgrowth of the germinated C. perfringens spores in rich medium. Furthermore, chitosan concentration of 1.0 mg/ml showed bacteriostatic activity against vegetative cells of C. perfringens strains in rich medium. Although chitosan showed strong inhibitory activities against C. perfringens in laboratory medium, higher levels were required to achieve similar levels of inhibition for C. perfringens spores inoculated into chicken meat. Thus, from this study, it has been shown that the inhibitory effects of chitosan against spore germination, outgrowth, and vegetative growth was concentration dependent, and no major differences in effects of different molecular weights of chitosan were observed. Our results also contribute to a better understanding on the potential application of chitosan in cooked meat products to control C. perfringens contamination. Bacterial spore germination is an essential step for the spores to restore their metabolic activities and lose their resistance properties. C. perfringens vegetative cells are easier to kill than the spore form. C. perfringens spores can initiate germination upon sensing a variety of compounds, termed germinants, via their cognate germinant receptors (GRs). In the third study, we identified 12 individual amino acids (aa) that triggered the germination of spores of C. perfringens NFB isolates in the presence of bicarbonate buffer. Unlike C. perfringens NFB isolates, spores of C. perfringens FP strain SM101 required potassium ions to germinate with all these amino acids. Surveying germination of spores of NFB isolates lacking one of the GR proteins with the newly identified aa germinants revealed that GerKC and GerAA play major roles in bicarbonate-aa germination. Furthermore, this study also supports the correlation of germination requirement of C. perfringens FP and NFB isolates and their environmental niches. Potassium ions are abundant in food environment and, thus, are required for FP spores to germinate. However, bicarbonate is a specific intestinal element and is essential for germination of NFB spores. In summary, these results should help developing a new spore-inactivation strategy where spore germination will be induced by aa germinants followed by mild chemical treatments. Collectively, this dissertation reports experimental results relevant to different inactivation approaches to control spores and vegetative cells of C. perfringens FP and NFB isolates in laboratory medium and chicken meat model system. These approaches include the incorporation of well-known GRAS-listed antimicrobial agents to food formulation, and the identification of nutrient compounds to trigger C. perfringens spore germination, which will allow spore-killing with mild treatments. Combination of these approaches should be effective to control the risk of C. perfringens associated food-borne and non-food-borne illnesses.
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  • 2017-08-04 to 2018-12-22

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