Optimising pre-incubation time and comparing sterility methods for the detection of thermophilic bacteria in UHT dairy products : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (Biological Sciences) at Massey University, Manawatū, New Zealand

Abstract

Globally, UHT milk and UHT dairy products are an important source of nutrition and therefore have the requirement to be safe for consumption. Commercial sterility testing of these products ensures their safety and reduces reputational/financial losses for the dairy companies. However, temperature abuse during storage and transport has been highlighted as a concern due to potential spoilage effects from possible thermophilic contamination. Traditional commercial sterility testing methods, established by governing bodies, are time-consuming and labour-intensive, and they are not within the boundary of rapid food testing. This study compared two rapid methods: the commercially available Charm Epic ATP bioluminescence method and a flow cytometry method (newly developed as part of this study), with the traditional plate method, for assessing the commercial sterility of UHT dairy products when contaminated with low numbers of obligate thermophiles. Four different UHT product types were investigated (milk, in-house cream, whipping cream and a medical beverage). In addition, the effect of shortening the UHT pack pre-incubation time on method performance was investigated. For instance, pre-incubation times of 6-24 hours were compared with 48 hours. It was found that a pre-incubation time of 48 hours had only 50% agreement when comparing the ATP bioluminescence method with the plate reference method when measuring UHT milk. Whereas pre-incubation times of 6-24 hours had >95% agreement, thus greatly improving the performance of the ATP bioluminescence method. With UHT milk, the flow cytometry method had >95% agreement with the plate method at all pre-incubation times tested, however overall, the readings tended to be highest at 24 hours, indicating that a 24 hour pre-incubation time would have the lowest chance of a false negative result. Neither the ATP bioluminescence nor the flow cytometry method had acceptable agreement (i.e. ≥95%) with plate counting when measuring the in-house cream or medical beverage products, regardless of the pre-incubation time. Thus, it was concluded that the plate count method would be most appropriate for these matrices. Given these findings, an adjusted approach was applied to improve the performance of the flow cytometry method when analysing the whipping cream product. Firstly, additional sample preparation was used which involved mixing samples with a cation chelator, followed by centrifugation to obtain a bacterial pellet, assisting the extraction of the bacteria from the matrix. Secondly, the gate to capture the live bacterial cells on the flow cytometry plots was positioned further away from the non-bacterial background particles. These two approaches prevented the spillover of non-bacterial particles into the live gate, thereby preventing false positive results, and improving method sensitivity. This resulted in >95% agreement between the flow cytometry method and the plate method, at all pre-incubation times tested with the whipping cream. Furthermore, this contrasted results with the ATP bioluminescence method, where the highest agreement achieved was 44%, with the whipping cream. As UHT milk is the most common type of UHT dairy product, the shorter pre-incubation time in commercial sterility testing identified in this study would be highly beneficial to dairy companies, particularly when using methods alternative to the plate method. Not only would a shorter pre incubation time have potential to improve the performance of the method, but it would also allow for a faster turnaround time. There needs to be recognition of the rapid transition into the death phase of the thermophilic bacterial growth cycle, as this has potential to impact rapid testing methods, such as those that measure ATP. These findings may be adopted into industry standards and regulations in the future.