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    The spore formation and toxin production in biofilms of Bacillus cereus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Huang, Yiying
    Bacillus cereus (B. cereus) is a foodborne pathogen causing diarrhoea and emesis which are the consequences of enterotoxin and emetic toxin production, respectively. Sporulation and biofilm formation are used as survival strategies by B. cereus protecting cells from harsh environments. However, these survival strategies also make B. cereus more difficult to control in the food industry. The aim of this study is to investigate the spore formation and toxin production in the biofilm of B. cereus. In this study, higher sporulation and higher spore heat resistance were demonstrated in biofilms grown on stainless-steel (SS) compared to planktonic populations. The structure of coat in spores isolated from biofilms, the upregulated germination genes in planktonic cells and upregulated sigma factor B in biofilm cells are possible explanations for these observations. The levels of dipicolinic acid (DPA) did not affect the heat resistance of spores harvested from biofilms in this study. Haemolytic toxin (Hbl) was mainly secreted by cells into surrounding media while emetic toxin (cereulide) was associated with cells. Higher Hbl toxin was observed in the presence of biofilms grown on SS compared to either planktonic culture or biofilm grown on glass wool (GW) using the Bacillus cereus Enterotoxin Reverses Passive Latex Agglutination test (BCET-RPLA). This was supported by the significant (P < 0.05) increase in HblACD expression in biofilm cells on SS, using both real-time quantitative PCR (RT-qPCR) and RNA sequencing. The transcriptomic analysis also revealed that biofilms grown on SS had an upregulated secretion pathway, suggesting biofilms of B. cereus grown on SS are more pathogenic than planktonic cells. Unlike the Hbl toxin, cereulide was associated with biofilm cells/structures and attached to the biofilm-forming substrates including SS and GW used in this study. The expression of cerA and cerB was similar between biofilms and planktonic cells using RT-qPCR. This project highlights the importance of biofilms by B. cereus in food safety through the enhanced heat resistance of spores, the higher Hbl toxin production and attached cereulide toxin.
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    Analysis of dairy cattle feed as source of heat resistant bacterial spores in milk and evaluation of contamination consequences for milk quality : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Flossdorf, David
    Spore-forming bacteria are resistant to heat treatments designed to control the growth of other bacteria and can impact the quality of milk. Bacterial spores in milk are hypothesised to originate from cattle feed, among other sources. In recent decades, supplementary feed usage and variability of used feed types has increased in New Zealand. To ensure low numbers of bacterial spores in dairy products, the number of spores in common New Zealand dairy cattle feed and milk were observed at psychotropic, mesophilic and thermophilic temperatures under aerobic and anaerobic conditions and the bacterial spore species found were identified. Differences were detected in the number of bacterial spores and variety of species present, between sample types. Tuber feed and palm kernel expeller (PKE) contained the greatest quantity of spores as well as the greatest diversity of bacterial spore species, while milk contained the lowest quantity and diversity. This supports a hypothesis that some cattle feeds are more likely to be a source of milk contamination than others. Almost all (96 %) of bacterial spore species found in milk were also found in cattle feed, indicating that spores may transfer from feed to milk. To provide more definitive evidence of the transfer of bacterial spores from cattle feed to milk, the genomes of 109 bacterial isolates from milk and feed were sequenced and compared. Clusters of similar genomes were found between milk and feed isolates, however within the set criteria of the study, no match was found. A greater genomic diversity was observed in cattle feed compared to milk. A lower genomic diversity was observed among Bacillus licheniformis and B. pumilus isolates compared to isolates from B. cereus, B. mycoides or B. thermoamylovorans. Not all spore-forming bacteria isolates cause issues in milk production therefore the potential of bacterial spores isolated from raw milk, to damage milk was determined. A set of 20 bacterial spore isolates were screened for their ability to grow in milk, form biofilms, produce milk damaging enzymes and resist heat treatment. All isolates grew in milk, almost all had some ability to produce biofilm on stainless steel and produce milk degrading enzymes. Differences in biofilm production and heat resistance of spores were found between the isolates. Out of all isolates, two B. licheniformis isolates were found to possess the greatest potential to damage milk and whole genome data was used to investigate the genes responsible for differences in the milk damaging abilities. A set of gene alleles was identified which might affect the potential of an isolate to influence milk quality. Highlights: • Determining the bacterial spore diversity in current New Zealand cattle feed and milk • The potential of comparative genomics to trace bacterial spores between cattle feed and milk • Characterisation of the milk damaging potential of common bacterial spore isolates from milk • Identification of spore-forming bacteria genes responsible for a high milk damaging potential in isolates. Limitations: • Only the most popular feeds were analysed. • Culture dependant methods were used for most of the work. • Tracking bacterial spores from feed to milk involved multiple species across multiple farms rather than a more intensive study of a single spore species on a single farm. • Milk damaging potential was assessed for only of subset of the most common isolates of bacterial spores found in milk.
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    Germination of psychrotolerant clostridia responsible for red meat spoilage : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2012) Adam, Katharine Helen
    Psychrotolerant clostridia are responsible for spoilage of fresh chilled vacuum-packed red meat (beef, lamb and venison). Red meat is one of New Zealand’s primary exports, and spoilage results in financial loss. Spoilage by psychrotolerant clostridia is difficult to control due to the ability of these bacteria to grow at cold temperatures, down to -1.5 °C. They can also form spores that have increased resistance to heat, chemicals, oxygen and desiccation compared to vegetative bacterial cells. As clostridia are strict anaerobes, it is considered highly likely that initial contamination of meat is primarily with spores. The main objective of this work was to determine the triggers of germination of spores, of those psychrophilic and psychrotrophic clostridia, associated with spoilage of New Zealand red meat. Germination of psychrotolerant clostridia was studied using a range of techniques including molecular, in vitro, and on meat methods. In this study in vitro germinant systems were identified for Clostridium frigidicarnis, and a New Zealand species designated LA1, consisting of lactate in combination with an amino acid. Some of the amino acids identified, including valine and cysteine, are naturally present on the surface of red meat. Failure to chill to, or maintain meat at, the recommended temperature, of -1.5 °C and a pH of above 5.5 were identified as being important factors leading to spoilage by Cl. frigidicarnis. Germination in Clostridium estertheticum was extremely poor in media, compared with meat slurry or fresh meat, preventing the identification of a specific germinant system(s), and indicating a non-nutrient factor may be involved. Two distinct nonchemical interventions, hot water wash (HWW) and cold water wash (CWW), were found to reduce spoilage of vacuum-packed chilled lamb inoculated with spores of Cl. estertheticum. Vegetative cells of psychrotolerant clostridia survived exposure to air longer than expected, upwards of seven days in the case of Cl. estertheticum subsp. estertheticum, suggesting that they play a greater role in initial contamination of meat than originally thought. From an industry point of view the results highlight the importance of preventing initial contamination and proper chilling, as well as the need for further investigation of HWW and CWW interventions.
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    The development of a chemical analogue of thermal destruction of bacterial spores : a thesis presented in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Massey University
    (Massey University, 1967) Packer, Gordon John Kitch
    Canning as a method of food preservation has its origin in the work of Nicolas Appert (1750-1841) who was the first to use heat as a means of preserving food in hermetically sealed containers. Although he did not understand the principles of his method, his systematic experimentation (and generous sharing of his discoveries) laid the foundations of thermal food preservation methods. Appert initially processed his food in cork sealed glass jars and bottles in boiling water baths for periods varying from 15 minutes to two and a half hours. Storage trials were the basis of his processing methods, some foods being kept up to ten years. His products ranged from meats, soups and vegetables to fruit and even cream and evaporated milk. He recognized the value of quick clean handling of good quality raw materials. Blanching was used with some products, and he was also aware of the distinction between acid and low-acid foods in regard to their length of processing. Appert's understanding of the process was that heating eliminated the "air" which was believed to be the cause of spoilage. This belief was to persist for nearly 100 years. Appert's work in glass containers led to the development in England about 1815-20 of tin containers for preserved foods. Appert himself used cans in some of his later work. Some time before 1830 the autoclave was introduced (apparently by Appert) as a means of cooking canned foods under pressure. By 1870, autoclaves were being used quite widely in industrial canning.