Evaluating the spoilage potential of a thermostable protease from Bacillus cereus 12–1 in fluid milk

Foodborne pathogen Bacillus cereus is widely distributed in dairy products and the processing environment. Beyond its threat to human health, the production of thermostable protease also poses a significant challenge to the dairy industry, particularly in pasteurised or ultra-high-temperature (UHT) processed milk products. Despite this, our understanding of dairy spoilage caused by B. cereus is very limited. This study aimed to evaluate the spoilage potential of a thermostable protease produced by B. cereus 12–1 in fluid milk. Specifically, crude protease characterisation, including optimum pH and temperature, the influence of Ca 2+ on enzyme activity and heat stability of protease, was studied. Moreover, the effects of this protease on the quality of both whole and skim UHT milk were evaluated, including zeta potential and particle size of milk, hydrolysis of milk protein, release of free amino acids and change of milk microstructure. This crude protease has relatively good stability at wide-ranging pH values from 5.0 to 10.0 (79.67–166.67 U/mL) and temperatures from 20°C to 70°C (92.67–165 U/mL), and maintained stability of 46.04% and 38.31% after heat treatment by 65°C/30 min or 100°C/10 min. Spoilage patterns in skim and whole UHT milk differ significantly due to variations in fat content. The protease caused protein hydrolysis in whole milk (15.25%) and skim milk (17.09%), effectively hydrolysed κ-casein and β-casein, caused the release of free amino acids (Lys, Val, Tyr, Phe, Ile, Met and Leu) in UHT milk and significantly increased particle size (increase by 940 nm for skim milk and 1319 nm for whole milk), and reduced zeta potential (reduction by 4 mV for skim milk and 3.83 mV for whole milk) of UHT milk. Results indicate the spoilage potential of crude protease from B. cereus and highlight the need to prevent dairy spoilage caused by B. cereus .