Effect of heat-resistant protease of Pseudomonas fluorescens on the quality of liquid milk: A physicochemical and proteomic perspective

Pseudomonas fluorescens is one of the common psychrophilic bacteria in liquid milk. The thermostable enzymes produced by P. fluorescens are an important cause of changes in quality and flavor of liquid milk during storage. In this study, 3 thermostable proteases derived from P. fluorescens, neutral metalloproteinase (Nm), extracellular alkaline metalloprotease AprA (AprA), and metalloprotease AprX (AprX), were added separately to liquid milk. The upper layer protein content of the 3 groups of samples continued to decline and the lower layer protein content first increased and then decreased. The upper layer fat content in AprA and AprX groups initially decreased then increased, while the lower layer fat content showed the opposite trend. In the Nm group, upper layer fat continued to decline and lower layer fat steadily rose. Meanwhile, the particle size, acidity, and viscosity of all the samples in the 3 groups increased and showed gelation. Hydrolyzed liquid milk produced color deterioration, exhibited a bitter and sour taste, and the main odor was attributed to methyl groups, ketones, and acids. Proteomic analysis revealed AprA had an obvious hydrolysis effect on liquid milk, generating numerous κ-CN fragments (Q95100, E7E1P7, K9ZUL) in accumulated proteins. This study highlights the role of thermostable proteases in accelerating quality deterioration of liquid milk, providing a theoretical basis for controlling psychrophilic bacterial thermostable enzymes.