The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

Abstract

ABSTRACTFree fatty acids are important flavor compounds in cheese.Propionibacterium freudenreichiiis the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis byP. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants ofP. freudenreichiiCIRM-BIA1Tfor each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of thepf279gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of thepf774gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of thepf279gene sequence, leading to a premature stop codon, whereas they harbored apf774gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase inP. freudenreichiiand a key agent of Swiss cheese lipolysis.