| Abstract: | ABSTRACT 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. |
