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Aims:  To determine the resistance of a variety of Bacillus species spores to a combined high pressure and heat treatment; and to determine the affect of varying sporulation and treatment conditions on the level of inactivation achieved. Methods and Results:  Spores from eight Bacillus species (40 isolates) were high pressure–heat treated at 600 MPa, 1 min, initial temperature 72°C. The level of inactivation was broad (no inactivation to 6 log10 spores ml−1 reduction) and it varied within species. Different sporulation agar, high pressure equipment and pressure‐transmitting fluid significantly affected the response of some isolates. Varying the initial treatment temperature (75, 85 or 95°C) shifted the relative order of isolate high pressure–heat resistance. Conclusions:  The response of Bacillus spores to combined high pressure–heat treatment is variable and can be attributed to both intrinsic and extrinsic factors. The combined process resulted in a high level of spore inactivation for several Bacillus species and is a potential alternative treatment to traditional heat‐only processes. Significance and Impact of the Study:  Sporulation conditions, processing conditions and treatment temperature all affect the response of Bacillus spores to the combined treatment of high pressure and heat. High levels of spore inactivation can be achieved but the response is variable both within and between species.

The Probelia™Salmonella sp. PCR amplification and detection kits (Sanofi Diagnostics Pasteur, Marnes La Coquette, France) were evaluated for the rapid and specific detection of Salmonella agona artificially inoculated into skim milk powder and ricotta cheese. The Probelia™ results were compared with those obtained using the Australian Standard Method. Using a pure culture of Salm. agona, the detection limit of Probelia™ was between 8 and 79 cfu ml−1, equivalent to 0·2–2 cfu per PCR reaction. Detection of Salm. agona inoculated in skim milk powder (at 5–10 cfu g−1, stored at 5, 15 or 25 °C) and ricotta cheese (at 1–2, 10–20 and 100–200 cfu per 25 g) was effected by using non‐selective enrichment prior to the PCR determinations. For all of the 40 milk powder samples and 12 ricotta cheese samples, the Probelia™ results were consistent with those using the Australian Standard Method. Using Probelia™, Salmonella was detected to genus level in the dairy products within 24–28 h, whereas the cultural technique required 3–4 d for presumptive positive isolates and further time for confirmation.

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During growth in raw milk, many psychrotrophic bacteria produce proteases that can retain activity following ultra‐high temperature (UHT) treatment. In this study, casein and skim milk powder assays for detecting very low levels of protease in UHT milk were optimized, and the suitability of azocasein and fluorescein isothiocyanate‐casein (FITC‐casein) as substrates was investigated. The strongest correlations of protease activity with proteolysis in stored UHT milk were observed when FITC‐casein was used as substrate in the assays. Assays using casein and FITC‐casein as substrates yielded the highest activities. To determine sensitivity, crude protease was added at low concentrations to UHT milk, and the milk was assayed for progress of proteolysis over 12 months and for protease activity using the casein and FITC‐casein assays. With long assay incubation times, the FITC‐casein assay was more sensitive than the casein assay and may be suitable for detecting very low levels of protease activity and predicting progress of proteolysis in stored UHT whole milk. PRACTICAL APPLICATIONS This study contributes to the development and evaluation of practical assays for the detection of protease activity in the industry to identify potential premature spoilage of contaminated UHT milk before it is distributed for sale. The developed assays are also useful for assessing the quality of milk powder as active protease can persist in milk powder to cause spoilage in reconstituted milk. Although the assays require up to 14 days to complete, this is not an excessive time, compared with the time required for microbiological clearance and total shelf life of the product. High protease activity can be identified with less incubation time. The cost of protease detection assays developed during this work is quite low and, although 20 min analysis time is required per sample, the tests can be very cost efficient when run in batches, as would be expected in a commercial testing facility.