Enumeration and discrimination of thermophilic bacilli in milk powder
Rückert, A. (2005). Enumeration and discrimination of thermophilic bacilli in milk powder (Thesis, Doctor of Philosophy (PhD)). The University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/13206
Permanent Research Commons link: https://hdl.handle.net/10289/13206
A survey of milk powders from eighteen different countries has established that the same cohorts of strains of thermophilic bacilli are the major contaminants regardless of source. In particular, Anoxybacillus flavithermus strain C emerges as the most commonly encountered contaminant especially in powders with a medium- to high thermophile count. Bacillus licheniformis strain F was the next most common contaminant overall which dominated low- to medium count powders. This organism was also the broadest distributed being present in 96% of the milk powders screened. The thermophilic Geobacillus stearothermophilus strain A represented the third most often occurring milk powder isolate. The presence of B. licheniformis strain G, Bacillus subtilis and A. flavithermus strain D as low-level contaminants of milk powder was reconfirmed. Methods have been developed for the rapid and efficient extraction of bacteria from milk powder and their enumeration using real-time PCR methodology based on the 16S rRNA gene or the spoOA gene. Further modifications to these methods allow discrimination between live and dead cells and between spores and vegetative cells. The former is important because the majority of vegetative cells in milk powder are dead as a result of processing stresses, yet their DNA remains available for amplification. Limits of detection of these methods for viable cells and spores are less than 1000 thermophiles per gram of milk powder. These methods can yield results within a time period of 90 minutes and thus are amenable to real-time monitoring of factory contamination in situ. A range of other methodologies and approaches to detecte and enumerate thermophilic bacilli were investigated but were not regarded as applicable. The use of antibodies raised against thermophilic bacilli did not produce sufficient discrimination between strains and did not allow enumeration of different strains with the same efficiency of detection, and could not differentiate between live and dead cells. DGGE-PCR performed on the highly conserved region of the 16S rRNA gene also exposed the presence of organisms other than thermophilic bacilli in milk powder compromising the resolution of the detection method. RAPD-PCR provided a high discrimination between the strains but was not applicable for the use in a factory setting. Overall, the discovery that virtually all milk powders produced internationally and in New Zealand are dominated by thermophilic strains representing four species is a fundamental finding regarding factory-derived contamination. This will have long-term effects on the future operation and design of evaporator lines, and has important economic implications with respect to optimizing day-to-day factory operation and storage of milk powders. In addition, the quantitative real-time PCR assays from this study should allow for a robust control of the drying process during extended run times and thus, are of important economical benefices to milk powder manufacturer.
The University of Waikato
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